WELCOME TO MIG WALS SYMPOSIUM SO WE'VE GOT A PRETTY PACKED SCHEDULE, AND WE'RE GOING TO START A FEW MINUTES LATE. IT HAPPENS ALL THE TIME SOMETIMES. AND WE WILL JUST CUT OFF A LITTLE BIT OF THE LUNCH AND THAT'S OKAY BECAUSE THE GUIDELINES NOW ARE TO EAT LESS FOOD, SO THAT WILL HELP YOU OUT. SO I WANT TO THANK YOU ALL FOR COMING. AND I AM GOING TO START OUT WITH INTRODUCING OUR EMCEE. MY NAME IS STEVE ZULLO I'M THE COORDINATOR MITOCHONDRIA INTEREST GROUP, AND I'M HEALTH SCIENCE ADMINISTRATOR WITH NIBIB AND I WANT TO START OUT BY INTRODUCING OUR EMCEE AND FIRST SPEAKER, DR. TRACEY ROUAULT, WHO RUNS THE LABORATORY OF IRONWORKS FOR MITOCHONDRIAL CONSTRUCTION. AND WITH THE NICHD SO TRACEY? OH, ONE OTHER THING. UNTIL THE DEDICATED TECH GETS HERE TO BE DOWN HERE TO SET UP THE MIKE, WHEN YOUR TALK IS COMING UP, IF YOU'D GO TO THE BACK SO THAT DENISE BACK THERE CAN SET YOU UP SO THEY WILL HEAR YOU. THANK YOU. >> OKAY, WELL, I WANT TO THANK STEVE FOR PUTTING TOGETHER A WIDE- RANGING PROGRAM AND I'M PLEASED TO BE PART OF IT IT. WHAT I'D LIKE TO TALK ABOUT TODAY IS THE WORK THAT ME AND MY COLLEAGUES HAVE BEEN DOING AND THE REASON THAT IT'S RELEVANT TO TODAY IS BECAUSE OF THE MITOCHONDRIAL RES PRPIRATORY STRAINS AND SOMETHING I FIND IS SOMETIMES IGNORED BUT THAT SHOULD BE NOT IS THE FACT THAT THERE ARE 12 ION SULFUR SPLICES IN THE MITOCHONDRIAL RES PRPIRATORY AND BY VIRTUE OF THEIR PLACEMENT IN COMPLEX 12 COMPLEX 1 AND COMPLEX 2, THEY FUNCTION AS E ELECTRONIC WIRES. THEY HAVE THE UNIQUE ABILITY TO SINGLE ELECTRONS AND YOU HAVE HAVE AN ELECHTRON DOUGNATED AND HAVE IT MOVE UP THIS WIRE-LIKE SET OF CLUSTERS AND THEN MOVE ALONG THE TRACKS OF THE ARROWS THAT I AM SHOWING HERE. THE HIRYDROJEGENASE HAS 3 AND I'LL BE TALKING A GREAT DEAL ABOUT THEM IN THIS TALK. AND THE COMPLEX 3 HAS ONE ION THAT IS ALSO CRUCIAL. AND COMPLEX 4 HAS ONLY HEN, BUT I THINK THAT FU TAKE INTO ACCOUNT ALL OF THE ION SULFUR AND HOW DEEPLY THEY MUST BE BURR BURIED, PARTICULARLY WITH COMPLEX 1, I HAS 45 SUBUNITS ON THE INSIDE SET NUP PROXIMITY TO WITH ONE ANOTHER SO THAT THEY CAN TRANSFER ELECTRONS POSES AN INTERESTING INTELLECTUAL QUESTION. HOW IS THAT ACCOMPLISHED DURING ASSEMBLY? ION CLUSTERS COME IN SIEVE -- DIFFERENT FUNCTIONS. A COMMON TYPE OF CLUSTER IS THE ION, THE SULFUR IS THE YELLOW, SHOWN HERE. OFTEN LIG AATED BY THE SULFUR IN PRO SDPEENTZ TWO OF THE MOST COMMON I IRONS ARE THE TWO IRONS AND SULFUR AND 4 IRON CLUSTERS. AND THEN YOU GET INTO A MORE COMPLICATED ONE, THAT I'VE SHOWN HERE AND THEY'RE IMPORTANT IN THINGS LIKE THE NIGHT RONALDING RONALDINGEN ANASE. THERE ARE 3 WHICH I MENTION -- MENTIONED, MITOCHONDRIAL CLUSTER CLUSTER. MANY OF THE PROTEINS AND ENZYMES IN DNA REPAIR OX DATES AND THE LIST CONTINUES TO GROW. ONE OF THE CONFUSING THINGS ABOUT IRON SULFUR CLUSTERS IS THEY WEREN'T DISCOVERED UNTIL THE LATE 19YOU 60S, WHICH IS LATE COMPARED TO OTHER GROUPS. AND THE REASON, FIRST OF ALL, THAT THEY HAVE A LACK OF A DISTINCTIVE COLOR. THEY TEND TO BE SLIGHTLY BROWN BROWNISH SCHOOL.^AND IS THEY TEND TO BE SED ATIATION SENSITIVE AND THE CLUSTER IS NO LONGER THERE. AND THIRD, IT'S VERY HARD TO COME UP WITH AN ASSAY FOR THEM. I THINK A LOT OF PEOPLE RARE LOOKING AT PROTEINS AND HE DON'T KNOW IT. ANOTHER FEATURE IS TO KEEP IN MIND BECAUSE THIS WILL COME UP LATER IS THE CLUSTERS HAVE AN ALMOST UNIQUE ABILITY TO INTER INTERCONVERT BETWEEN ONE FORM AND ANOTHER. AND ONE OF THE CONVERSIONS THAT IS MOST EASILY ACHIEVED IS TEHE COV LESS ENENS OF TWO CLUSTERS IN A 4-IRON CLUSTER. AND IT HAS TO DO WITH QUANTIUM MECHANICS AND DELOCALIZATION OF ELEC TROOPS AND A VARIETY OF THINGS. BUT IT'S A VERY DESIRABLE PROPERTY AND . ELECTRONS. NOW I'D LIKE TO GO OVER THE GENERAL THEME FOR THE BIOGENESIS CLUSTERS IN MAMMAL IIAN CELLS. AND THIS WAS REALLY TRULY A BLACK BOX UNTIL 1994. AND THEN AT THAT POINT COLLEAGUES AT VIRGINIA TECHNIC INSTITUTE FOUND A CLUSTER BY A APPARATUS AND BACKED UP IN ECO. CLI. AND BASED ON THEIR WORK, WE BEGAN SEARCHING IN THE EXPRESS SEQUENCE DATABASES THAT WERE AVAILABLE AT THAT TIME FOR HOMO HOMOLOGS. SO QUITE SURPRISING LY WE WERE ABLE TO FIND QUITE A FEW. AND THEY INCLUDED, WHAT IS NOW REFERRED TO AS M SLESF, WHICH STANDS FOR NITROGEN FIX ATIATION, FROM THE ORIGINAL NITROGEN FIX ATIOATION OPER AND. AND FS 1 IS MAKES THE SULFUR IN AVAILABLE FORM SO THAT IT CAN INTRODUCE ITSELF INTO AN IRON SULFUR CLUSTER. TH THEN THERE IS A SCAFFOLD PROTEIN REFERRED TO AS ISTU AND THERE IS AN IST 11, WHICH MAKES ITS FUNCTION POSSIBLE BY MEANS THAT ARE NOT REALLY CLEAR TO US. AND ALONG THE WAY, IRON ARRIVED, PERHAPS CARRIED BY ANOTHER PRO PROTEIN, PRO TAXIN, WHICH IS KNOWN TO MANY PEOPLE AS PROTEIN THAT IS LOST IN THE DISEASE PRO TAX IA. AND YOU END UP WITH THE FORMATION OF 2-IRON SULFUR CLUST CLUSTERS ON ISTU PROTEIN HERE AND HERE IN THE COMPLEX IN WHICH THE CENTRAL FORM IS BETWEEN THE SULFUR AASE. AND THEN OTHER THINGS HAPPENED, WHICH IS A PROTEIN -- THE COCHAP ERRONE, KNOWN AS HSC 20. IT HAS A FEW OTHER NAMES BUT WE'LL STICK WITH THIS ONE FOR THIS TALK. BINDS TO ISTU, THEN ENTERS INTO ANOTHER COMPLEX. THIS IS THE COHAPPEN ER ONONE BOUND WITH C 9-TYPE PROTEIN THAT HAS ATPH ACTIVITY AND IT CARRIES IST ISTU WITH IT. AND THE ATP AASE ACTIVITY THEN LEADS TO A CONFIRMATION AL CHANGE IN THIS COMPLEX. AND THAT CONFORMATION AL CHANGE, WE THINK IS ASSOCIATED WITH THE ABILITY OF THE ISTU TO DEFORM ITSELF AND ALALLOW ITS IRON SUL SULFUR CLUSTERS TO BE TRANSFERRED TO A WHOLE HOST OF RESCIPIENT PROTEIN THAT COULD INCLUDE SXAFLDZ CANDIDATES FOR A PROTEIN CALLED NFE 1 OR DIRECTLY TO SOME RESCIPIENT IRON SULFUR PROTEIN. NOW WE WEREN'T INTERESTED IN THE SECOND PART OF THE PROCESS, WHICH IS AN INTERESTING QUESTION -- HOW DOES A MAMMALAL CELL KNOW WHERE TO PUT AN IRON SULFUR CLUSTER? WE THOUGHT THERE HAD TO BE SOME SORT OF INFORMATION CONTAINED IN THAT. AND REASON THAT WE REALLY THOUGHT THAT WAS THAT OTHERWISE THESE WOULD HAVE IRON SULFUR CLUSTERS. ZINC PROTEINS ARE OVEREXPRESSED IN ECO. CLI. IN FACT, THE ECO. CLI MAKE A MISTAKE. HOW DO MAMMAL IIAN CELLS KNOW TON MAKE THAT MISTAKE? A LOT OF THINKING HAS BEEN ALONG THE LINES THAT THE COCHAPRONE THAT I SHOWED YOU IN THE TRANSFER IS THE ONE THAT IS RESPONSIBLE FOR SPEC FIIFICITY IN THE PROCESS. AND WE DECIDED TO USE A YEAST HYBRID APPROACH TO IDENTIFY PRO PROTEINS THAT DIRECTLY BIND THE HSE 20. AND HERE, I THINK MANY OF PEOPLE HERE ARE FAMILIAR, BUT JUST TO REMIND YOU, THESE TWO HYBRIDS INVOLVE TAKING -- EXCUSE ME -- HUMAN PROTEINS TO PART OF A YEAST TRANSCRIPTION FACTOR. IN THIS CASE, HSC 20. AND THEN TAKING A LIBRARY THAT'S MADE OUT OF HUMAN PROTEINS WITH THEIR MITOCHONDRIAL SEQUENCES AND ALL THAT SORT OF INFORMATION REMOVED, THAT HAS TO BE ACTIVATE ACTIVATED DOMAIN FOR THIS TRANSCRIPTION FACTOR. WHEN THEY COME TOGETHER BY VIRTUE OF A PROTEIN-PROTEIN INTEROAACTION. NOW THEY ACTIVATE TRANSCRIPTION AND THAT CAN BE THE BASIS FOR SELECTION FOR PROTEINS THAT ARE PHYSICAL LY INTERACTING. AND IN OUR ASSAY, THE WAY THAT IT WORKS IS WE LOOKED FOR GROWTH GROWTH-ON PLATES AND IF WE SAW THE GROWTH, THAT MEANT THAT THIS TRANSCRIPTION HAD BEEN TURNED ON ON. AND WHEN WE SAW THAT, WE DID DNA SEQUENCING, AND THEN TO IDENTIFY BINDING PARTNERS. WE FOUND -- WE SCREENED A MILLION HUMAN GENE FORMS UNDER VERY STRINGENT CONDITIONS AND IDENTIFIED 54 PREY PLASMAS. AND THE LIST OF WHAT WE FOUND WAS CONTAINED IN A PAPER THAT WAS JUST PUBLISHED. SO I WON'T DISCUSS THAT EXHAUST EXHAUSTIVELY. BUT ONE OF THE THINGS THAT CAUGHT OUR EYE SIGNIFICANTLY WAS THE FACT THAT WE FOUND STHB AND WE POUND IT IN -- FOUND IT IN MULTIPLE FORMS AND THAT IS EN ENCOURAGE BECAUSE IT'S KNOWN TO CREATE IRON SULFUR CLUSTERS AND THAT LETS US TO KNOW THAT WE WERE ON THE RIGHT TRACK. THEN THE QUESTION IS IF WE HAD A CANDIDATE, THEN THE VERY WELLED VALIDATIONS-OF-THAT WOULD BE TO TAKE -- VALID ASIATION OF THAT WOULD BE TO TAKE A LIVING CELL AND LOOK TO SEE IF IT BINDS THE SCH D AND SEE IF WE FIND BIND HSC 20 20. AND WE WERE ABLE TO KNOW SHOW HA IT DOES INTERACT BOTH IN VI VITRO AND IN VIVO, WHICH IS THE MORE COMPELLING EVIDENCE. AND THAT IS SHOWN HERE. HERE IS THE IP WITH AN ANTI-STHB OSHOWN HERE AND YOU SEE HSC 20 COMING DOWN AND A N UNIT OF MATURE SDA. HERE 20 BRINGS DOWN B, JUST AS WE WOULD PREDICT BUT A DIFFERENT COCHAPRONE DOES NOT HAVE THAT EFFECT. AND THEN FURTHER, WE FOUND THAT THERE WAS A COMPLEX FORMATION BETWEEN HSC 20, ISB AND WE DO RE RESCIPROCAL COR COMMERCIALITC'S. SO HERE IS HSC 20 AND HERE IS A CONTROL. AND THEN HCSB AND CA 9 AND HAC 20. AND WE PUT THE COMPLEX TOGETHER LIKE THIS, WHERE WE HAVE HSBA 9, SCH B FIND BIND ING ING TO A PORTION OF THE AH C 20 WITH THE CLUSTER IN THIS COMPLEX. AND THEN THIS SUMMER, AS WE THINK ABOUT HOW HSCB -- AND HERE IS WHAT WE CALL A C 2 B COMPLEX THAT I JUST SHOWED YOU. AND UPON IRON SULFUR CLUSTER IN INSERTION, WE THINK IT OCCURRED ONE AT A TIME. THERE WERE THREE. WE THINK THAT PERHAPS EVEN SO IN INSERTION ALLY THE FIRST CLUSTER IS INSERTED AND THEN FOLDING BECAUSE IT'S LIG AATED BY THE SECOND AND THEN THE THIRD. AND THEN ONCE IT'S COMPLETELY FOLDED, IT ASSOCIATES WITH A TO FORM THIS COMPLEX, WHICH WE CALL P 2 A. THESE ARE INTERESTINGLY STILL IN THE MATRICX. AND THEN A FINAL STEP IS FOR THEM TO HOOK UP WITH H BCHBHC AND B WHICH ARE INTERMEMBRANE PROTEIN AND NOW THE COMPLEX IS COMPLETE AND YOU CAN SEE THE IRON SULFUR CLUSTERS COME IN. THERE IS AGAIN THE EFFECT OF A WIRE FORMING IN THE PROTEIN. NOW THE BINDING OF HSC 20 AND FORMATION OF THE COCHAPRONE COMPLEX IS REQUIRED FOR ASSEMBLY FUNCTION. WE DID A SERIES OF STUDIES WITH GEL ASSAYS AND THEN SEPARATED ON A SECOND DIMENSION. AND HERE YOU CAN SEE COMPLEX 2 B B, WHICH CONSISTS OF SBHB AND IS ISBU, AND JUST AS YOU I SHOWED YOU. BUT AS THE COMPLEX SXHACHZ GETS ITS IRON SULFUR CLUSTER, YOU END UP WITH COMPLEX 2, WITH S BCHBH C IN IT. BUT THESE OTHER ENABLING FACTORS HAVE DROPPED OUT. IF WE KNOCKED DOWN 20 AND 9, WE LOSE COMPLEX 2 ASELECT. AND HAVING LY -- ACTIVITY. AND INTERESTINGLY IF WE LOOKED AT LABELING, WE ALSO LOSE THE ABILITY OF IRON TO BE INSERTED, AS YOU CAN SEE CLEARLY HERE AFTER FIVE DAYS OF SILENCEING OF THESE TWO INSERTIONS. THEN FURTHER, HSC 20 HAS A REGION THAT ACTIVATES ATPA. WE MUTEIJENIZED TO SEE WHAT EFFECT IT HAD ON THE ASSEMBLY OF THE CLUSTER. AND INTERESTINGLY, IT HAS A PRETTY SIGNIFICANT EFFECT. HERE WE SEE THE IMPORT OF INVI INVITRO HC SB INTO MITOCHONDRION MITOCHONDRION, AND WE CAN SEE THE COMPLEX FORMING. AND AT THE END WE HAVE, AFTER 30 MINUTES, A MATURE COMPLEX. AND THE COMPLEXES ARE DROPPED OUT BY THAT TIME. IF YOU COMPARE THAT TO THE SCHB IN THE SETTING WHERE HSC, YOU CAN SEE THAT THE COMPLEX STARTS TO FORM, BUT IT CAN'T GET THE JOB DONE AND IN THE END THERE IS NO COMPLEX. IT MAKES SENSE THAT ENERGY WOULD BE REQUIRED FOR THE THINGS THAT WE'RE TALKING ABOUT. AND DOWN HERE, YOU CAN SEE BY THE IRON RADIO GRAM, WE SEE IRON IN COMPLEX 2B AND THEN WITH MATURIZATION INTO A MORE MATURE COMPLEX SCOMBRUCHLT NO SUCH IRON INCOP RATION INTO OUR HSC 20. THE C TUMOR AL DOMAIN BINDS TO BOTH D AND ISDU. AND IN MUTATIONS AGGREGATE THAT. AND THEN WE WANT TED TO KNOW EXACTLY WHAT PORTION OF HSC 20 -- WHAT PORTION B WAS BEING RECOGNIZED BY HSC 20. AND THAT WAS A FEN APHENOMENAL LLY COMPLICATED QUESTION BECAUSE IN THE END IT TURNED OUT THAT WE HAD THREE MONTIFS TO WHICH HSC 20 BINDS. AND THE ONLY WAY THAT WE WOULD WORK TOUT BY GOING BACK SCAAND DIV DIVIDE NG THE HSC B INTO MULTI MULTIPLE TEXTURES, BETWEEN 50 AND 100, IN WHICH WE LOOKED FOR THOSE THAT WOULD INTERACT WITH H HSC 20 AND THOSE THAT DIDN'T. ALONG THE WAY WE GOT CONFUSED BECAUSE INITIAL LY WE DIV WHITIDED IT IN HALVE AND BOTH HALVES WERE POSITIVE. AND THEN YOU'LL SEE WHEN I POINT OUT THE MONTIFS HOW IT WOULD BE CONFUSING. WE FINALLY GOT DOWN TO THIS IN WHICH WE SAW THIS MONTIF IY RCHLR AND THIS MONTIF LY RCHLR. AND THROUGH MUTEIGENESIS OF THESE INTO TRIPLE CANCER, THE R -- WE KNOW THAT DISTINCTION OF THESE CAUSES TERRIBLE TROUBLE IN THE CELLS AND THEY LOSE HC SB. PUT TOGETHER, WHAT WE THINK WE KNOW AND UNDERSTAND ABOUT IRON SULFUR CLUSTER ACQUISITION AND THE MONTIFS THAT ARE IMPORTANT. THE FIRST LY RCHR MONTTIF OWCCURRED IN AN UNSTRUCTURED REGION NEAR THE ENTERUS AND THAT BINDS THE COMPLEX THAT CONTAINS THE IS ISBCHISBU AND IT INSERTS AN IRON SULFUR CLUSTER, WHICH IS A 2-IRON SUL SULFUR CLUSTER INTO THIS HCB. THEN FOR THE 3 IRON SULFUR CLUST CLUSTER, THE SECOND MONTIF LYR IS LOCATED HERE AND WE THINK THAT THAT IS ENABLING THESE CLUSTERS TO BE POSITIONED. AND IF THEN FOLDING OCCURS, PARTLY IS DRIVEN BY THE FACT THAT THE SIFTINES LIG AATE THE CLUSTER. INTERESTINGLY, THESE MONTIFS ARE VERY HIGHLY CONSERVED AND WE WERE INTERESTED TO SEE, AS FAR AS THE IY RCHR AND LY RCHLR GOES, THAT IT GOES DOWN NOT ONLY TO PLANTS BUT ALSO TO ECO. CLI. THIS, THEREFORE, IS A MONTIF OF IMPORTANCE THAT EXTENDS THAT FAR IN EVOLUTION. AND WE ALSO WERE AWARE THAT GOL ERMO FAMILY AND YOU CAN FIND IT AS A COMPLEX WHERE ONE SOUPER FAMILY ON DATABASES AND YOU CAN SEE THESE ARE ONLY ANNOTATED BECAUSE THEY'RE IN U CAR IOT. FOR THE MOST PART IS A DESCRIPTION, FRONTS, ONE OF THE ASSOCIATED COMPLEX 3 DEFICIENIFICIENCY. BUT NOTE EXACTLY HOW THEY WOULD BE WORKING. WE DID A LITTLE BIT OF WORK ON L LY RCHR M 8, WHICH IS KNOWNING AS SB SBH 1 AND IT INTERACTS WITH 20 AND ENDS WITH SH -- SCH B. AND IT'S A CONTACT POINT THAT WE HAVE NOT YET DEFINED COMPLETELY. BUT CERTAINLY, THE PORTION OF SD SDHB IS IT INTERACTS CLEARLY BECAUSE IT DIVIDES AND IT INTER INTERACTS WITH THE SECOND HALF, AS IS SHOWN HERE IN COLONO COLONOPRESCRIPTION OF THE SDH D. AND THEN HERE IS OUR MODEL. WE HAVE THE IY RCHR MONTTIF. WE GET THE INSERTION OF THE 2 IRON SUL FER CLUSTER. AND THEN SD SHELTH A FCHLF IS 1 BINDS TO A NON-LY RCHR SITE BUT ITTARIANS A -- CONTAINS A MOTEEF AND BRINGS AMONG THIS ENTIRE COMPLEX. AND LY RCHR ITSELF BINDS THE MOTEEF. AND THEN WHAT MIGHT -- MONTIF. AND THESE TWO MAY BE BOUND AT THE SAME TIME AND IN PROXIMITY SUCH THAT THIS IRON SULFUR CLUST CLUSTER AND THIS CAN DO THE MAGICAL TRANSFORMATION THAT I MENTIONED EARLIER, WHICH IS TO FORM A 4-IRON SULFUR CLUSTER, WHAT IS WHAT WE INHERITED AT THE THIS POINT. AND HERE AGAIN IS A WORKING MODEL. AND FINALLY, WHEN WE PUT ALL OF THESE VARIOUS THINGS TOGETHER, WE END UP WITH A CLUSTER, WHICH CONTAIN THE 3-IRON SULFUR CLUST CLUSTERS. ONE OF THE IMPORTANT THINGS IS THE SENSE OF DIRECTIONALITY THAT ONE OCCURS FIRST. AND FU DON'T GET THE FIRST IRON CLUSTER, PROTEIN IS IMMEDIATELY DEGRADED. THERE IS NO HOPE FOR A SECOND AND THIRD. FINALLY, THEN YOU GET ASSOCIATION WITH SD SHELTHA. AND ANOTHER PROTEIN L 7, APPEARS TO BE INVOLVED IN OBTAINING ITS IRON SULFUR CLUSTER. YOU CAN SEE COPRESCRIPTION BETWEEN BINDING OF 7 AND WE'RE WORKING ON THAT. AND HERE IS A MODEL. HERE IS THE PROTEIN THAT WILL RECEIVE THE IRON SULFUR CLUSTER. AND IT OCCURS LATE. IT DOESN'T HAVE TO BE PUT INTO THE ACTUAL INSIDE OF THE COMPLEX COMPLEX, SO NOT NEARLY AS CHALLENGING AS S BCHLBH D OR COMPLEX 1. AND THEN TWO ANNOTATED MEMBERS OF THE LY RCHR FAMILIES ARE COMPLEX 1 AND H 6, WHICH IS ALSO CALLED POLYMER M 6 AND D 9, WHICH IS M 3. AND WE CAN SHOW THAT THERE IS AN INTEROAACTION BETWEEN N 9 AND HSC 9 AND 20, SHOWN HERE. AND WE FOUND ANOTHER, IN WHICH THE LY RCHR, UNLIKE THE DEFINING FEATURE IN THESE PROTEINS, WHERE THE LY RCHR IS IT'S SUPPOSED TO BE ENTERMNUS. THIS IS ACTUALLY AT THE END. WE THOUGHT IT MIGHT BE FUNCTION FUNCTIONAL. AND IN FACT, IF WE DO AN IT OF IT, WE DO AGAIN GET A CA 9 AND 20 TO COPRECIPITATE. SO TO SUMMARIZE, WHERE MONTIFS ARE BINDING SITES FOR HSC 20, IT IS THE COCHAP PER ONONE THAT CHAP EPER ONES THE MOVEMENT OF THE IRON SULFUR CLUSTER INTO POSITION TO FACILITATE TRANSFERRING CLUSTERS TO SPECIFIC RESCIPIENTS. HC SB HAS TWO SUCH MONTIFS AND REQUIRES TWO CLUSTERS. WE FIND THAT THE PRINCIPLE HERE IS MORE GENERAL I'IZABLE AND NOW THREE COMPLEX ONE SUBUNITS A APPEAR TO UNDERGO THE EXACT SAME BINDING OF THIS TRANSFER COMPLEX COMPLEX. AND THAT IS REALLY VERY INTERESTING, BECAUSE ONE OF THE MYSTERIES ABOUT COMPLEX 1 AND ITS 45 SUBUNITS IS HOW THE CLUST CLUSTERS MAKE THEIR WAY TO THE VERY MIDDLE OF THE COMPLEX. IT WOULD HAVE TO OCCUR IN STAGES AND PERHAPS WE'LL BE ABLE TO DETECT SOME OF THESE STAGES USING WHAT WE KNOW ABOUT CANCER. AND THEN MONTIFS CLEARLY APPEAR TO BE IMPORTANT IN SULFUR CLUST CLUSTER ACQUISITION IN THE SES RES PRPIRATORY CHAINS BUT WE HAVE A LONG LIST OF PROTEINS THAT ARE NOT RES PRPIRATORY THAT CONTAIN THE MONTH TIFS AND ARE BOUND TO HSC 20. WE ARE WORKING ON THEM AND SOME OF THEM ARE THOUGHT-PROTECTING AND WE THINK THAT ONE WAY TO THINK ABOUT THIS IS IN DIFFERENT WAYS TO FIND PREVIOUSLY UN UNIDENTIFIED IRON SULFUR PRO PROTEINS. BECAUSE IT'S SO HARD TO WORK WITH, IT'S HARD TO SEE -- VERIFY THEM. BUT IF THEY HAVE A MONTIF, THEN MOST LIKELY WOULD BE A GOOD RE RESCIPIENT AND PERHAPS FIND MORE. THE TREND, ESPECIALLY IN THE LAST YEAR, IS FOR MANY, MANY MORE PROTEINS IN WHICH CASES ABOUT DNA REPAIR AND SCIENCE LAST YEAR. MANY, MANY MORE IRON SULFUR PRO PROTEINS THAN IS PRESENT RECOGNIZED AND IS IMPORTANT. I'D LIKE TO STOP HERE AND THANK MY GROUP AND ESPECIALLY MYO, SHOWN HERE, WHO DID MOST OF THE WORK THAT I SHOWED YOU IN THIS TALK. DID HYBRID ASSAY AND MANY OF THOSE STUDIES AND THESE TWO DID MOST OF THE WORK, BUT WITH MUCH HELP FROM MANY OTHER PEOPLE IN THE GROUP. I DO URGE YOU TO CHECK HEMOTOSIS HEMOTOSIS, IN AND YOU'LL HAVE ALL OF THE DETAILS OF THIS AND HER PAPER IN METABOLISM FROM JUST A COUPLE WEEKS AGO. SO THANK YOU. I'D LIKE TO TAKE QUESTIONS. APPLAU [APPLAUSE] INAUDIB[INAUDIBLE] >> WELL, ALL OF THEM WERE NUCLEAR ENOLD AND THEY CONTAINED MITOCHONDRIA TARGET. ALTHOUGH SOME OF THEM ENDED UP BEING IN THE NUCLEUS BY VIRTUE OF ALTERNATIVE CLUSTERING. THERE ARE NONE THAT WE KNOW OF THAT IS MITOCHONDRIAL. >> VERY INTERESTING TALK, THANK YOU. FROM AN INTELLECTUAL POINT OF VIEW, I WAS WONDERING ABOUT THE OXIDATIVE. DO YOU SEE ANY CHANGES IN THE RATIO OF THE COMPLEX FORMATION AND/OR IF YOU SEE IT IMPAIRED SO THAT THE COMPLEX IS -- ENABLES SIMILAR TO THE TRIPLE -- THAT YOU ARE REFERRING. >> WELL, THAT'S AN INTERESTING QUESTION. WE HAVEN'T DONE THAT SORT OF STUDY, BUT CERTAINLY WE WOULD PREDICT THAT THE EXPRESS WOULD BE BAD FOR THIS, BECAUSE WE NEED NEED, FIRST OF ALL, -- AND EVEN THOUGH THE IRON SULFUR CLUSTER IS ENSHROUDED BY A CHAP PER ONONE AND APPEARS TO BE VERY WELL PROTECTED, PROBABLY NOT I WOULD SAY PERFECTLY. SO I THINK IN THE END THERE WOULD BE A PROBLEM, YEAH. >> I HAVE ANOTHER QUESTION. IT'S NOT A QUESTION. THERE ARE SOME CELLS THAT ARE VERY DEPENDENT ON MITOCHONDRIAL ACTIVATED METABOLISM AND GLY COL SIS-DEPENDENT. DID YOU SEE THE SIMILAR ITY IN MAYBE DAMAGE, OR OXYGEN A ASSIMILATION IN DIFFERENT TISSUES? >> WELL, THE MITOCHONDRIA -- WE HAVE ONE CELL LINE THAT HAS A MUTATION IN THE FIRST IY RCHLR MONTTIF AND THE R IS A GLUT AMINE CELL AND THOSE CELLS, IN THE MITOCHONDRIA, ARE REALLY A DISASTER. THEY'RE BROKEN UP. THE EXTENT OF THE HARM IS EVEN PAST WHAT WE WOULD SUGGEST. SO A LOT OF TROUBLE. AND THAT CELL IS ALMOST PERFECTLY AN AROBEROBIC. WELL, IT DOES NOT REQUIRE. >> THANK YOU VERY MUCH. >> YES. YOU'D POINTED OUT THAT THERE ARE TWO LY RCHRS WITH THE COMPLEX 2 AND THEN YOU HAVE THREE IN COMPLEX 1 WITH A YR. AND THERE ARE, I THINK, 6 TO 8 IRON SULFUR CELLS IN COMPLEX 1. DO YOU THINK AS IN COMPLEX 2, WHERE TWO LY RCHRES ARE IN THE CENTERS, THAT MAYBE THOSE THREE SUB UNITS WILL GIVE YOU OTHERS OR DO YOU THINK THERE ARE OTHER SYSTEMS THAT ARE REQUIRED TO MAKE ALL THE COMPLEXES? >> WELL, IT'S A GOOD QUESTION. WE HAVEN'T REALLY DELVED INTO IT VERY FAR AT ALL. WE'RE JUST PLEASED THAT THERE ARE ANY, IN WHICH LY RCHR APPEARS TO BE A KEY. 3 COULD BE ENOUGH. IF THEY HAVE A REPEATING MECH MECHANISM. BUT WHAT WE, OVER THE LONG TERM TEND TO DO IS BREAK DOWN AT SEM ASSEMBLY INTO ITS PARTS, WHICH IS A MUCH EASIER GOAL. AND TRY TO FIND THE SUBCOMPLEXES SUBCOMPLEXES. AND THAT MAY HELP US IN THE END TO FIGURE OUT IF SOMETHING IS DOUNATING TO MORE THAN TWO OTHER SUBUNITS OR NOT. >> DO YOU KNOW -- DO YOU KNOW IF THE TIYROSINE RESIDUE CAN BE FOPHOSPHORYLATED IN LY RCHLR, ARE THEY IN A POSITION WHERE THEY CAN BE FOPHOSPHORYLATED? IN OTHER WORDS, IS IT POSSIBLE TO FOPHOSPHORYLATION -- IN FOSS PHOSPHORYLATION TO REGULATE THE FUNCTION OF THESE CELLS >> WELL, I DON'T THINK WE CHECKED AS AS MUCH AS WE COULD. SO WE THINK PROBABLY NOT, BUT WE NEED MORE DATA. >> OKAY. WELL THEN NOW I AM GOING TO SWITCH ROLES AND INTRODUCE MARK MATTSON. MARK IS A CHIEF OF THE LABORATORY OF NEWUROSCIENCES AT THE NATIONAL INSTITUTE ON AGING IN BALTIMORE, WHERE HE'S ALSO A PROFESSOR IN THE DEPARTMENT OF NEWUROSCIENCE AT JOHNS HOPKINS. HE'S A DIRECTOR IN COURSE IN NEWUROBIOLOGY AND AGING. HE'S LED A MULTIFACETED TEAM IN TECHNOLOGY AND RESEARCH AND CELL CELLULAR AGE AND ALZHEIMER'S, PARKINSON'S. HIS WORK HAS ELOUCIDATEED HOW THE BRAIN RESPONDS TO CHALLENGES CAN AND HE HAS USED THAT INFORMATION INTO THE DEVELOPMENTAL INTER INTERVENTION FOR OPTIMAL BRAIN FUNCTION. >> OKAY. SO WE KEEP ON -- TO KEEP ON TIME TIME, I AM GOING TO MOVE TO 20 MINUTES. I HAVE MY STOP WATCH, AND I'LL START IT. THIS IS A HIPPOCAMPUS IN THE NEWUROCIRCUITRY WHICH IS AN IMPORTANT BRAIN REGION IN LEARNING AND MEMORY AND MY LAB AND HENRY'S LOB HAS DONE A LOT OF WORK ON EXERCISE, ON PLAQUE PLAQUEITY IN THE HIPPOCAMPUS. WE'VE DONE WORK ON -- AND MANY LABS HAVE DONE WORK ON ENVIRONMENTAL ENRICHMENT. AND ALL THESE MANIPULATIONS WOULD SEEM TO BE GOOD FOR FUNCTION OF HIPPOCAMPUS, BUT MAY PROTECT AGAINST THIS FUNCTION OF HIPPOCAMPUS DURING AGE, RESULT RESULTING IN INCREASED ACTIVITY AND ALL SORTS OF PRODUCTION OF NEWUROPATHIC PATHS. IT SEEMS TO PLAY A CRITIC AL ROLE IN THE ENHANCEMENT OF LEARNING AND MEMORY AND RESPONSE TO THESE ENVIRONMENTAL CHALLENGES BY IN INCREASING THE NUMBER OF SYNAPSE SYNAPSES, INCREASING THE STRENGTH OF SYNAPSES, AND ALSO BY PROMOTING THE DIFFERENTIATION OF STEM CELLS, WHICH RESIDE IN THIS REGION OF THE HIPPOSAMPSON AND THE NEW NEWURONS WHICH THEN INTEGRATE INTO THE OF THE HIPPO HIPPOCAMPUS. I AM GOING TO TALK ABOUT TWO DIFFERENT STUDIES DONE BY CHANG, WHO IS A SENIOR MEMBER OF MY LABORATORY. THE FIRST ONE ASKING THE QUESTION WHETHER EXERCISE AFFECTS ON THE -- EXERCISE EFFECTS ON THE BRAIN ARE SIMILAR TO EFFECT ON MUSCLE WITH REGARDS TO MITOCHONDRIAL CHANGES. AND THEN THE SECOND STUDY THEY'LL GOING TO TALK ABOUT IS WORK WE'VE DONE ON SUPEROXIDE PRODUCTION AND ITS ROLE IN PLAQUEITY, PARTICULARLY IN NEWURO NEUROGENESIS. PLASTICITY. SO I CAN ADVANCE THE SLIDES HERE OR NOT? OKAY. OKAY, I GOT IT. OKAY, PEOPLE WHO STUDIED THE EXERCISE IN THE MUSCLE HAVE SHOWN THAT EXERCISE WILL IN INCREASE THE NUMBER OF MITOCHONDRIA IN MUSCLE CELLS, IF THAT MAKES SENSE. SO THAT THEY CAN HAVE MORE AT. AND MORE ENERGY TO FUNCTION. AND THE MECHANISM INVOLVES PRODUCTION OF REACTIVE SPECIES AND KIN AASE, WHICH IN TURN UP UPREGULATES A TRANSCRIPTION FACTOR CALLED PGC 1 ALPHA AND THIS INDUCES THE EXPRESSION OF MULTIPLE GENES THAT ENCODE PRO PROTEINS THAT ARE CRITIC AL FOR THE GROWTH AND VISION OF MITOCHONDRIA. SO IT WOULD -- CHANG ASKED A SIMPLE QUESTION. WE STARTED WITH HIPPOCAMPUS NEWURONS DERIVED FROM THE BRAIN. THESE NEWURONS -- FIF YOU LOOK AT THEM OVER TIME AND CULTURE, THIS IS A NEWURON 3 DAYS IN CULTURE, STAINED WITH MIGTOTRACK EER REDS AND WE ARE LABELING MITOCHONDRIA MITOCHONDRIA. THERE IS ANOTHER NEWURON TEN DAYS IN CULTURE. AND WHAT YOU SEE IS THE NEWEURONS ELABORATE AND AS THEY ELABORATE, THE NUMBER OF MITOCHONDRIA IN INCREASE AND THE MITOCHONDRIA ARE DISTRIBUTED INTO THE GROWING DENDRITES AND THAT'S QUANTIFIED NIR COUPLE OF WAYS. AS, AS THESE NEWURONS GROW, AND I SHOULD SAY AS THEY FORM SYNAPSE S WITH OTHER NEWURONS IN THE CULTURE, THE ATP LEVELS INCREASE INCREASE. OKAY, SO THE ROLE OF PB C 1 AND MITOCHONDRIA BIOGENESIS IN THE FORMATION OF SYNAPSES IN THESE DEVELOPING HIPPOCAMP O NEURONS IN CULTURE, THEY GENERATED A DENO DENOVIRUS CONSTRUCTS. THESE ARE KNOCKDOWN PBC ALPHA USING SMALL INTERFERING RNAS OR SHE OVEREXPRESS THE C DNA OF PGC 1 ALPHA, WHICH SHOWS (M)RNA LEVELS IN URONS WHEN SHE KNOCKS DOWN OR OOVEREXPRESSES ALPHA. THIS IS A PROTEIN LEVEL. THIS IS A BASAL LEVEL AND NEWER NEURONS INFECTED WITH CONTROLLED VIRUS. AND HERE IS KNOCKDOWN AND OVER OVEREXPRESSIONS. SHE KNOWS WHICH NEWURONS ARE IN INFECTED WITH THE VIRUS AND SHE SHOWS THAT INDEED, WHEN SHE IN INFECT THE NEWURONS WITH HRAN, THE IMMUNOINTERACTIVITY IS GREATLY REDUCED, COMPARED TO UN UNINFECTED NEWURONS AND WHEN SHE OVEREXPRESSES, INDEED THE NEWER NEURONS THAT TAKE UP THE VIRUS HAVE RELATIVELY GREATER LEVELS OF PG C 1 ALPHA IMMUNOACTIVITY. ACTUALLY, SHATHAT'S SHOWN HERE. OKAY, SO THEN WE SEE WITH INFECT INFECTED NEWURONS 3 DAYS IN CULTURE AND QUANTIFIED SYNAPSES AT 10E DAYS IN CULTURE BY COUNT COUNTING THE NUMBER OF DEDRINDRITIC SPINES WHICH ARE SYNAPSE SIGNIFICAYNAPTIC REGIONS OF SYNAPSES THAT HAVE FORMED. SO THIS IS A DENDRITE HERE IN HIGH MAGNIFICATION. THESE PERP DIENDICULAR PROJECTIONS ARE THE SIGNIFICAYNAPTIC REGIONS OF THE SYNAPSES. WHAT SHE FINDS IS SHE KNOCKS DOWN PGC ALPHA, THE NUMBER OF DEDRNDRITIC INCREASES. WHETHER SHE KNOCKS DOWN ALPHA, THE NUMBER OF DENITY -- DENSITY INCREASES. THE BEAUMOOTTOM LINE IN THE DEVELOPING NEURONS AND CULTURE, FU KNOCK DOWN PG C ALPHA, WHICH WILL REDUCE MITOCHONDRIAL BIO BIOGENESIS. IT'S IN THE PAPER -- THAT WILL DECREASE SYNAPSE FORMATION. SO THAT SUGGESTS THAT PGC 1 ALF ALPHA AND BIOGENESIS ARE CRITIC AL FOR THE FORMATION OF SYNAPSES DURING DEVELOPMENT. THEN WE ASKED THE QUESTION WHAT ABOUT IN THE ADULT HIPPOCAMPUS? HOW WILL MANIPULATING PGC 1 ALF ALPHA AFFECT SYNAPSES, AND SHE INJECTED ADENOVIRUS WITH PGC 1 ALPHA WITH HRAN INTO THE -- AND SHOWN HERE ARE MANY NEWURONS IN INFECT WITH THE VIRUS. SHE SHOWED BY IMMUNOSTAINING THAT NEWURONS INFECTED WITH THE VIRUS HAVE REDUCED LEVELS OF PGC 1 ALPHA IMMUNOACTIVITY, COMPARED TO NEWURONS IN THE GYRUS INFECTED WITH A CONTROLLED VIRUS. THEN A NUMBER OF BRANCHES AND NUMBER OF DEDRINDRITIC SPINES, WHICH WERE ESSENTIALLY SYNAPSES. AND WHAT SHE FOUND THAT OVER A PERIOD OF WEEK AFTER INFECTION THERE IS A DECREASED NUMBER OF SYNAPSES, INDICATING THAT PGC 1 ALPHAA IS CRITIC AL FOR THE MAINTENANCE OF SYNAPSES IN THE ADULT HIPPOCAMPUS. I SHOWED IN THE FIRST SLIDE THAT EXERCISE, FASTING AND HOPEFULLY ALL OF YOU ARE DOING NOW IS THINKING WHAT ABOUT WHAT I AM DOING. SO YOUR NEWURONS ARE ACTIVE IN THE HIPPOCAMPUS RIGHT NOW. EVERYBODY IS UPREGULATING BDF. WILL IT AFFECT BIOGENESIS? SHE MADE LOSEIFER AATES TO LOOK AT PGC 1 ALPHA MOTOR ACTIVITY AND HAD A CONSTRUCT WITH AND WITHOUT BINDING JIESHTHS WHICH IS KNOWN TO UPREGULATE PGC 1 ALPHA AND SHE FINDS THAT BDNF INCREASES CG CGC 1 ALPHA, IS UPREGULATED. PGC 1 ALPHA AND THAT DEPENDS ON THE KREBS BINDING SITES. AND SHE DID MANIPULATION OF BIND BINDING PATHWAYS OF THE TRACK P BY USING DRUGS THAT COLLECTIVELY BLOCK THE KIN AASE WITH THE LY COMPOUND OR THE IRK MAP KIN AASES, AND SHE FOUND THAT IF SHE IN INHIBITS THE IRK, B NCHNF ABILITY TO UPREGULATE PGC 1 ALPHA IS ATTEN ATTENUATEED, INDICATING THAT THE IRKS ARE PRETTY COOL. THEN WE FOUND THAT BD NCHNF LIN CEASE THE NUMBER OF SYNAPSES IN HIS EMBRYONIC NEWURONS, SO THESE ARE TREATING THE NEWURONS WITH BD BDNF, BEGINNING ON DAY 3. AND IN THIS CASE, SHE'S USING ANOTHER WAY TO LOOK AT THE NUMBER OF SYNAPSES BY IMMUNO IMMUNOSTAINING WITH AN ANTIBODY TO POST SIGNIFICAYNAPTIC DENSITY PROTEIN 95. SO IT UPREGULATES OR INCREASES THE NUMBER OF SYNAPSES. BUT IF YOU KNOCK DOWN PGC 1 ALF ALPHA, THE ABILITY TO INCREASE SYNAPSES IS ESSENTIALLY A ABOLISHED. AND MODIFIED THIS BY AIMMUNO AIMMUNOBLOCK. SO ALTOGETHER, THESE DATA SUGGEST THAT PERHAPS, ALTHOUGH WE HAVEN'T SHOWN THIS YET, BUT PERHAPS SIM LYE THAT UPREGULATE BD NCHNF -- ACTUALLY THERE IS ONE PUBLISHED PAPER FROM A DIFFERENT LAB. BUT WE FIND THAT BD NCHNF, THROUGH IRK AND KREBS PATH WWAY, UP UPREGULATES PGC 1 EVILLY AND WILL INCREASE THE NUMBER OF MITOCHONDRIAL NEWURONS. AND THIS IS ASSOCIATED WITH THE FORMATION OF SYNAPSES IN DEVELOPING HIPPOCAMPUS AND MAINTENANCE OF SYNAPSE IN THE ADULT AND NOT YET KNOWN, PATHI -- PATH WWAY ON LEARNING AND MEMORY, WHICH ONE WOULD PREDICT THERE MAY BE A SYNAPSE -- SYNAPSE ARE THE SUBSTRATE, WE THINK, OF LEARNING AND REMEMBER. OKAY, THE SECOND HAS TO DO WITH SUPEROXIDE PRODUCTION IN MITOCHONDRIA. IN COLLABORATION WITH HIS OLDER BROTHER, WE DEVELOPED A PROBE THAT WE THINK IS A FAIRLY GOOD REPORTER OF MITOCHONDRIAL SUPER SUPEROXIDE. IT'S A FLUORESCENT PROBE THAT IS TARGETED TO MITOCHONDRIA. IT HAS A MITOCHONDRIAL TARGETING SEQUENCE. SO WE CAN INSERT CELLS WITH THIS CIRCUMSTANLAR PER MUTED YFP. AND TO SHOW THAT IN RESPONSE TO CONDITIONS THAT ARE AFFECT EXPECTED TO INCREASE SUPEROXIDE PRODUCTION, FOR EXAMPLE OXIDATE, WOULD GET AN INCREASE IN SIGNAL. THIS PARTICULAR DATA ARE FROM CULTURE AND CARDIAC SITES. AND THEN IF WE ADD, IT WILL A ATTENUATE THE ABILITY OF OXIDASE TO INCREASE THE SIGNAL. AND IN THIS PAPER WHICH WAS PUBLISHED IN "CELL NOW" SIX YEARS AGO, THERE IS STENEXTENSIVE CHARACTERIZATION AND PROBABLY MORE SINCE THEN. THIS IS SOME WORK THAT WE DID IN THIS INITIAL PAPER. THIS IS IMAGEING MITOCHONDRIAL SUPEROXIDE IN DENDRITES OF CULTUREED HIPPOCAMPONEWURONS. SO THIS IS A MITOCHONDRIA, FOR EXAMPLE, A DIFFERENT -- AT DIFFERENT TIME POINTS. AND WHAT WE FIND IS THAT THE MITOCHONDRIA WILL EXHIBIT INTER INTERMITTENT BURSTS OF SUPEROX SUPEROXIDE PRODUCTIONS WITH THIS KIND OF KIN EIGHETIC SHOWN HERE OVER A PERIOD OF SECONDS AND THEN RECOVERY. THESE SUPEROXIDE, WHAT WE CALL FLASHES, ARE IMMEDIAMEDIATED BY THE TRANSITION PART BECAUSE THEY CAN BE BLOCKED WITH SIGHCLO SPORIN OR ENHANCED BY DRUGS THAT OPEN THE PERMABILITY TRANSITION. WE DON'T KNOW EXACTLY WHAT'S GOING ON AT THE MOLECULAR LEVEL, BUT WE THINK THAT THE PERM PERMABILITY TRANSITION FOR PRO PROTEINS MAY BE CLOSELY, PERHAPS EVEN PHYSICAL LY ASSOCIATED WITH ELECTRON TRANSPORT CAME IN COMPLEX WHERE THE SUPEROXIDES GENERATE. THIS JUST SHOWS HERE A STUDY WHERE WE KNOCKED DOWN -- ACTUALLY KNOCKED DOWN WE CAN KNOCK DOWN PSYCHELO PHILIN D WHICH IS INVOLVED IN TRANSITION PORES AND WE FIND IF WE KNOCK KNOCKDOWN PSYCHELO PHIL D, IT IN INCREASED THE PHRENFREQUENCY OF THE SPONTANEOUS SUPEROXIDE FLASHES. OKAY. SO THE IDEA HERE IS THAT OPENING OF THESE PERMABILITY TRANSITION PORES LEADS TO SUPEROXIDE PRODUCTION. SO WHEN THE PORES OPEN, THERE IS A BURST OF SUPEROX IITE PRODUCTION BUT THEN RECOVERS. SO THERE SEEMS TO BE A ROLE FOR CASTLCIUM IN THIS PROCESS IN THAT IF WE MANIPULATE AND UPDATE INTO THE MITOCHONDRIA, IT CAN AFFECT THE SOUPER OXIDE FLASHES. SO NOW WHAT I AM GOING TO TELL YOU IS A STUDY DONE BY HAO, WHO WAS A POST DOC FELLOW IN THE LAB AND WAS CLOSELY MENTORED BY CHANG AND ME. AND SO WHAT WE WERE INTERESTED IN WHAT HAPPENS IN THE DIFFERENT DIFFERENTIATION OF NEWUROPRO GENT NEUROPROGENITOR CELLS IN THE NEWURONS. AND HE DID A LOT OF WORK LOOKING AT, FOR EXAMPLE, B NCHNF SIGNALING AND HOW IT PROMOTES DIFFERENTIATION OF THESE NEWURO NEUROPRO GEGENITOR CELLS AND THE STEM CELLS. SO HERE WHAT WE HAVE IS THIS TIME-WRAPPED IMAGEING OF MITOCHONDRIAL SOUPER OXIDE. AND FOR EXAMPLE, HERE IS A SING SINGLE NEWUROPRO GENGENITOR CELL AND CULTURE OVER TIME. AND IF WE LOOK AT THE MITOCHONDRIA IN THESE NEWURONS AND THEN LOOK AT THE FLUORESCENCE, WE FIND THAT THESE FAILULUOROPRO GENGENITOR CELLS DO PROHIBIT EXHIBIT SUPERER OXIDE FLASHES BUT ACTUALLY THEY HAVE A VERY LOW FREQUENCY. FOR EXAMPLE, IF WE IMAGEED 10 CELLS IN CULTURE OVER A PERIOD OF 10 MINUTES, WE MAY SEE ONE FLASH AND ONLY -- IN ONLY HALF OF THOSE CELLS. SO THAT WAS INTERESTING, BECAUSE IN THE MATURE NEURONS, WHICH AS YOU KNOW, ARE EXCITABLE CELLS, AS WELL AS IN CARDIAC MYOCITES, WHICH WE REPORTED IN THE CELL PAPER, THE FREQUENCY OF THE SUPERER OXIDE FLASHES IS MUCH GREATER, THAT WE'LL SEE WITHIN A PERIOD OF SEVERAL MINUTES MULTI MULTIPLE SOUPER OXIDE FLASHES. OKAY, WE FOUND THAT BY MAN MANIPULATING MITOCHONDRIAL SOUPER OXIDE WITH DRUGS -- I AM NOT GOING TO GO INTO DETAILS OF THIS -- BUT THERE ARE DRUGS THAT ARE MITOCHONDRIAL SOUPER OXIDE SKA SCAVENGEERS AND WE FIND THAT THAT WILL REDUCE THE INCIDENCE OF THE FLASHES. IF WE TREAT THE CELLS WITH SêCLO SPOR ARANE IRE DRUG THAT OPENS THE PERMABILITY TRANSITION PORES, IT WILL INCREASE THE FREQUENCY. OKAY, THEN WE ASKED THE QUESTION IF WE MANIPULATE MITOCHONDRIAL SOUPER OXIDE AND CHANGE THE FREQUENCY OF THE FLASHES, HOW WILL THAT AFFECT THE PROLIFERATION AND DIFFERENT DIFFERENTIATION OF THESE PRO GENTOR CELLS? AND WE FOUND THAT IF WE REDUCED MITOCHONDRIAL SOUPER OXIDE LEVELS WITH THESE VARIOUS MANIPULATIONS OR IF WE BOUGHT THE PERMABILITY TRANSITION PORES, THAT WILL IN INCREASE THE PROLIFERATION, ESSENTIALLY, OF THESE NEWUROPRO GENTOR CELLS, WHERE THE GROWING NUMBER OCCURS HERE. AND LOOKING AT THE NEWUROSPHERE NUMBER WHICH INCREASES AND THEN WE CAN ASSOCIATE THESE CELLS AND THEN LABEL THEM WITH BROMODEOX Y YOU AREIDIANE AND GET WHAT WE CALL A PROLIFERATION INDEX. AND IF WE REDUCED SOUPER OXIDE FLASHES EITHER BY SCAVENGING THE SOUPER OXIDE OR IF WE BLOCKED THE PERMABILITY TRANSITION PORES, THAT INCREASES THE PROLIFERATION OF THE PRO GEGENITOR CELLS. CONVERSELY, WHICH I DIDN'T SHOW, IF WE INCREASED SUPERER OXIDE PRODUCTION, WE CAN REDUCE THE PROLIFERATION OF THE NEWUROPRO GENTOR CELLS AND THEN DIFFERENT DIFFERENTIATION IN NEWURONS. THEN WHAT WE DID IS WE TOOK MANG MANGANNESE SUPERER OXIDE KNOCK DO YOU THINK MICE. THESE MICE WILL DIE DURING DEVELOPMENT, BUT WE CAN LOOK DURING EMBRYONIC DEVELOPMENT AT DEVELOPING ANTRIL CORTEX. AND WHAT IS SHOWN HERE IS THE IN INJECTION OF THE PREGNANT MICE, WHICH IS SHOWN IN GREEN HERE. AND SO PROLIFERATEING CELLS ARE LABELED GREEN. THE RED CELLS ARE JUST THE NUKE NUCLEI OF THE CELLS THAT ARE NOT PROLIFERATIVE. THE WAY THE CORTEX DEVELOPS IS THAT THE STEM CELLS PROLIFERATE HERE, AS A VENTRICULAR WALL AND AS THEY DIFFERENTIATE IN THE NEWURONS, THEY MIGHT -- MIGRATE OUT PERIPHERAL LY. SO THESE RED CELLS OUT HERE, MOST OF THESE CELLS THAT ARE DIFFERENTIATING IN THE NEWEURONS AND THAT ARE NO LONGER PROLIFERATEING. AND WHAT HE FOUND IS BY COUNTING THE NUMBER OF BR DU-LABELED CELLS, THE SOD-MUTANT MICE PRESUME LABILE ELEVATED SOUPER OX OXIDE HAVE REDUCED PROLIFERATION OF THE STEM CELL BUSY. AND ALSO -- STEM CELLS. AND ALSO INCREASE DIFFERENTIATION. AND SO HERE IS WHAT HAO WAS DOING IS TO TAKE THESE EMBRYONIC STEM CELLS, WHICH ARE MAINTAINED IN A STATE BY KEEPING THEM IN THE PRESENCE OF F DCHDF 2 AND EGF. IF YOU WITHDRAW F DCHDF 2 AND EGF, THE STEM CELLS WILL STOP DIVIDE DIVIDEING AND DIFFERENTIATE IN THE UR NEWURON. SO THIS IS LOOKING AT DIFFERENT DIFFERENTIATION AFTER WITHDRAWAL OF F COMMERCIALIC F AND EGF, DAY ONE, DAY TWO, DAY ONE. AND HIS IMMUNOSTAINING HERE NIN GREEN WITH AN ANTIBODY AGAINST A NEWURON-SPECIFIC MICROTUBULE LAR PROTEIN. SO YOU SEE, AS YOU WITHDRAW A NUMBER OF NEWURONS INCREASE. AND THEN WHAT SHE FOUND IS BY LOOKING AT THE FLASHES, WHAT SHE FINDS IS THAT, AS THESE CELLS DIFFERENTIATE IN THE NEWURONS, THE FREQUENCY OF THE SUPEROXIDE FLASHES INCREASES AND IN FACT IF SHE AND -- LOOKED AT FLASHES -- THIS IS MORE SHOWING THAT THE SOUPER OXIDE PRODUCTION AND THE CELLS WILL DIFFERENTIATE. SHE ALSO LOOKED AT MEMBRANE POTENTIAL, WHICH INCREASES AS THE CELL DIFFERENTIATE. AND THEN FINALLY, BY EITHER DE DECREASING SOUUPER OXIDE, OR BLOCKING PERMABILITY TRANSITION PORES, WHEN SHE DOES THAT, SHE WILL DECREASE THE DIFFERENT DIFFERENTIATION OF THE CELLS. SO THIS IS EG 1 NEWUROMARKER, THE PERCENTAGE OF CELLS THAT ARE ESSENTIALLY NEWURONS. IF YOU BLOCKED SUPERER OXIDE PRODUCTION, THAT WILL DECREASE DIFFERENTIATION OF CELLS IN THE UR NEWS NEWURONS. IT WILL ACCELERATE THE DIFFERENT DIFFERENTIATION OF THE NEWURONS. AT LEAST TRANSIENT LY. OKAY, I AM ONE MINUTE OVER TIME. SO WHAT I AM GOING TO DO IN THE LAST 30 SECONDS. I HAVE TWO MORE SLIDES. I JUST WANT TED TO SAY THAT WE'VE DONE A STUDY THAT THE WORK IN THE LAB IS RELATED TO WHAT GOES WRONG WITH NEWUROPLASTICITY DURING AGEING AND NEWURODEJERNT NEURODEGENERATIVE DISORDERS. SO IF THEY DEJERGENERATE AND BECOME DYSFUNCTION AAL? WHY MIGHT NEWUROGENESIS BE IM IMPAIRED? AND IN ALZHEIMER'S DISEASE THE MET BETA PEPTIDE ACCUMULATES. AND I'LL JUST SHOW YOU ONE SLIDE FROM A STUDY WE RECENTLY PUBLISHED, WHERE WE FOUND, IF WE EXPOSED CULTURED NEWURONS TO BETA BETAPEPTIDE, THERETWILL INCREASE THE FREQUENCY OF THE SOUPER OXIDE FLASHES EARLY ON AND THEN. WITH CONTINUED EXPOSURE OF THE NEURONS TO ABETA, WE GET KIND OF A GLOBAL INCREASE IN REACTION OF THE SPECIES. AND THE NEWURONS WILL DEJERNGENERATE. SO SORRY I WAS A LITTLE LONG. BUT IF YOU WANT TO READ ABOUT THE DISEASE-RELATED STUDY, IT WAS RECENTLY PUBLISHED. OKAY. SO THAT'S THE END OF MY TALK. I'LL TAKE QUESTIONS. THANKS. APPLAU [APPLAUSE] OKAY. SO I DIDN'T HAVE TIME TO SHOW IT IT. BUT IN THE STUDY OF THE NEWUROPRO GENTOR CELLS, WE FOUND THAT THE SOUPER OXIDE PRODUCTIONS INHIBIT THE IRK GINGERS HIBITS THE KIN A KINASES THAT ARE INVOLVED IN PROLIFERATION OF THE NEWUROPRO GENTOR CELLS. SO AT LEAST IN THAT CASE, THAT SEEMS TO PLAY A ROLE. SO THE IDEA IS THAT THE SOUPER OX OXIDE IS AFFECTING SIGNALING BY MODULATEING KIN AASES. AND THOSE -- THERE IS ALSO A LITERATURE ON REACTIVE OXYGEN SPECIES MODIFYING THE PATHWAYS.^ A LOT OF THEM IN THE CANCER LITERATURE, FOR EXAMPLE. >> INAUDIB[INAUDIBLE] >> YES, YES. THAT'S CORRECT. YES. >> INAUDIB[INAUDIBLE] >> THAT'S A GOOD QUESTION AND SOMETHING WE HAVEN'T LOOKED AT. ALTHOUGH MITOCHONDRIAL BIO BIOGENESIS -- MY UNDERSTANDING IS IT CAN OCCUR IN AX OONS AND DEN DENDRITES. AND YOU CAN IMAGINE, FOR EXAMPLE JUFSHGS A MOTOR NEWURON IN YOUR SPINAL CORD AND WITH -- IT^-- THE TERMINAL IS DOWN IN YOUR BIG TOE AND THERE IS MITOCHONDRIA DOWN THERE. IT WOULD MAKE SENSE TO ME THAT YOU CAN HAVE LOCAL SIGNALING THAT MIGHT AFFECT MITOCHONDRIAL. THERE IS CERTAINLY EVIDENCE THAT YOU CAN GIVE. BUT YOU'RE RIGHT, FOR THE PGC ALPHA, WHICH REQUIRES TRANSCRIPTION, THAT TRANSCRIPTION IS OBVIOUSLY OWE OCCURRING IN THE NUCLEUS. YES. >> SO THE PAPER YOU CITED FROM 2006 SHOWS VERY NICELY THAT THE YFP BLIPPINGING IS INDEPENDENT OF CASTLCIUM. ARE YOU GUYS CONFIDENT THAT IT'S SPECIFIC COMPARED TO PH HENCE SENSING? >> SINCE THE CELL PAPER CAME OUT, THERE HAVE -- BAIN NUMBER OF PAPERS. AND WHAT CHANG HAS DONE IS THAT WHILE THE PGC -- EXPRESSES CAN CHANGE WITH PG -- HV SCLEREX THE MAGNITUDE OF THE HV CHANGES ARE NOT OCCURRING UNDER THESE KINDS OF FIPHYSIOLODGGICAL CONDITIONS. >> ALL RIGHT, THANK YOU. >> I WAS THINKING ABOUT THE CASTLCIUM. SO WHEN YOU GET THE MEMBRANE FROM THE TRANSITION FORMATION, YOU ALSO GET THE SIGNALING MOLL MOLECULE? >> NO. SO THAT'S AN INTERESTING QUESTION. BECAUSE IT'S -- IT HASN'T BEEN LOOKED AT IN DETAIL. AS WE KNOW FOR EXAMPLE, IF WE MANIPULATE CASTLCIUM IN THE CELL, IT CAN AFFECT FLASHES. BUT AS FAR AS THE COUPLEING OF PERMABILITY OF TRANSITION PORE TO SYSTEMS WITHIN THE MITOCHONDRIA THAT REGULATE MOVEMENT OF CASTLCIUM ACROSS THE MITOCHONDRIAL MEMBRANE, WE DON'T KNOW. OKAY? SO OUR NEXT SPEAKER IS RICHARD Y YU Y ULE FROM NIM DF AND HE'S BEEN LOOKING AT PARKINSON'S DISEASE AND PARKINSON'S DISEASE HAS LED TO A FOCUS ON MITOCHONDRIA AND THAT'S WHAT HE'S GOING TO TALK ABOUT. >> THANK YOU, MARK. AND THANK YOU, STEVE, FOR IN INVITING ME. SO THERE ARE MANY LINKS BETWEEN MITOCHONDRIAL DYSFUNCTION AND PARKINSON'S DISEASE. IT'S BEEN KNOWN FOR MANY YEARS. PARKINSON'S DISEASE IS LARGELY LATE-ONSET AND SPRAORADIC, WITHOUT OBVIOUS GENETIC LINKAGE. AND THE PROBLEM FOR PARKINSON'S DISEASE IS THERE IS NO TREATMENT THAT WILL MITIGATE OR STOP THE PROGRESSIVE LOSS OF NEWURONS. AND PART OF THE PROBLEM IN DEVELOPING THERAPEUTICS IS WE DON'T KNOW THE FLAWS. NOW THERE IS A SUBSET OF FULFILL FORMS OF PARKINSON'S DISEASE -- FAMILIAL FORMS OF PARKINSON'S DISEASE AND CERTAIN GENES INVOLVED IN THESE FAMILIAL FORMS THAT HAVE BEEN IDENTIFIED. THERE IS DOMINANT MUTATIONS, FOR EXAMPLE, THAT ARE LIKELY TO GAIN A FUNCTION THROUGH CELLS. AND THERE IS TWO I AM GOING TO TALK ABOUT TODAY IN MUTATIONS YOU HAVE TO USE -- LOSE FUNCTION OF BOTH, INDICATING THAT THEIR LOSS OF FUNCTION. AND THE NORMAL FUNCTION OF THESE PROTEINS WAS WE MIGHT HAVE A BETTER INSIGHT INTO THE CAUSE OF THE DISEASE IN BECAUSE THEESHTS AND IT MIGHT ALSO LEAD TO SOME BETTER UNDERSTANDING OF SPRATT SPORADICS. -- OF IT BEING SPRAORADIC. SO THIS IS THE WORK WE'VE BEEN DOING FOR ABOUT SIX YEARS NOW. AND WE AND OTHERS HAVE DEVELOPED THIS PATH WWAY THAT LINKS PARKINSON'S DISEASE TO MITOCHONDRIA. WHEN THESE WERE FIRST IDENTIFIED IDENTIFIED, IT WAS CLEARLY OBVIOUSLY -- SXOUB HAS BEEN SHOWN AND YOU CAN TELL BY THE SEQUENCE THAT THIS HAS MITOCHONDRIAL IMPORT SEQUENCE. AND ALSO LOOKING AT THE SEQUENCE SEQUENCE, YOU CAN DETECT IF IT'S LIKELY TO BE A KIN AASE. MITOCHONDRIAL KIN AASE. PARKIN'S, BASED ON THE SEQUENCE IS AN E 3 LIG ATATE AND EARLY EVIDENCE THAT PARKIN WAS INVOLVED IN MITOCHONDRIAL FUNCTION CAME FROM SLIDEWORK AND MADE KNOWN MUTATIONS IN THE PARK PARKIN GENOME SHOWS THAT MITOCHONDRIA WAS SLOW AND DIS DYSFUNCTION AL. AND THEN CHUNG IN KOREA SHOWED MUTATION S IS IN FLIES. AND WHEN YOU HAVE EXPERIMENTS WHERE YOU HAVE MUTATION S IS IN BOTH GENES WERE NO WORSE, INDICATING ANY EVIDENCE THAT THEY SEEM TO WORK IN THE SAME PATH SIGHT.^ SO THEN WE ANDS WILL HAVE IDENTIFIED CELL BIOLOGICAL AND BIOCHEMICAL PATHWAYS, WHERE PINK 1 AND PARKIN WORK TOGETHER AND I'LL JUST SUMMARIZE A LOT OF THE ROSHING WORK FROM THE LABS. BUT LET'S SEE HOW THE CELLS AND ONE OF THOSE FIVE BECOME -- BY A NUMBER OF DIFFERENT -- WITH IT'S QUITE INTERESTING THIS KIN AASE, PINK 1, ACCUMULATES COLLECTIVELY ON THE DAMAGED MITOCHONDRIA, NOT -- AND PINK 1 WILL RECRUIT THIS E 3 LIG AASE PARKIN FROM THE MITOCHONDRIA AND ACTIVATE IT IN PARKIN AND OUTER MITTOKINOMEM MITTOKINOMEMBRANE PROTEIN WHICH CAN INDUCE ENGULFMENT OF THE MITOCHONDRIA AND DISINTEGRATION. SO THIS SUGGESTS A MODEL THAT THESE TWO GENE PRODUCTS MAY WORK TOGETHER IN A PATHWAY AS MITOCHONDRIA QUALITY CONTROL AND I'LL SHOW YOU SOME OF THE DATA THAT LED TO THIS IDEA. ONE OF THEM IS IN A FIRST PAPER THAT DEREK PUBLISHED. HE IS A GRADUATE STUDENT IN A PROGRAM WHEN HE DID THIS. AND THIS STEMS FROM WHERE HE KNOCKED OUT MIGTOFUSION. AND CHANG SNOWED ONE OF HIS PAPERS THAT FU KNOCK OUT TWO, THERE WAS NO FUNCTION. BUT FU KNOCKED OUT BOTH OF THEM, A SUBSET OF THE MITOCHONDRIA WAS SPECIAL AND WE CONFIRMED THAT. SO WHAT DEREK DID IS HE EXPRESS EXPRESSED PARKINS IN THESE CELLS AND HE FOUND AS IN NORMAL CELLS, PARKIN, FROM THE LITERATURE AND ALL THE CELLS. AND THE SAME M SLES 1 AND M SLEES 2 KNOCK KNOCKOUTS. BUT THE DOUBLE KNOCKOUTS WHERE THERE IS DAMAGE TO THE MITOCHONDRIA, IN ALMOST ALL THE CELLS, PARKIN WAS LOCALIZED IN THESE FOESI. AND IF YOU LOOK AT THIS UNDER HIGHER MAGNIFICATION WITH THESE OTHER MARKERS, HERE IS THE MSN 1 AND 2 KNOCKOUT CELLS. IF YOU LOOKED AT ALL THE MITOCHONDRIA, HERE IS ALL THE MITOCHONDRIA. AND YOU CAN SEE RIGHT UP TOP THAT PARKIN DOES NOT LOOK LIKE ALL THE MITOCHONDRIA. LOOKS LIKE A SUBSET OF THE MITOCHONDRIA. AND IF YOU USE MITTOTRACK ER TO USE WHICH WHAS HIGHER OR LOWER, YOU CAN SEE SOME OF THE MOUNDS. AND WHAT'S INTERESTING -- MITOCHONDRIA. WHAT'S INTERESTING IS RIGHT HERE IF YOU LOOK AT WHERE THE PARKIN IS, IT'S ON THE DAMAGED ONE, OR THE LEFT ONE. AND THAT'S ONE WAY OF DOING IT. ANOTHER WAY OF DOING IT IS A VERY ROBUST AND EASY TO DO. YOU CAN ADD -- N [INDISCERNABLE[INDISCERNIBLE] SO ANYWAY, IN GREEN IS PARKIN AND THE CIYTOSXOLT RED ARE MITOCHONDRIA. AND FU ADD IT WITHIN 30 MINUTES YOU CAN SEE A GREEN PARKINS RE RECRUITED TO MITOCHONDRIA. VERY ROBUST. AND A LOT OF PEOPLE HAVE USED THIS. IT'S A VERY EASY WAY TO FIGURE OUT THE PROCESS. NOW WHEN YOU LOOK AT KNOCKOUT CELLS, THERE IS 100% BLOCK IN THAT. THAT MOVEMENT OF PARKIN ABSOLUTELY STEMS FROM 1 PINK. PINK 1. FU LOSE MEMBRANE TRANSFER, PINK 1 ACCUMULATES ON THE DAMAGED MITOCHONDRIA. YOU SEE IN 1 NORMAL CELLS. THERE IS ALMOST UNDETECTABLE LEVELS OF PINK 1. BUT THEN IF YOU ADD FOR 3 HOURS, YOU VERY SEE VERY ROBUST AC ACCUMULATION OF PINK 1. AND IF YOU WASH -- IT RECOVERS AND YOU CAN SEE THAT THE PINK 1, WITHIN 2 1/2 MINUTES OR 5 MINUTES IS ELIMINATED. THIS EXPERIMENT, WITH A LOT OF OTHERS, INDICATED THAT LINK PINK 1 IS BEING TURNED OVER VERY RAPIDLY P. IT'S NORMAL IMPORTED, INTEGRATED INTEGRATED. WE SCREENED MITOCHONDRIAL TO SEE WHICH WERE INVOLVED IN THESE EL ELIMINATION OF PINK 1. AND WE FOUND KNOCKOUT CELLS TO SHOW THAT WITHOUT ANY TREATMENT WHATSOEVER COMPARING LANE 1 AND LANE 1, YOU CAN FIND A LOWER BAND OF PINK 1. AND NEN YOU ADD C COMMERCIALIC T, YOU GAIN IMPORT. THERE IS A CLEAR DIFFERENCE IN THE KNOCKOUT. IN THIS AND OTHER EXPERIMENTS FROM OTHER LABS LEAD TO THIS MODEL OF HOW PINK 1 IS A STENN EXTENSOR OF MITOCHONDRIAL DAMAGE DAMAGE. IN A HEALTHY NORMAL CELL ADHERES TO PINK 1. THIS IS THE MITOCHONDRIAL IMPORT SEQUENCE AND IT'S NORMALLY IMPORTED TO THE COMPLEX, CLEAVEED BY MTT AND THEN -- IT'S PUT BACK OUT INTO THE CIYTOSOL AND GENERATES INTERNAL FEN PHENOMENALAMINE. THROUGH DEGRADATION AND D 3 AND THIS SEEMS TO BE THE NORMAL PATH WAY, WHICH KEEPS PINK 1 ALSO UN UNDETECTABLE. AND FU BLOCK IMPORTS, ALMOST ANYTHING THAT BLOCKS IMPORTS, I THINK, IS A CHANNEL SUCH AS BLOCKED -- BE IT'S BLOCKED FROM THE MEMBRANE WHERE IT'S LOCAL LOCALIZED. AND INSTEAD PINK 1 STIMULATES AND BINDS TO THE SELECTION AND IT REQUIRES AN ABSOLUTELY REQUIREMENT FOR ONE OF THE SMALLER COMPONENTS OR ITS AC ACCUMULATION BOUND ON THE OUTER MIGTOKIN ANAL MEMBRANE. NOW, SOME PEOPLE HAVE SAID TO ME ME, WELL THIS IS INTERESTING BUT IT REQUIRES C COMMERCIALIC T. I WANT TO EXPRESS THAT IT'S NOT THE CASE. IN THAT FIRST SLIDE SHOWED YOU IN DEREK'S FIRST PAPER HE SHOWED IN THE KNOCKOUT CELLS PARKINS LOOK LIKE MITOCHONDRIA. YOU CAN ADD CHEMICALS AND SOME OF THEM MITOCHONDRIAL-SPECIFIC. BUT A NUMBER OF DIFFERENT TYPES OF GENETIC DAMAGE WITHOUT CHEMICAL TREATMENT -- ALSO OX OXIDATIVE DAMAGE WHICH HAS BEEN LINK ED TO ENVIRONMENTAL PARKINSON'S DISEASE. OUR NEW WORK IS SURPRISING BECAUSE WHAT THESE ALL HAVE IN COMMON IS THEY ELIMINATE POTENTIAL OF MITOCHONDRIA. WE PUBLISHED LAST YEAR AND FOUND THAT UNFOLDED PROTEINS IN THE MITOCHONDRIA CAN ALSO CREATE THIS PATH WWAY WITHOUT LOSS. SO I THINK THE COMMON DEFOMENT DENOMINATOR APPEARS TO BE IN INHIBITION OF THE INNER MEMBRANE AND IT'S DEREGULATED. I WANT TO SHOW THIS INTERESTING UNFOLDED PROTEIN RESULT. WE DEVELOPED INDUCTION OF EXPRESSIONS. WILD-TYPE TRANSFER, WHICH IS A FEATURE OF THIS. AND DELTA OPC, INTERNAL DELUSION RESULTS IN A MIXED FOLDING OF THIS. THIS WAS DISCOVERED BY NICK IN AUSTRALIA AND SHOWN TO INDUCE A MITOCHONDRIAL PROTEIN RESPONSE. AND HE CONFIRMED THAT. SO IF YOU DO A SOLUBLE EXPRESSION, YOU SEE THAT ALL OF THE DELTA OCC IS INSOLUBLE. IF YOU LOOK AT PINK 1, IT'S STABILIZEED WITH THE PROTEIN. SO WITHOUT ANY -- IN [INDISCERNABLEIBLE] AND WE'VE SHOWN THAT IN A NUMBER OF DIFFERENT WAYS. HERE IS ANOTHER DIRECT COMPARISON OF PINK 1 AND DELTA OTP ACCUMULATION OF PINK 1. WHAT'S INTERESTING IS THIS AC ACCUMULATION OF PINK 1 WILL IN INDUCE PARKIN IN THE MITOCHONDRIA AND THE MIGTOPHASE GEFILTE ONE OF THE BIG MYSTERIES HAS BEEN HOW TO DOES PINK 1 RE RECRUIT PARKINS TO THE MITOCHONDRIA? SO THIS IS A KIN AASE THAT AC ACCUMULATES AS YOU SHOWED YOU AND CAUSES THIS ACTIVATION. AND WE AND OTHERS HAVE LOOKED FOR MANY YEARS FOR THIS. I FOUND OUT THIS MORNING OUR PAPER HAS BEEN ACCEPTED. A POST DOC FROM MY LAB WAS RETAIN ED FRED FROM THE LAB. AND SHE DEVELOPED A FAIRLY SFIFT SOPHISTICATED STRATEGY TO IDENTIFY THE PINK 1 SUBSTRATE. WE TOOK WILD-TYPE FEPHENOCELLS THAT EXPRESS AND ACCUMULATE, AS I SHOWED YOU EARLIER, AND WE ALSO DAYCARE POST DOC IN MY LAB KNOCKED OUT PINK 1 USING THE CELL LINE. SO WE HAD MATCHED CELL PAIRS -- WILD TYPES AND PINK 1 KNOCKOUTS. HERE YOU GET PINK 1 AC POPULATION AND HERE YOU DON'T. AND SHE ISOLATED THE MITOCHONDRIAL CELLS WHERE THE PINK 1 WAS STIMULATED AND PRESUME ABEABLY FOSPHOSPHORYLATEING IN SUBSTRATE. AND PARKINS WAS RECRUIT ED ED TO MITOCHONDRIA, WE -- SHE GENTLY DIGESTED THE TRIYPSIN OUTER MITOCHONDRIAL PROTEIN AND IT'S DIGESTED. ANOTHER MEMBRANE PROTEINS AND LEFT THE INNER MEMBRANE PROTEINS IN TACT FROM THIS AND REMOVED THIS AND ANALYZED THIS CRISTOPEP CRISTOPEPTIDE THAT WAS RELEASED. AND WE LOOKED FOR FOPHOSPHOPEP PHOSPHOPEPTIDES AND SHE FOUND ONE STRIKING PEPTIDE OF THE WILD TYPE CELLS. AND IT'S QUITE INTERESTING. IT TURNED OUT TO BE THIS PEPTIDE IN RED FROM A UBIQUITINOUS CELL. AND I TOLD PEOPLE IN MY LAB FOR MANY YEARS. WHEN ONE FINDS A SUBSTRATE, YOU ARE GOING TO KNOW IT. AND PART OF THE REASON THEY WERE VERY OPTIMISTIC WITH THE PEPTIDE IS AN EARLIER PAPER BY A GROUP HAD SHOWN THAT PINK 1 FOSS PHOSPHORYLATES PARKINS. IT HAS A CONVENIENCE HOM OOLOGOUS AND WE'VE CONFIRMED IT. OTHER PEOPLE CONFIRMED IT AND IN INDEED PINK 1 WILL FOPHOSPHORYLATE PARKINS AT EXACTLY. AND WE AND OTHERS HAVE SHOWN THAT, ALTHOUGH PINK 1 CAN FOSS PHOSPHORYLATE PARKINS, THAT'S NOT SUFFICIENT -- THERE ARE OTHER SUBSTRATES THAT WILL A APPEAR OTHER THAN THE FOSS PHOSPHORYLATION. THIS REALLY SEEMED LIKE A LIKELY CANDATE AND WE WERE ABLE TO CONFIRM IT ALSO WITH A KIND GIFT GIFT. CANDIDATE. WE INCUBATEED WILD TYPE PINK 1 AND A KIN AASE PINK 1 WITH P 32 AT ATP AND PURIFIED RECOMBINANT PRO PROTEIN SYSTEM. AND AS YOU SEE THAT THE RADIO GRAM SHOWS THAT P 32 IS BEING LABELING? A PINK 1-DEPENDENT WAY. SO THEN WE MOVED TO THE BIGGER QUESTION OF OKAY, SO PINK 1 CAN FOPHOSPHORYLATE UBIQUITIN. SO WHAT WE DID IS WE PRODUCED THE -- WE ALSO INCUBATEED UB UBIQUITIN, WHICH IS A SITE RIGHT HERE. SO THAT IT CANNOT BE FOSS PHOSPHORYLATED. AND WE TREATED THEM WITH PINK 1. THEN WE REMOVED THE PINK 1. AND THIS IS THE EXPERIMENT. WE TOOK THE FOPHOSPHOUBIQUITIN AND ADDED IT TO PARKINS. AND LOOKED AT ENZYME ACTIVITY AND SAW THAT INDEED THE WILD TYPE UBIQUITIN STIMULATES CHAIN FORMATION FROM PARKINS. WHEREAS THE A 55 MUTANTS DID NOT NOT. WE ALSO SHOWED IN CELLS THAT F 6 65A OVEREXPRESSION IN CELLS HAVE A MUTANT UBT QUINN FOSPHOSPHORYLATE AND WILL LARGELY INHIBIT TRANS TRANSPORTATION IN MITOCHONDRIA. AND SHARON DID THIS VERY NICE EXPERIMENT SHOWING BY WESTERN BLOCK IN CELLS THAT A FOPHOSPHO-- WHERE WE PUT A RESIDUE AT 55. IT BY ITSELF ACTIVATES PARKIN. PARKIN HAS A VERY NICE PAPER SHOWING THAT THIS RESIDUE 431 FORM AN ENZYME INTERMED WITH UBT UBIQUITIN TRANSFORM REACTION. AND IT WILL FORM A STABLE BOND ATTRACTING THE ENZYME. IT HAS A SLIGHTLY HIGHER BAND THAN PARKIN. YOU SEE THAT BAPARK JASON SINGLE BAND. AND FU ADD 52 TO THAT, YOU CAN CONFIRM THAT AN OX Y ESTER BECAUSE YOU CAN ADD SODIUM HIGH HYDROXIDE AND IT WILL REMOVE THAT BAND. SO HERE IS THE EXPERIMENT. SO COMPARING THAT MATCH, YOU CAN SEE THIS EXPRESSION IS UBIQUITIN IN CELLS THAT HAVE A FOPHOSPHOGENT PHOSPHOGENETIC MUTATION. SO THIS, I THINK, EXPLAINS THE SUBSTRATE OF PINK 1 THAT ACT ACTIVATES PARKINS AND IT MAKES AN INTERESTING -- A SUBSTRATE OF PINK 1, WHICH IS UBIQUITIN, ACT ACTIVATES PARKINS, WHICH IS A UBIQUITIN LIG AATE. ON PROTEINS AND ON THE MITOCHONDRIA BUT THEN FORMS MORE SUBSTRATES TOR ACTIVATE MORE PARKINS AND A C 4 AMPLIFICATION AND THAT PROBABLY EXPLAINS WHY YOU CAN GREATLY OVEREXPRESS PARK PARKINS IN CELLS AND ALL WILL TRANSLOCATE BECAUSE OF THIS. AND THEN THE LAUGHST THING I WANT TO TOUCH ON IS ANOTHER IMPORTANT ISSUE IS WHAT'S THE EVIDENCE OF THIS PATH WWAY IN VIVO? SO WHAT WE'VE DONE IS WE'VE CROSSED THE MUTATEOR MOUSE, WHICH YOU ARE PROBABLY WELL AWARE THAT THE MOUSE MADE BY LARSEN AND TOM, THAT HAS A DEFECT IN THE MITOCHONDRIAL DNA AND THIS WILL STIMULATE MUTATION MUTATIONS IN PREMATURE AGING FEPHENOTYPES. NOW THE KNOCKOUT MOUSE HAS NO STRONG FEPHENOTYPE AND THAT'S INTERESTING. I JUST WANT TO POINT OUT TO THIS AUDIENCE WHO UNDERSTANDS MITOCHONDRION THAT HUMANS THAT HAVE MUTATION S IS IN PINK 1 AND PARKINS FEPHENOCOPY ONE ANOTHER AS PARKINSON'S DISEASE. MICE THAT HAVE KNOCKOUT AS PINK 1 IN PARKINS, THEY FEPHENOCOPY ONE ANOTHER WITHOUT ANY CLEAR FEPHENO PHENOTYPES. AND FLIES FEPHENOCOPY ONE ANOTHER WITH A VERY STRONG CHEMICAL MUSCLE. SO WHAT YOU CAN IMAGINE THE REGULATION IN PARKINS. MITOCHONDRIA ARE DIFFERENTIAL LY IMPORTANT IN DIFFERENT TISSUES IN DIFFERENT SPECIES AND THAT MIGHT EXPLAIN. THE KNOCKOUT MOUSE HAS NO STRONG FEPHENOTYPE. SO WE LOOKED AT A NUMBER OF TISSUES. SO IF YOU COUNT THE NEWURONS IN WILD TYPE MOUSE, COMPARE IT WITH THE KNOCKOUT POWS, AS IN THE LITERATURE THERE IS NO DEFECT. MOUSE. AND THE MUTATEOR HAS NO DEFECT. BUT WHEN YOU ELIMINATE PARKINS FROM THE MUTATEOR MOUSE, IT DE DECREASES. WE'RE BEGINNING TO LOOK AT OTHER BRAIN LEGIONS AND THE STRATUMS INTO LOSS OF NEURONS. SO THIS INDICATES THAT INDOCX -- INDODENDOGENOUS PARKINS IS COMPENSATE COMPENSATEING FOR THE MUTATEING FEPHENOTYPE. AND WE LOOKED AT A MOTOR FEPHENO PHENOTYPE THAT ONE CAN ASSESS HOW FAST MICE CAN CLIMB DOWN A POLE. AND WILD-TYPE MICE CLIMB DOWN . [INDISCERNABLE]. BUT THE MUTATEOR THAT HAS A LOSS OF SUBSTANTIATEED NEURONS HAS A CLEAR DEFECT IN FEPHENOTYPE. AS I MENTIONED, UNLIKE A LOT OF MOUSE MODELS WITH PARKINSON'S DISEASE WITH SPECIFIC KNOCKOUTS, THESE ARE COLD--- WHOLE-BODY KNOCKOUTS. SO PMANY DIFFERENT TISSUES COULD BE AFFECTED. DIABETES, ET CETERA. SO WE TESTED WHETHER THIS ABILITY IS DUE TO THE LOSS BY CREATING THESE MICE. INDICATING THAT PARKIN IS INDEED FUNCTIONING IN VIVO TO COMP COMPENSATE FOR A EXPREN EXPRESS. AND I JUST WANT DO CLOSE WAY COUPLE OF SUMMARY SLIDES. THIS PATH WWAY SHOWS. WE'VE ALREADY KNOWN THERE IS A GENETIC LINK BETWEEN PARKINS AND THIS WORK SCAAND MANY OTHERS SHOWS A BIOCHEMICAL AND SUBBIOLOGICAL RELATIONSHIP BETWEEN THESE TWO GENE PRODUCTS AND PARKINSON'S DISEASE. AND THIS FUNCTION MAY LEAD TO PARKINS SCOMBRICHLT JUST WANT TO THANK -- I JUST WANT TO THANK MY POST DOC FOR OVER SEVEN YEARS. DEREK, THE GRADUATE STUDENT I MENTIONED WHO INITIAL LY DISCOVERED THIS AND IN COLLABORATION WITH MARK IN OUR BUILDING. AND MIKE LAZARO, SHOWING PINK 1 BINDS TO COMPLEX AND ENTOMOLOGY OF PARKINS ACTIVATION. THAT'S THE GUY WHO FOUND THAT -- SAM WHO FOUND POTENTIAL AC ACCUMULATION. AND LESLIE CANE IS THE ONE WHO GOT A PAPER ACCEPTED TODAY SHOWING THAT PINK 1 UBIQUITIN -- SHE GOT A LOT OF HELP FROM MIKE MICHAEL AND ADAM SCAAND I SHOWED THE DATA FROM SARAH. AND THE MOUSE WORK WAS DONE BY A ARILICIA. AND KOJI IS THE ONE WHO DISCOVERED -- THANK YOU. APPLAU [APPLAUSE] ANY QUESTIONS? >> INAUDIB[INAUDIBLE] >> SO I WAS SAYING THAT YOU DAY COUPLE OF EXPERIMENTS WITH ATP TRIED TO COUNTERACT THAT EFFECT. SO HAVE YOU EVER EXPLORED THE POSSIBILITY THAT, FOR INSTANCE, OVEREXPRESSED ATP DECREASED THE PINK 1 ACCUMULATION? >> YES. THAT'S TRUE. FOR EXAMPLE, OTHERS AND WE HAVE NOW USED INHIBITS FOSS SDPORGS THAT WILL NOT VERY ROBUST LY FIGURE THIS PATH WWAY. SO IF YOU HAD OLDER MICE, THEN IT WILL. AND ALSO BECAUSE THERE IS A GUY IN CANADA WHO DID A FUEL GENE M (M)RNA IN MICE AND HE FOUND THAT KNOCKDOWN OF IF 1 WOULD INHIBIT. AND IF 1 IS THE SUBUNIT OF THE ATP SYNTHASE THAT PUTS THE BRAKE ON THE PROCESS. SO IF YOU CAN'T BREAK IT, THEN IT WILL STOP THE PROCESS. >> I WANTED TO ASK A QUESTION. FIRSTLY, WHAT IS THE RELATIVE EFFICIENTLY OF FOSPHOSPHORYLATEING UBIQUITIN? SECONDLY, WHAT ABOUT THESE IN INTRAMEMBRANE REGIONS? >> WELL, WE HAVEN'T -- I THINK THAT COULD BE COMPLICATED. I'M NOT SURE IF IT'S KNOWN, BECAUSE ACCORDING TO HELEN WALD WALDEN, FOR EXAMPLE, I HAVEN'T REALLY LOOKED TO SEE WHERE IN THE 3-DIMENSIONAL STRUCTURE OF PARKINS THAT F 55 IS BUT IT'S IN INHIBITED RELATIVE TO PRE-UB PRE-UBIQUITIN. WE HAVEN'T DELVED TOO MUCH INTO PARKINS INFORMATION PHORYLATION, OTHER THAN CELL EXPERIMENT TO SHOW THAT YOU DON'T SEE -- THAT'S HOW LESLIE GOT INTO THIS. WE ALLERGY THOUGHT WE COULD FORM MUCKE'S DATA. BUT WE MUTATED USING 65A IN PARK PARKINS AND IT WAS DEFECTIVE. BUT NOT COMPLETELY DEFECTIVE. YOU STILL COULD HAVE ROBUST PINK 1-DEPENDENT -- THAT'S WHAT -- A AND ALSO WE'VE SHOWN IN THIS CURRENT MANUSCRIPT THAT IN THE ABSENCE OF 55 IN PARKINS, YOU CAN GET FULL ACTIVATION -- YOU CAN GET GOOD ACTIVATION WITH UBIQUITIN. BUT MAYBE THERE IS SOME KIND OF ADVANCE. AND SECOND QUESTION WAS ABOUT THE PAPER. WELL, PART OF THE REASON IS HE HAD AN INTERVIEW ON AND SAID THIS PATH WWAY USES -- WELL, IT'S NOT ACTUALLY THE CASE. BUT HE ALSO ACKNOWLEDGED IN THAT INTERVIEW THAT THEY COULD NOT SHOW PINK 1 DIRECTLY FOSS PHOSPHORYLATES WITH INTERMEM INTERMEMBRANE. SO IT COULD BE INDIRECT AND I GUESS I WOULD THAT I IT IS IN INDIRECT. AND FU DON'T HAVE PINK 1, LIKE IN KNOCKOUT CELL LINES, YOU MIGHT ACCUMULATE A LOT OF MITOCHONDRIAL DAMAGE BECAUSE YOU DON'T HAVE THIS GLOBAL PATH WWAY AND TO COMPENSATE FOR THAT DAMAGE, THERE MIGHT BE FOSS PHOSPHORYLATION OF THE PROTEIN MEMBRANES BY OTHER KIN AASE. >> ONE LAST QUICK QUESTION AND THEN WE'RE GOING TO TAKE A TEN TEN-MINUTE BREAK. >> THANK YOU. SOME OF THE QUESTION I HAD WAS ALREADY ASKED. BUT MY QUESTION IS DOES THE DAMAGE TO THE PINK 1 AC ACCUMULATION HAVE ANY IMPACT ON THE MITOCHONDRIA IN THE BRAIN SUCH AS MICRO-- >> I DON'T KNOW. BUT RIGHT NOW WE'RE LOOKING AT OLFACTORY NEURONS THAT MICE. REMEMBER, THE MICE HAS A MUTATE MUTATEOR. BECAUSE OLFACTION IS LINK ED TO PARKINSON'S DISEASE. SO WE'RE LOOKING AT OTHERS. COULD BE. >> THANK YOU. >> OKAY. THE BREAK IS SPONSORED BY THE RESEARCH ALLIANCE. AND THE BREAK -- REFRESHMENTS ARE BACK BY THE POSTERS. GO OUT THE BACK DOOR. GO UP A COUPLE OF HALLWAYS DOWN AND FOLLOW THE SIGNS AND THE REFRESHMENTS. AND COME BACK IN 10 MINUTES, WHICH WOULD BE ABOUT 10:00. THANK YOU. >> OKAY, WE'RE GOING TO GET STARTED. THE NEXT SPEAKER IS MARIE HARDWICK FROM JOHNS HOPKINS UNIVERSITY. SHE'S DONE FASCINATING WORK ELUCIDATING FUNCTIONS OF FAMILY MEMBER PROTEINS THAT ARE NOT NECESSARILY RELATED TO CELL DEATH. SHE'S GOING TO TALK I THINK A AT LEAST PARTLY ABOUT THAT TODAY. >> THANK YOU. THEY TELL US THAT THERE'S SOME BILLIONS OF CELLS THAT DIE IN THE HUMAN BODY EVERY DAY INCLUDING TWO MILLION RBC'S PER SECOND, 40,000 SKIN CELLS, IN AN HOUR, AND ABOUT ONE NEURON A SECOND, ROUGHLY. AND THIS PROCESS IS THOUGHT TO OCCUR LARGELY BY APOPTOSIS, SO I'M SUMMARIZING THE PREVAILING MODEL, A DEATH TRIANGLE WHERE THE ANTIDEATH -- THIS IS CONTROLLED BY THE FAMILY MEMBERS, BUT THE ANTIDEATH PROTEINS BIND AND INHIBIT THE MOLECULES AND THEY RESPOND TO THE STRESS SIGNALS IN THE CELL AND THEY WORK BY BINDING AND INHIBITING THE ANTIDEATH, OR THEY CAN BIND AND ACTIVATE THE PRO DEATH MOLECULES WHICH THEN BIND BACK -- THAT'S WHY I CALL IT A TRIANGLE, PEOPLE ARE TRYING TO SORT OUT THE DETAILS. ALL OF THIS IS THOUGHT TO PLAY OUT ON THE OUTER MITOCHONDRIAL MEMBRANE WHERE THE ANTIDEATH PROTEINS CAN PREVENT. THE NEW MODELS NOW SUGGEST THAT IT'S RATHER THAN PENETRATING THE MEMBRANE AS HAS BEEN SUGGESTED AND YOU GET ACTIVATION OF THE CASSPASES IN SELECTED PROTEINS AND DEATH. THAT'S THE PREVAILING MODEL. MAYBE IT'S TRUE BUT MY LAB WAS FINDING THINGS NOT IN AGREEMENT IN PART BECAUSE WE WERE LOOKING AT THE NERVOUS SYSTEM. HERE IS ONE EXAMPLE WHERE THE BAK KNOCKOUT WHICH SHOULD BE PROTECTED INSTEAD HAS MORE DEATH IN THIS MODEL, THE SEIZURE MODEL, KNOWN TO KILL HIPPO CAMPAL NEURONS, IT LOOKS LIKE BAK IS PROTECTED IN THIS MODEL. OUR VIEW IS ESSENTIALLY ALL OF THESE PROTEINS HAVE PRO DEATH FUNCTIONS, BUT THEY DID SOMETHING ELSE, BEFORE THEY WERE CONVERTED INTO DEATH FACTORS. AND HOW DO THEY GET CONVERTED? ONE OF THE ONES WE'VE STUDIED AND CONTINUING TO STUDY IS CASPASE, THEY MAKE KEY CUTS IN CELLS, CLOSED TO SPOTS, 61 AND 76, BY CASPASES, AT LEAST THREE AND MAYBE OTHERS. THIS GETS CLEAVED, THE POINT MUTATIONS, IT'S A KNOCKIN MOUSE, TO LOOK AT THE ROLE OF THE SINGLE SUBSTRATE. THE ANSWER IS THAT ONE OF THE INTERESTING THINGS IN SUZANNE'S LAB AT EINSTEIN, YOU GET SURVIVAL OF NEURONS THAT OTHERWISE WOULD DIE BY MUTATING THOSE SITES. THERE'S THE QUANTIFICATION. THIS RAISES THE QUESTION, ARE WE JUST PRESERVING THE FULL LENGTH PROTEIN OR ARE WE PREVENTING THE GENERATION OF THIS REALLY TOXIC FRAGMENT? IF YOU TRANSSECT IN THE EQUIVALENT, IT'S VERY LETHAL. ALL RIGHT. SO WE THINK WE CAN ANSWER THAT QUESTION AND TO DO THAT WE USE THIS MOLECULE, DESIGNER MOLECULE, ABT-737 WHICH BINDS INTO THE CLEFT OF THE BCH-XL AND MIMIC BH 3 PROTEIN OR MOTIF OF KILLER PROTEIN, DETAILED CELL IN PINK. AND BAD IN YELLOW. ABT, THE VERSIONS ARE IN CLINICAL TRIALS FOR CANCER, TO KILL TUMOR CELLS BY INHIBITING ANTIDEATH PROTEINS. BUT IN THIS MODEL ISCHEMIA MODEL, IN SUZANNE'S LAB, WE GET THE OPPOSITE EFFECT. ABT PROTECTS AGAINST THE DEATH IN ISCHEMIC INJURY. AND SO OUR INTERPRETATION OF THIS IS THAT SINCE ALL OF THE PARTS THAT BIND ABT ARE STILL PRESENT, IT'S WORKING TO PROTECT BY PREVENTING THE CLEAVAGE FRAGMENT FROM DOING DAMAGE. OKAY. SO THAT'S ABOUT DEATH. ACTUALLY I CAN THINK OF NONAPOPTOTIC MECHANISMS, BUT FOR THE MOMENT WE'LL LEAVE THAT THERE. WHAT ARE THEY DOING BEFORE THEY GET CONVERTED INTO DEATH FACTORS? PEOPLE REFER TO THIS AS NONAPOPTOTIC FUNCTIONS, LIKE IT'S A LOCALIZED APOPTOTIC PROCESS, USING THE SAME MACHINELMACHINERY TO BUILD ANOTHER ONE AND SO FORTH. THAT MAY BE TRUE, THAT CASPASES ARE IMPLICATED, BUT OTHER THINGS SEEM TO BE DIFFICULT TO EXPLAIN BY THAT MECHANISM. WE SAW DETAIL XL INCREASES, RATES OF FIGURES AND FUSION, CHANGING BIOMASS, AND MULTIPLE OTHER PARTNERS IN CELLS. I'M NOT GOING TO ANSWER THAT QUESTION. I CAN JUST TELL YOU WHAT DIRECTIONS WE'RE GOING IN. SO TO LOOK AT THIS THESE ARE OLDER DATA LOOKING WITH DIFFERENT VIEW POINTS. WE MADE A DETAILED KNOCKOUT. STRAIGHT KNOCKOUT IS LETHAL. THIS IS IN THE CORTEX AND HIPPOCAMPUS, BUT SPARES INTERNEURONS AND GLEUS. IT'S KNOCKED OUT AT ABOUT E-12. SO I THINK OUR EARLIEST WAS AROUND E-16. THERE'S NO DEATH YET UNTIL ABOUT, YOU KNOW, E-19. OKAY. SO SEVERAL DAYS HAVE GONE BY, YOU CAN THINK OF LOTS OF REASONS WHY THAT MIGHT OCCUR, BUT TAKING THE OBVIOUS, IS THAT WE'VE BEEN MISSING THIS PROTEIN FOR A WHILE AND ALL OF A SUDDEN NOW WE GET A BURST OF DEATH. IT'S NO THE JUST DEATH HERE AND THERE. IT'S DEATH IN A SPECIFIC PATTERN, SO AS YOU HEARD FROM MARK MATTSON, THE NEURONS WERE BORN AND MIGRATE TO THE LAYERS OF THE CORTEX AND WE SEE THE LINES OF NEURONS, AND ANOTHER LINE THAT KEEPS GOING, DEFINITIVE LOOKING STRUCTURES WE PRESUME CORRESPOND TO A VERY SPECIFIC TYPE OF DEVELOPMENTAL SIGNAL. AND EVEN, YOU KNOW, DEATH CURTAILS AS THE WILDTYPE STARTS CATCHING UP, THE KNOCKOUT STARTS HAVING LESS DEATH, AND YET, YOU KNOW, THERE WILL BE A BIG CHANGE IN BRAIN SIZE, BUT IT HASN'T HAPPENED YET. SO AGAIN THERE SEEMS TO BE A DELAY, THIS ISN'T JUST SUMMARIZING THE RATIO OF THE KNOCKOUT AND CONTROL, EITHER IN THE NUMBER OF TUNEL CELLS OR CORTEX THICKNESS, BUT NOT MUCH IS GOING ON. AT LATER TIMES YOU HAVE BIG DIFFERENCES. THESE DO MAKE MICE AND THEY WALK AROUND, AND DO A NUMBER OF NORMAL THINGS BUT THEY HAVE SMALL BRAINS AS ADULTS. BUT THEY DO STILL HAVE TAR HE GETTED NEURONS, JUST TO CONVINCE YOU THAT LABELED WITH CRE, MANY ARE MISSING IN THE CONDITIONAL KNOCKOUT THERE ARE MANY CRE POSITIVE CELLS REMAINING. I THINK THAT SUGGESTS AT LEAST THAT IT'S NOT -- IT MIGHT NOT BE DEATH MECHANISMS AT ALL. MAYBE IT'S DOING SOMETHING IMPORTANT AND THAT THE DOWNSTREAM CONSEQUENCE IS ABOUT DEATH. AND SO HERE IS ONE PIECE OF EVIDENCE TO SUPPORT THAT, IN THE HIPPOCAMPUS, YOU CAN SEE THE MICE PROCESSES ON NEURONS IN THE CONTROL, BUT IN THE CONDITIONAL KNOCKOUT, THESE PROCESSES DON'T SEEM TO KNOW WHERE TO GO. THEY ARE COMING IN AND OUT OF THE PLANE OF THE TISSUE SO YOU CAN SEE THE CUT EDGES, LIKE THEY ARE MISDIRECTED. AND ALSO YOU CAN SEE THE MITOCHONDRIA ARE NOT GETTING TO THE TERMINUS, THAT LOOKS LIKE THE CASE IN THE BRAIN. SO HERE IS AN ADULT BRAIN, WILDTYPE KNOCKOUT MUCH SMALLER, BUT IT HAS ALL THE LAYERS. AND IT LOOKS LIKE THE DEVELOPMENTAL FORMATION OF THE LAYERS IS RELATIVELY NORMAL, AS YOU WOULD EXPECT FROM THE SMALLER BRAIN, FEWER SYNAPSES, AN EVEN GREATER DECREASE IN THE NUMBER OF PRE-SYNAPTIC TERMINALS AND MANY OTHER FEATURES AND SO FORTH. FUNCTIONAL DIFFERENCES. SO WITH WE LOOK WHERE BCL-X IS LOCALIZED, THE OUTER MITOCHONDRIAL MEMBRANES, WHEN WE TOOK THE KNOCKOUT AND WILDTYPE, IT WAS PRETTY STRIKING THAT A LOT OF THE BCL-X LABEL WAS INSIDE MITOCHONDRIA. IN ADDITION TO THE OUTER AND ADJACENT E.R. THERE'S A WHOLE LITERATURE LONG FORGOTTEN AND DISBELIEVED BY OTHERS. HOW THEY GOT THERE WE'RE STUDYING THAT. WE'VE ONLY IDENTIFIED ONE SO FAR THAT HAS A GENUINE IMPORT SIGNAL ON THE END TERMINUS, THAT'S BID, AND OTHERS REPORTED BCL-1 HAS ONE THAT WORKS PARTLY. SO WE DON'T KNOW EXACTLY HOW THEY GET INTO MITOCHONDRIA. OR WHEN, HOW, AND THOSE DETAILS. BUT ONE THING WAS CLEAR, AND THAT IS THAT THE BCL-X SUFFICIENT NEURONS IN CULTURE NOW HAVE VERY LEAKY INNER MEMBRANES INDICATED BY THE FLUCTUATION IN TMRM, AND MY COLLABORATOR HAS DONE MUCH MORE EXTENSIVE ANALYSIS OF THIS. AND INSPIRED BY OURS AND OTHER RESULTS THROUGH A NUMBER OF BIOCHEMICAL RESULTS WHICH I WON'T GO THROUGH BCL-X IS ENRICHED, AND SO LIZ HAS A PAPER IN THE WORKS CURRENTLY LOOKING AT THE SUBUNIT REGULATIONS AND SO SHE BELIEVES THERE'S A CHANNEL HERE THAT IS REGULATED BY CCL-XL, AND THAT -- BCL-XL THAT MAY CONTRIBUTE TO THE WASTED LEAKAGE OF IONS ACROSS THE INNER MEMBRANE. IF BCL-X DOES THAT AND PREVENTS CELLS FROM WASTING ENERGY, I WOULD THINK IT WOULD BE INCREDIBLY POWERFUL SURVIVAL MECHANISM, COMPLETELY INDEPENDENT OF CLASSICAL APOPTOSIS. AND THEN ONE MORE EXPERIMENT WITH YEAST, BCL TENDS TO MAKE YEAST SURVIVE BETTER EVEN THOUGH THEY DON'T HAVE THE PROTEIN BY AMINO ACID SIMILARITY. WE PUT BCL-X IN YEAST AND TREATED IT WITH DEATH STIMULI AND THEY SURVIVED BETTER. OUR POSITIVE CONTROL HERE IS A DNM-1 KNOCKOUT, DRP-1 HOMOLOG BETTER WITH MAMMALIAN STUDIES, SUGGESTED TO BE LINKED TO MITOCHONDRIA. I'LL COME BACK TO THAT IN A MINUTE. THE RESULT IS THAT IF YOU USE AN ATP 2 OR BAIT BETA KNOCKOUT YOU CANNOT PROTECT THE YEAST USING BCX L. OUR MODEL, WE'RE NOW PURSUING THAT IN A VARIETY OF DIRECTIONS, INCLUDING THE DIRECT EFFECT ON MEMBRANE CURVATURE. I WON'T TALK ABOUT THAT ANYMORE. INSTEAD I WANT TO TELL ANOTHER STORY ABOUT HOW THINGS AREN'T ALWAYS THE WAY THEY APPEAR TO BE AT FIRST. WE BECAME ENAMORED WITH THE YEAST TOOL BECAUSE PEOPLE HADN'T EXPLOITED WHAT I WOULD SAY IS THE BEST GENETIC SYSTEM AVAILABLE TO SCIENTISTS, HASN'T BEEN EXPLOITED FOR THE STUDY OF CELL DEATH. IT'S AN ARGUMENT WE CAN HAVE. I ALWAYS WELCOME THE SKEPTICS. OKAY. SO AT ANY RATE WE HAD INVENT THE THE ASSAYS BECAUSE THEY HADN'T BEEN DONE. WE USED THE KNOCKOUTS AS CONTROLS INSTEAD OF ASSAYS, BOTH ARE REQUIRED FOR MITOCHONDRIAL FIGURES IN YEAST. WHAT WE DID WAS WE PUT THEM IN A PCR MACHINE TO CONTROL HEAT PRECISELY SO WE DON'T HEAT SHOCK THEM, WE HEAT RAMP THEM. AND THEN PLATE THEM, AND REVEAL THEIR PHENOTYPES. WILDTYPE DEATH RESISTANT AND WHILE THIS IS SUPPOSED TO BE DEATH RESISTANT, IT WOULD BE PREDICTED BY MOLECULAR INTERACTION, BUT WE WERE USED TO BCL PROTEINS HAVING INVERTED FUNCTIONS, BUT WE FINALLY GOT AROUND TO IT AND DID ALL THE GENETICS AND FOUND THAT THERE WAS ANOTHER GENE IN THE F OF PHY-1 KNOCKOUT. WE GOT THOSE MADE BY OTHERS BUT THAT THE SAME GENOTYPE. WE DID THE -- WE SEQUENCED THE GENOMES AND FOUND ALL THREE DEVELOPED A POINT MUTATION IN THE SAME GENE. THERE'S ALMOST NOTHING KNOWN ABOUT THIS GENE, WHI-2. WHAT DOES IT DO? THIS IS A PETITE FREQUENCY ASSAY, MITOCHONDRIA IN YEAST, IT'S VERY HIGH, LOOKING AT RAPIDLY REPLICATING CELLS, HERE IS WILDTYPE AND FIS-1. NOW WHEN YOU COMBINE THE MUTATIONS YOU INCLUDE THE MITOCHONDRIAL FUNCTIONS. THIS COULD BE ONE REASON THEY NEED TO SELECT FOR A WHI-2 FUNCTION. WE HAVEN'T GOTTEN BACK TO DOING E-M. THIS IS A PHENOTYPE OF THE FIS-1 WHERE MITOCHONDRIA ARE GLUED AND CANNOT FUSE AND I KNOW THERE'S A COUPLE NEW REPORTS OUT LOOKING AT THE ROLE OF FIS-1 WHICH IS VERY INTERESTING. KNOWN IF THIS IS FIS-1 OR WHI-2 OR BOTH. THIS IS THE WILDTYPE FRAGMENTING. WHAT ELSE DOES WHI-2 DO? IT HAS A VERY INTERESTING OTHER PHENOTYPE CONCENTRATE OVER HERE, THIS IS A CONTROL MEDIA. WE FOUND THIS BY ACCIDENT BY BORROWING A PLATE FROM ANOTHER LAB. MEDIUMS BY THE SAME NAME, DIFFERENT RECIPE. IT TURNS OUT THE DIFFERENCE BETWEEN THE MEDIUM IS IT HAS LOWER -- WILDTYPES AND LOW LUCENE STOP GROWING, IT'S NOT LIKE IT DOESN'T HAVE ENOUGH FOOD, BECAUSE THE STRAINS KEEP GROWING. THEY OUTGROW WILD TYPE, MEANING THEY EITHER CAN'T SENSE OR CANNOT RESPOND TO LOW LUCENE, WHICH IS A NEW FUNCTION FOR WHI-2. THIS IS A RESISTANT MUTANT. WE CAN RESTORE WHI-2, WE DID DEMONSTRATE IT'S A FUNCTION, THAT IMPLIES WHI-2 COULD BE A SUPPRESSOR OF TOR, WITH INHIBITION OF PROLIFERATION OR REGULATION AND REMOVE AMINO ACIDS OR REMOVE WHI-2 AND NOW YOU DON'T -- YOU FAIL TO TURN OR TOR, YOU KEEP GROWING, THE PREDICTION WOULD BE YOU WOULD SUPPRESS AUTOPHAGY. WE ASKED ARE THERE ANY OTHER KNOCKOUT GENE STRAINS THAT ALSO DEVELOP A WHI-2 MUTATION? OKAY. AND THE ANSWER IS YES. A LOT OF THEM. WE SCREENED THE ENTIRE KNOCKOUT SELECTION OF ESSENTIAL GENES, WE FOUND A SHOCKING 750 OF THEM OVERGROW, AND 250 OR SO OF THOSE HAVE A WHI-2 CONSTELLATION OF PHENOTYPES, THE NUMBER IS NOW 145. 145 DIFFERENT GENES, WHEN KNOCKED OUT, GET A WHI GENE MUTATION. MAYBE SOME OF YOU ARE THINKING THAT'S RANDOM, YOU MAKE ANOTHER KNOCKOUT COLLECTION, YOU'LL GET ANOTHER DIFFERENT SET OF 145 GENES, WHATEVER, AND THAT'S NOT TRUE. IF YOU DO KNOCK OUT THOSE SAME GENES IN A DIFFERENT COLLECTION, YOU GET REMARKABLE -- AT LEAST HALF, I FORGET THE NUMBERS, ALSO ACQUIRED A WHI-2 MUTATION AND MANY ACQUIRED THE SAME PHENOTYPE THROUGH A DIFFERENT MUTATION. SO I THINK THIS IS A VERY IMPORTANT GENE. BUT IT'S PUBLISHED TO BE -- OH, I DIDN'T MENTION THIS. THIS IS JUST A MAP OF ALL OF POINT MUTATIONS WE FOUND IN THE KNOCKOUT STRAINS IN YEAST AFTER SEQUENCING. SO TO FOLLOW UP THIS QUESTION, IT'S IMPORTANT TO INVESTIGATE FURTHER, NOW WE'RE ASKING IS THIS TRUE, DOES IT REGULATE DOWNSTREAM TARGETS OF TOR AND REGULATE AUTOPHAGY? WE TOOK OUT, AGAIN, WHI-2 AND WE ADDED A REPORTER, THIS IS AN ATG-8 PROMOTER GIVING THIS REPORTER. YOU GET A ROBUST INDUCTION WHEN YOU TAKE OUT LUTENE, IT'S NOT ANYTHING ELSE, NOT GLUCOSE, NOT ANY AMINO ACIDS, ONLY LUTENE. SORRY, I GOT AHEAD OF MYSELF. THIS IS NORMAL, ON THE WHI-2 YOU GET A FAILURE TO INDUCE AUTOPHAGY AND FLUX AUTOPHAGY. THIS IS NORMAL. THIS IS IMPAIRED. YOU SEE A LITTLE BIT OF INDUCTION. I THINK THAT'S BECAUSE THEY ARE RUNNING OUT OF MAYBE SOMETHING ELSE LIKE A LITTLE BIT OF GLUCOSE OR WHATEVER. IF YOU TAKE OUT SOMETHING BESIDES LUTENE IT LOOKS LIKE THIS. WE BORROWED THE MAMMALIAN ANTIBODY. WITH HELP FROM OUR INFORMATIC COLLEAGUINGCOLLEAGUES AT HOPKINS IT WAS EASY TO IDENTIFY THE MAMMALIAN. ALMOST NOTHING IS KNOWN ABOUT THIS, VERY LITTLE INFORMATION IN LITERATURE, MOST OF THEM HAVEN'T BEEN PUBLISHED, OR NO CHARACTERIZATION LAB DONE, BUT NOW A NUMBER ARE STILL SHOWING UP IN DISEASE STATES, THIS IS IMPLICATED IN SEVERAL REPORTS, AND THIS IS THE MOST DEFINITIVE, INCLUDING WORK DONE HERE AT NIH, THE RARE DISEASE WHERE THEY SEQUENCE A FAMILY AND ALSO FOUND KCTD-7 IN EPILEPSY AND NEURODEGENERATIVE DISEASE. HERE IS THIS START. WE EXPRESS CCTD-11 AND ASK IF IT CAN SUPPRESS TOR ACTIVITY. SO HERE IS -- TAKE OUT AMINO ACID FOR ALMOST AN HOUR AND YOU DECREASE TOR IN THE CONTROL. YOU REALLY DECREASE TOR IF YOU OVERCOMPRESS KCTD-11. IT TAKES TEN MINUTES TO TURN TOR BACK ON. YOU MAY HAVE AN INCREASE IN THE S6K TOTAL. HERE IS THE NIH EFFORT, IN THEIR HOMETOWN. TWO OF THE FOUR CHILDREN HAD MUTATIONS IN THE GENE, A STUDENT IN MY LAB HAS BEEN COLLECTING THE MUTATIONS AND HAS ABOUT 24 PATIENTS NOW AROUND THE WORLD THAT HAVE KCTD-7. HE PASS A POSTER HERE. HE HAS A POSTER HERE. YOU CAN TALK TO HIM FOR MORE INFORMATION. AS SOON AS WE EXCHANGE THESE CELLS WITH ANTI-TOM20, IT WAS OBVIOUS THAT THERE WAS SOMETHING DIFFERENT ABOUT THE NETWORK AND THEN WE SAW WITH KYLE DOING A COMPUTATIONAL METHOD TO ANALYZE THESE NETWORKS, COLOR-CODED BY THEIR TOTAL NETWORK LINK. THEY HAVE MANY OTHER MITOCHONDRIAL DEFECTS IN ATT PRODUCTION AND AUTOPHAYY, SO KCTZ-7 LOCALIZES TO BIZARRE MEMORIAL RAINS THAT KYLE HAS PICTURES OF. I LIKE TELLING THE STORY. PURE BASIC SCIENCE ON A YEAST, WHICH THERE'S VERY LITTLE NEW FUNDING ON IN NIH, SO MANY YEAST PEOPLE HAD TO MOVE TO OTHER PROJECTS, LED TO THE IDENTIFICATION OF WHAT I THINK IS SOMETHING WRONG WITH THESE KIDS WITH EPM-3. THANK YOU. [APPLAUSE] >> DO YOU KNOW OF ANY LINK BETWEEN, IN THE FIRST PART OF THE TALK, BCL-XL KNOCKOUT AND MITOPHAG JOBS. >MITOPHAGY? >> I HAVEN'T DONE THAT EXPERIMENT. YOU CAN PROTECT THE MITOS FROM PARKIN, FOR EXAMPLE, GOING TO A SUBSET BUT NOT TO MOST OF THEM. I DON'T KNOW HOW THAT WORKS. THEY ARE REPORTED TO INTERACT TO REGULATE AUTOPHAGY IN A NEGATIVE DIRECTION. I THINK THERE'S MORE TO THAT PROBABLY. THERE ARE GOING TO BE POSITIVE REGULATION OF AUTOPHAGY BUT I'M SPECULATING FROM VERY LITTLE EVIDENCE SO I DON'T KNOW THE ANSWER TO YOUR QUESTIONS. >> YES. BECAUSE WHEN YOU SHOW THAT THERE WAS A SORT OF KILL DEATH, ONE CAN IMAGINE THAT IF YOU SOMEHOW AFFECT THE MITOPHAGY IN KNOCKOUTS COULD LEAD TO CELL DEATH AFTERWARDS AS A PHENOMENON. >> I DON'T -- I THINK AUTOPHAGY IS NOT THE UNDERLYING BIOCHEMICAL FUNCTION, THAT BCLX CARRIES OUT, BUT I THINK IT'S IN THE PATHWAY. >> EVEN IN THE QUESTION THAT WAS ASKED TO DR. YOULE, THAT CAN COUNTERACT THE EFFECT OF, YOU KNOW, MITOCHONDRIAL AND PINK1 AND SO FORTH. >> I KNOW. THANK YOU. A LOT OF PEOPLE ARE RESISTANT TO THIS IDEA BECAUSE THEY KEEP TELLING ME WE PURIFIED IT, BCLX IS NOT IN THERE. YOU CAN PURIFY OUTER MEMBRANES, AND NOT FIND BCXL. I KEEP ASKING DO YOU FIND THOSE PROTEINS? THEY ARE NOT IN THERE. I DON'T KNOW HOW THEY MOVE. I DON'T THINK THAT DETERS ME. YES? >> SO THE LOCALIZED BCXL, I'M WONDERING WITH SPECIFIC NEURONS OR GENERAL TO OTHER CELLS? >> RIGHT. I HAVEN'T LOOKED. I'LL PREDICT THERE ARE MANY OTHER PLACES WHERE IT GETS INTO THE MATRIX, BUT I HAVEN'T LOOKED EXCEPT IN THE BRAIN AND NEURONS. THE END TERMINUS IS -- IT ENGAGES THE IMPORT MACHINERY, BUT PROBABLY IS NOT SUFFICIENT TO IMPORT. >> IN OUR EXPERIMENTS WE FIND COMPARATIVE DETAIL TO THE CLOSE MEMBER OF THE DETAIL PROTEIN, BCXL IS MORE FUNCTIONAL IN NEURONS THAN BCL-2. >>> BCL-2 CAN KILL NEURONS PRETTY NICELY BUT IN THE BRAIN BCXL IS BY FAR THE MOST ABUNDANT OF THEM SO THAT MAKES SENSE. SOMETHING SPECIAL. OKAY. >> A QUICK QUESTION. DOES A WHI-2 KNOCK OUT MITOCHONDRIA IN YEAST? >> WELL, YOU KNOW, WE HAVEN'T COME BACK TO LOOK AT THAT CAREFULLY. I JUST KNOW IT DOESN'T CAUSE A PHENOTYPE LIKE THE FIS-1 KNOCKOUT. WE USE THAT AS PART OF THE ANALYSIS. WHI-2 DOESN'T HAVE THAT FUSION PHENOTYPE, BUT DOES IT HAVE OTHER MITOCHONDRIAL MORPHOLOGY CHANGES? I WOULD BET SO. I HAVEN'T LOOKED CAREFULLY. >> I'VE GOT TWO QUICK QUESTIONS. ONE WAS RELATED TO YOU CITED A PAPER THAT YOU DID, HAVE YOU DONE ANY MORE WORK WITH THE BCL OR XL IN IT RELATION TO MEMBRANE POTENTIAL AND COUPLE EFFICIENCY? >> IN WHAT SITUATION? >> COUPLING EFFICIENCY AND MEMBRANE POTENTIAL. >> CATALYTIC. >> YEAH. >> BCXL INCREASES ATP OUTPUT OF MITOCHONDRIA. HOW, I GUESS WE'RE THINKING ABOUT PERMEABILITY, BUT -- >> OKAY. >> THAT'S AN OPEN QUESTION. >> YEAH, IT'S A VERY INTERESTING AREA I'M INTERESTED IN. SECOND QUESTION, WHEN YOU FIRST STARTED AS WELL, YOUR TALK, HAVE YOU CONSIDERED THIS IN RELATION TO AUTISM? >> NOT SO FROM BCL-2 BUT ONE WAS POSED IN "NATURE" AS BEING A NEW AUTISM GENE. WE HAVEN'T CLONED IT OUT YET. >> THANK YOU. >> IN THE SAME REGARD, THAT GENE, YOU MENTIONED 11 AND 7 I THINK -- >> THAT'S IT'S ONE. >> WHEN THAT WAS KNOCKED DOWN, WITH ZEBRA FISH, IT GOT A BRAIN-SPECIFIC DEFECT. THE QUESTION IS DO YOU THINK THAT ALL OF THE KCT -- I CAN NEVER SAY IT, KCTD -- >> IT'S A TERRIBLE NAME. >> PEOPLE THINK IT MUST BE ABOUT THE POTASSIUM WHICH HAS NOTHING TO DO WITH ANYTHING I CAN READ. DO YOU THINK ALL HAVE A SIMILAR EFFECT ON BIOENERGY ETHICENERGY ECICS AND TOR? >> NOT REALLY. I CAN'T FIND ANYBODY ELSE WHO RECOGNIZES THEM. SO THEY HAVE AN END TERMINUS, BUT MOST OF THE FAMILY HAS UNIQUE SUBTERMINUS, LONG, SHORT, DIFFERENT MOTIFS, I'M GOING TO GUESS THE END TERMINUS HAS A COMMON MEMBRANE, BCD MEMBRANES ARE KNOWN TO BIND, MAYBE THAT'S INVOLVED, THE C-TERMINUS IS TARGETING, YOU KNOW, FINDING DIFFERENT THINGS IN THE CELL TO DEAL WITH. WE'RE JUST STARTING TO FIGURE OUT WHAT THOSE MIGHT BE. SO -- YEAH, COMPLETELY -- IT WAS AMAZING TO ME WHEN THEY SEQUENCED THE GENOME, THERE WERE ZERO PAPERS ON CTD-7 IN THE LITERATURE. >>> THE NEXT SPEAKER IS GARY FISKUM FROM UNIVERSITY OF MARYLAND SCHOOL OF MEDICINE. HE'S BEEN A PIONEER IN TRYING TO UNDERSTAND THE ROLE OF ALTERATIONS IN MITOCHONDRIA IN BRAIN INJURY, PARTICULARLY ISCHEMIC BRAIN INJURY AND TRAUMATIC BRAIN INJURY IN NEONATES, AND THAT'S WHAT HE WILL TALK ABOUT TODAY. >> THANKS. >> THANKS, MARK AND STEVE FOR ORGANIZING THIS WONDERFUL SYMPOSIUM AND ALSO FOR HONORING PROFESSOR WALLACE, WHO IS REALLY ONE OF THE GIANTS IN THE FIELD MITOCHONDRIAOLOGY. THIS IS A SHIFT IN GEARS HERE. I'M NOT GOING TO TALK MOLECULAR BIOLOGY OR TRYING TO PROVE ANY SPECIFIC MITOCHONDRIAL MECHANISM OF CELL INJURY OR DEATH, BUT REALLY GIVE SOME EXAMPLES OF ADVANCES IN NEURO PROTECTION FOR ACUTE NEUROLOGIC DISORDERS THAT AT LEAST WERE INSPIRED BY THE THOUGHT OF TARGETING MITOCHONDRIA. NO DISCLOSURES AND I APPRECIATE THE SUPPORT FROM NIH. THIS IS A BROAD OVERVIEW OF THE MITOCHONDRIAL TARGETED NEURO PROTECTION, PARTICULARLY THAT HAS BEEN APPLIED IN DIFFERENT AN POSTAL MODELS OF EITHER ISCHEMIC OR TRAUMATIC BRAIN INJURY. AT THE ONSET, I'D LIKE YOU TO APPRECIATE THE FUNDAMENTAL DIFFERENCE BETWEEN THESE ACCUSE NEUROLOGIC DISORDERS, COMPARED TO NEURODEGENERATIVE DISEASES. THE CELL DEATH AND NEURONAL DEATH THAT OCCURS IN TRAUMA OR ISCHEMIA, WHETHER IT'S FOCAL OR GLOBAL, IS VERY RAPID AND VERY EXTENSIVE. IT CAN HAPPEN, START WITHIN AN HOUR, AND COME TO FRUITION WITHIN DAYS IN THE BRAIN, AND SO THE MECHANISMS HAPPEN VERY RAPIDLY. THERE ARE ALSO CHRONIC MECHANISMS OF INJURY SUCH AS INFLAMMATION THAT FOLLOW BUT THE INITIAL MECHANISMS OF CELL INJURY AND DEATH ARE ACUTE AND EXTENSIVE. ONE OF THE FIRST RECOGNIZESSED WAS FAILURE AND CEREBRAL ENERGY MMETABOLISM. DIFFERENT FUELS, ALTERNATIVE BIOFUELS, THE ADULT BRAIN USES ALMOST EXCLUSIVELY GLUCOSE, WITH GLUCOSE METABOLISM INHIBITED, THERE'S EVIDENCE OTHER FUELS WITH COMPENSATE FOR THAT INHIBITION. MORE RECENTLY, THE WHOLE CONCEPT OF INCREASING NUMBERS OF ACTIVE MITOCHONDRIA THROUGH BIOGENESIS, PG-1 ALPHA ACTIVORS. OXIDATIVE STRESS BECAME APPARENT VERY EARLY ON IN THE STUDY OF ACUTE ISCHEMIC AND TRAUMATIC BRAIN INJURY. AND THIS IS A FIELD CERTAINLY THAT WE'VE WORKED ON, THERE HAVE BEEN A NUMBER OF ATTEMPTS AND SOMEWHAT SUCCESSFUL IN ANIMAL MODELS USING MITOCHONDRIALAL TARGETED ANTIOXIDANTS, MITOCHONDRIAL TARGETED CO-ENZYME-Q, SPIN AND THE WHOLE FIELD OF UNCOUPLER INDUCED NEURO PROTECTION IS FOCUSED ON ACCUSE BRAIN INJURY, STARTING OUT WITH LABS USING PROTON IONOPHORES LIKE FCCP, A SLIPPERY SLOPE BECAUSE IF YOU OVERDOSE YOU HAVE ENERGY FAILURE AND RAPID CELL DEATH. IN FACT, A NUMBER OF WEIGHT LOSS CLINICS OVER THE YEARS HAVE ACTUALLY USED THESE UNCOUPLER MOLECULES FOR INDIVIDUALS, A NUMBER OF DEATHS OCCURRED. THE CONCEPT IS BY MILD UNCOUPLING, REDUCING THE MEMBRANE POTENTIAL, A SMALL INCREMENT, THAT ONE WOULD NOT HAVE THE REDUCING ENERGY FORCE TO PROMOTE THE FORMATION OF SUPEROXIDE, AND METABOLITES. ENDOGENOUS PROTEINS ARE LESS LIKELY TO BE TOXIC, AND A RECENT PAPER I SAW JUST THE OTHER DAY ABOUT A HUNGER HORMONE HAS BEEN SHOWN TO STIMULATE UCP STIMULATION, THE WHOLE FIELD OF ACTIVATING MITOCHONDRIAL KATP CHANNELS IS BASED ON MILD UNCOUPLING. IN ADDITION TO APPROACHING FORMATION OF REACTIVE OXYGEN SPECIES THERE'S THE DETOXIFICATION AND ONE CLASS OF AGENTS WE'VE WORKED ON THAT I'LL MENTION TODAY ARE THOSE THAT ACTIVATE THE NRF 2 TRANSCRIPTIONAL FACTOR. A LOT OF EXPRESSION HAS GONE INTO INHIBITING THE PERMEABILITY TRANSITION. YOU HEARD FROM DR. MATTSON EARLIER, BUT CERTAINLY THERE'S VERY GOOD EVIDENCE IN BOTH CARDIAC AND CEREBRAL ISCHEMIA THAT PERMEABILITY TRANSITION OPENING NOT JUST FLICKERING BUT OPENING TO THE POINT WHERE THERE'S LITERALLY OSMOTIC PLAYING AN IMPORTANT ROLE. I THINK MORE NEEDS TO BE INCLUDED, SOME APPROACHES HAVE INCLUDED THE HIV TAP PROTEIN CONJUGATED TO GET IT INTO CELLS IN THE CENTRAL NERVOUS SYSTEM, SYNTHESIZED BAX ANTAGONISTS, DRP INHIBITORS LIKE MDIVI-1 WHICH PLAYS A ROLE IN APOPTOSIS, AND ONE WAS THE ABT-737 FROM THE JONAS LAB MARIE MENTIONED EARLIER. THERE'S A CLOSE RELATIONSHIP BETWEEN OXIDATIVE STRESS AND MITOCHONDRIAL ENERGY METABOLISM. OF COURSE, UNDER NORMAL CIRCUMSTANCES, 99% OF OXIDANT USED IN CELLS IN THE BODY, CERTAINLY THE NEURONS, IS CONVERTED TO WATER THROUGH RESPIRATION BUT THERE'S A SIGNIFICANT LEVEL THAT IS PRODUCES SUPER-OXIDE AND OTHER FREE RADICALS SUCH AS NITRIC OXIDE FOR PHYSICA PHYSIOLOGICAL PURPOSES. THOSE ARE METABOLIZED TO VARIOUS DIFFERENT METABOLYTES. THESE ARE PROMOTED BY CONDITIONS WHERE THERE'S AN INCREASE IN THE AVAILABILITY OF REDUCED IRON OR ACIDOSIS THAT INCREASES IRON'S AVAILABILITY. AND THOSE ULTIMATELY OXIDIZE LIPIDS, PROTEINS, DNA AND RNA, ULTIMATELY EFFICIENT ENOUGH TO CAUSE ENZYME DYSFUNCTION, METABOLIC INHIBITION, AND JUST AS IMPORTANT AS THE FORMATION ARE THE MECHANISMS OF DETOXIFICATION THAT INVOLVES REDUCING POWER, FUEL AT SOME LEVEL, THE CO-FACTOR NADPH WHICH WORKS IN ENZYMES THAT KEEP GLUTOTHIONE REDUCE AND VARIETY OF ENZYMES RESPONSIBLE FOR DETOXIFICATION. I GOT THIS A LITTLE OUT OF ORDER. I'VE GOT TO GO BACKWARDS. SO THE MAIN ASPECT OF ACUTE BRAIN INJURY THAT WE WORKED ON, AND THAT HAS BEEN CLINICALLY TRANSLATED HAS TO DO WITH CARDIAC ARREST. IN THE UNITED STATES ALONE THERE'S OVER 250,000 INDIVIDUALS SUCCESSFULLY RESUSCITATED BUT VIRTUALLY ALL OF THOSE PEOPLE ARE NEUROLOGICALLY IMPAIRED TO SOME DEGREE OR ANOTHER. SOME VERY SEVERELY, SOME NOT SO SEVERELY BUT STILL IN A STATE WHERE THEY ARE NOT NECESSARILY ABLE TO GO BACK TO WORK, ET CETERA, ET CETERA. THE PRIMARY CAUSE OF THIS BRAIN INJURY IS THE ISCHEMIA AND REPER FUSION, THE ISCHEMIA AND CARDIAC ARREST CAN ONLY BE A SHORT TIME OR THE VICTIM IS NOT RESUSCITATIVABLE. 20 DO 30 MINUTES, IT'S ALMOST IMPOSSIBLE TO RESUSCITATE. LITTLE A BRIEF PERIOD OF COMPLETE I ISCHEMIA FOLLOWED BY A PERIOD REPERFUSION DRIVEN BY PRIMARY AND SECONDARY OXIDATIVE STRESS. CAUGHT EARLY ON, IN THE MID-'80s WHEN I WAS AT GEORGE WASHINGTON UNIVERSITY WORKING WITH AN EMERGENCY MEDICINE PHYSICIAN, BOB ROSENTHAL, WHO RESUSCITATED A LOT OF PEOPLE, IN DISCUSSIONS ABOUT RESUSCITATION, AND IN PLANNING OUR ANIMAL MODELS, HE SAID WE'RE GOING TO GIVE A HUNDRED PERCENT OXYGEN WHEN WE RESUSCITATE THE INDIVIDUALS. OH, IS THAT RIGHT? WHEN WAS THAT PROVEN TO BE EFFECTIVE? NOBODY HAD EVER QUESTIONED IT. IT JUST SOUNDED GOOD. SO IT STRUCK A CHORD WITH ME. IT MIGHT BE THE RIGHT THING TO DO BUT IT MIGHT BE OVERDOING IT AS WELL AND IT MIGHT BE EXACERBATING THE OXIDATIVE STRESS. AND THAT WAS PARTICULARLY BECAUSE JUST A COUPLE YEARS EARLIER, I REMEMBERED READING AN ARTICLE IN ARCHIVES OF BIOPHYSICS, WITH LIVER AND BRAIN MITOCHONDRIA SHOWING THAT THERE IS NO SATURATION OF REACTIVE OXYGEN SPECIES PRODUCTION WITH AMBIENT OXYGEN CONCENTRATES. CON -- CONCENTRATION. IN THE BRAIN, THE PERCENT OXYGEN IS DOWN IN THE ORDER OF 3 TO 5%, BUT IT DOESN'T SATURATE, WHEREAS THE SATURATION FOR CEREBRAL ENERGY METABOLISM IN THE BRAIN IS ABOUT, OH, 1% OXYGEN. SO IF YOU ELEVATE OXYGEN BEYOND THAT 1% IN SITU LEVEL YOU'RE NOT DOING ANYTHING TO SIMULATE ENERGY METABOLISM BUT THE HIGHER YOU GO, THE HIGHER OF OXYGEN SPEAKS. SPECIES. IN THIS INITIAL ARTICLE THEY LOOKED AT THE SAME WITH MICRO SOMES, AND THE SAME THING OCCURS WITH THEM. I THINK IT'S PARTICULARLY RELEVANT FOR NONNEURONNAL CELLS SUCH AS MICRO GLEA KNOWN TO BE ACTIVATED STIMULATING ENZYMES RESPONSIBLE FOR THOSE REACTIVE OXYGEN SPECIES. SO THE INITIAL SET OF EXPERIMENTS I'D LIKE TO SUMMARIZE HAVE TO DO WITH THOSE DEALING WITH PROTECTION AGAINST EARLY POST ISCHEMIC MITOCHONDRIAL BIOENERGETIC DYSFUNCTION AND DELAYED NEUROLOGIC INJURY BY AVOIDING UNNECESSARY HYPEROXIA, MITOCHONDRIA CAN INCREASE FREE RADICAL PRODUCTION WITH HIGH LEVEL OF OXYGEN BACKUP IT WAS KNOWN ENZYME WAS SENSITIVE TO OXIDATIVE STRESS. SUBSEQUENTLY WE KNOW NOW THERE ARE A VARIETY OF MITOCHONDRIAL PROTEINS AND ELECTRON TRANSPORT CHAINS THAT ARE SENSITIVE TO THIS. SO THE BASIC HYPOTHESIS, WE COULD IMPROVE OUTCOME BY AVOIDING UNNECESSARY HYPEROXIA. THIS SUMMARIZES A NUMBER OF STUDIES OVER THE YEARS. THESE PARTICULAR DATA WERE OBTAINED FROM A CANINE CARDIAC ARREST AND RESUSCITATION MODEL. I CAN TALK TO YOU AFTERWARDS ABOUT THE NECESSITY FOR USING A LARGE ANIMAL MODEL LIKE THAT. IT'S CERTAINLY FAR, FAR, FAR MORE REPRODUCIBLE THAN A RODENT MODEL, MORE CLINICAL RELEVANT IN THE WAY IT IS CONDUCTED THAN THAT OF RODENT MODELS. WE'VE ALSO USED RODENT MODELS AND CONFIRMED THAT, THE SAME RESULTS, IN RODENTS BUT WE FEEL THE DOG OR PIG ARE A MUCH BETTER MODEL. EARLY ON WE FOUND THAT IF WE -- IN THIS CASE IT WAS ROOM AIR COMPARED TO 100% OXYGEN DURING RESUSCITATION AFTER A 10-MINUTE DEFIB INDUCED CARDIAC ARREST. INSTEAD OF THE HIGH OXYGEN LOWERING LACTATE PRODUCTION IT INCREASED TISSUE LACTATE, TRUE EARLY ON, UP TO 24 HOURS AFTERWARDS. SO THAT KIND OF PUT A DENT IN THE IDEA YOU NEED TO RESUSCITATE WITH A LOT OF OXYGEN. IT MIGHT BE A DAMAGE TO METABOLIC ENZYMES AND LOOKED AT PYRUVATE D.H. AND GLUTAMATE. IT'S A DIFFERENT DISORDER BUT SINCE IT FORMS THE BRIDGE BETWEEN AN AEROBIC AND AEROBIC, IT COULD BE A SITE OF INHIBITION, IN THE HIPPOCAMPUS, AT TWO OR 24 HOURS AFTERWARDS, THE HYPEROCIX ANIMALS, IT DIDN'T REALLY PROVE THE ENERGY METABOLISM WAS DOWN, THIS IMPLIED IT WAS DOWN, SO WE INFUSED THE ANIMALS WITH C-13 GLUCOSE, AT AN HOUR INTO THE RESUSCITATION, AND THEN REMOVED THE BRAINS AT TWO HOURS IN THIS EXPERIMENT, PROCESSING THE TISSUE FOR C-13 NMR TO LOCALIZE THE CARBON INTO VARIOUS MA METABOLITES, LOOKING AT THE DIFFERENT CARBONS, A BIG SIGNAL IN THE BRAIN, YOU KNOW, THE TCA CYCLE, AND I THINK IN THE C-5 POSITION WE FOUND THE HYPEROXIC RESUSCITATION DECREASED THAT INCORPORATION INTO GUTOMATE. REALLY THE FIRST DIRECT EVIDENCE THE AEROBIC METABOLISM WAS WORSENED BY HIGH OXYGEN. WE THEN ISOLATED MITOCHONDRIA FROM DIFFERENT LOCATIONS IN THE BRAIN, HIPPOCAMPUS MORE DAMAGE, CORTEX LESS DAMAGE, THERE WAS A SELECTED INHIBITION IN THE HIPPOCAMPUS, WHEN WE LOOKED AT SUBSTRATES PARVATE MALATE SHOWED A DROPPED IN MAXIMAL STAGE 3 RESPIRATION, A TREND IN INHIBITION WAS NOT SIGNIFICANT, SUPPORTING THE CONCEPT PARVATE MEDIATED RESPIRATION WAS SELECTIVELY SENSITIVE. A COUPLE EXAMPLES OF RESULTS SUPPORTING THE HIGH BOTH 'TIS IT HYPOTHESI S IT WAS STRESS, INDEED SUBSTANTIALLY HIGHER IN THE HYPEROXIC COMPARED TO NORMOXIC GROUP. WE LOOKED AT DNA/RNA OXIDATION, SO THIS IS WHERE THE DATA STARTS TO KIND OF GET JUMBLED UP TOGETHER. WE DID A NUMBER OF PARADIGMS, AND THE MOST CLINICALLY RELEVANT PARADIGM, WHICH INCLUDES THESE DATA, DID NOT JUST USE ROOM AIR. CLINICALLY THAT ONE WOR WOULDN'T WORK FOR A LOT OF PEOPLE ARRESTED, THEY ARE 80-YEAR-OLD PEOPLE WHO HAVE BEEN SMOKING, BAD PULMONARY FUNCTION, THEY WOULD BE HYPOXIC, SO WE CAME UP WITH A SCHEME TO TIGHT RATTITRATE OXYGEN TO REACH A TARGET HEMOGLOBIN SATURATION BETWEEN 94 AND 96% SO THE INDIVIDUAL IS NEITHER HYPOXIC OR HYPEROXIC. WE FOUND WITH HISTO PATHOLOGY THERE WAS A REDUCTION IN THE SHORT TERM NEUROLOGY IR NEUROLOGIC INJURY, THE ANIMALS WERE AWAKENED AND REANEST THA --AND REANESTHETIZED. WITH THE DOG MODEL, A COUPLE WITH THE RAT MODEL AND ANOTHER LAB THAT CONFIRMED QUALITATIVELY THE RESULTS WITH PIG MODEL, BACK IN DECEMBER OF 2010 THE AMERICAN HEART ASSOCIATION ADVANCED CARDIAC LIFE SUPPORT GUIDELINES QUOTING PARTICULARLY OUR WORK CAME TO THE CONCLUSION THE GUIDELINES NEEDED TO BE CHANGED, AND THAT PROVIDED APPROPRIATE EQUIPMENT IS AVAILABLE, PULSE OXYMETRY, RESUSCITATION PERSONNEL SHOULD ADJUST THE OXYGEN TO MINIMUM CONCENTRATION, NOT THE MAXIMUM NEEDED TO ACHIEVE HEMOGLOBIN SATURATION GREATER THAN 95% WITH THE GOAL OF AVOIDING HYPEROXITY. THEY REITERATED THAT IN 2013. WE'RE HAPPY OUR RESULTS HAVE LED TO A CHANGE IN GUIDELINES THAT WE THINK ARE MORE STRATEGIC AND REASONABLE. I MUST ADMIT THOUGH THIS IS NOT BASED ON ANY PROSPECTIVE CLINICAL TRIAL. WE'RE A LITTLE BIT ANXIOUS ABOUT THAT. THERE IS ONE IN PROGRESS. THERE HAVE BEEN SEVERAL RETROSPECTIVE TRIALS INDICATING THAT AT LEAST MORTALITY IS WORSE WITH CARDIAC ARREST VICTIMS WITH HYPEROXIA. NOW I WANT TO SWITCH OVER TO ANOTHER TACK, AND THIS GETS BACK TO THE DETOXIFICATION, SO OUR INITIAL APPROACH WAS TO SIMPLY REDUCE THE AMOUNT OF OXYGEN, THEREFORE REDUCING THE OXIDATIVE STRESS, BUT IS THERE A ROLE OF INTERVENTIONS IN INCREASING DETOXIFICATION? AND THIS GETS TO THE HYPOTHESIS GENOMIC PRECONDITIONING AGAINST OXIDATIVE STRESS REDUCES MITOCHONDRIAL DYSFUNCTION AND BRAIN INJURY. AND WE USED THIS NRF-2 TRANSCRIPTIONAL FACTOR AS AN APPROACH, FOR THOSE NOT AWARE, NRF 2 IS BOUND TO KEAP-1, AND IS TARGETED FOR DEGRADATION, NORMAL LEVELS ARE LOW. WHEN SPECIFIC GROUPS ARE OXIDIZED BY FREE RADICALS OR A SHIFT IN THE REDUX STATE OR REACTION WITH SPECIFIC CHEMICALS LIKE THIS COMPOUND FOUND IN CRUCIFEROUS VEGETABLES, ALLOWED TO ENTER THE NUCLEUS, TURNING ON GENES. WE USED THE SAME ANIMAL MODEL, THESE ARE UNPUBLISHED RESULTS, RECENT AND HERE WE'RE LOOKING AT THE HIPPOCAMPUS COMPARING SHAMS, ANIMALS THAT SHOW DAMAGE AND OXYMETRY ANIMALS THAT HAD SULFORAPHANE IN ADDITION TO OXYMETRR, REDUCING THE DNA OXIDATION, AND A PROFOUND DECREASE IN THE NEUROLOGIC DEFICIT SCORES, 100 BEING BRAIN DEAD, ZERO BEING NORMAL, ANY ANIMALS IN THIS ARE COMATOSE, THEY COULD WALK AROUND, THEY WOULD RESPOND TO A WHISTLE, ET CETERA. AND A SIMILAR PROFOUND REDUCTION IN CELL DEATH WITH THE SU LFPOFHORAPHANE. WE KNEW IT HAD EFFECTS BUT NOBODY LOOKED AT THE MITOCHONDRIA. WE THOUGHT, WELL, THIS MIGHT INVOLVE THE MITOCHONDRIA. IT DOESN'T HAVE TO BUT LET'S SEE WHAT SULFORAPHANE DOES TO MITOCHONDRIA. FOR MANY YEARS, WE'VE WORKED WITH REGULATION OF THE PERMEABILITY TRANSITION, AND OUR CONTRIBUTIONS ARE MAINLY IN THE REDUX SENSITIVITY OF PERMEABILITY, DUE TO CYCLOPHILIN-D, WITH AN INFLUX OF CALCIUM WITH OXIDATIVE STRESS. THAT'S KEPT REDUCED. SO A TRANSCRIPTIONAL ACTIVATING FACTOR WOULD BE EXPECTED TO PROTECT AGAINST PERMEABILITY TRANSITION AND I WON'T GO INTO GREAT DETAIL, THIS IS A MEASUREMENT OF CALCIUM RELEASED FROM THE MITOCHONDRIA, THIS IS A MEASUREMENT OF NADPH REDUX STATE. WE SEE AN OXIDATION DUE TO THE METABOLISM OF THE PEROXIDE FOLLOWED BY RELEASE OF CALCIUM TOTALLY INCORRECTED BY CYCLOSPORIN. IF WE TREAT THE RATS PREVIOUSLY, A SINGLE INJECTION WITH SULFORPHOPHANE, WE BLOCK OR REVERSE THE OXIDATION THERE. SO SUBSEQUENTLY WE FOUND, THIS HAS BEEN PUBLISHED, A SERIES OF DIFFERENT MITOCHONDRIAL PROTEINS AND GLUTATHIONE LIKELY RESPONSIBLE FOR THE INHIBITION. DR. MATTSON HAS GIVEN ME THE HAIRY EYEBALL OVER THERE, I BETTER WRAP UP. AND JUST TO SAY THERE'S A THIRD APPROACH WE'VE USED AND A NUMBER OF OTHER PEOPLE ARE USING IN THE PAST, AND CURRENTLY, USING THESE ALTERNATIVE BIOFUELS, OTHERS FOCUSED HE ON KETONE BODIES. ALSO PYRUVATE AND LACTATE, NEURO PROTECTIVE IN A NUMBER OF DIFFERENT PARADIGMS. AN EXAMPLE WOULD BE RECENTLY IN AN IMMATURE ANIMAL TRAUMATIC BRAIN INJURY MODEL WE FOUND IMPROVEMENT IN NEUROLOGIC OUTCOME, THIS HAS TO DO WITH -- HERE WE FOUND A REDUCTION IN THE LESION VOLUME OF THE ANIMALS, THIS IS ALL WITH L-CARNATENE, RATIONALIZED AS WE SEE IN THE TRAUMATIC BRAIN INJURY MODELS. THE LAST SLIDE IS SHOWN HERE, DATA RECENTLY OBTAINED BY TYLER DEMOREST, A Ph.D. STUDENT HERE WITH US TODAY, LOOKING AT THE EFFECTS IN A NEONATAL PARADIGM, A VERY IMMATURE BRAIN PARADIGM, AND WE FIND THAT IN MALES, THERE IS A SUBSTANTIAL DECREASE IN MITOCHONDRIAL RESPIRATION, STAGE 3 RESPIRATION, AT 20 HOURS FOLLOWING THIS ISCHEMIA REPERFUSION PARADIGM. WE SEE ON THE CONTRA-LATERAL SIDE, HYPOXIA ONLY SIDE OF THE BRAIN, A REDUCTION. WE'RE COMPARING MALES AND FEMALES, WE SEE NO EFFECT ON FEMALES ON THE CONTRA SIDE. WE SEE 50% LESS EFFECT ON MITOCHONDRIA ON THE IPSILATERAL SIDE, BRINGING UP THE POINT THERE CAN BE SEXUALLY DIMORPHIC RESPONSES EVEN AT THE IMMATURE LEVEL, POSTNATAL DAY 7 RATS, THE SEX HORMONES FROM NOT AT ALL INCREASED AT THIS POINT, THEREFORE ONE OF THE CONCLUSIONS IS THAT MORE ATTENTION SHOULD BE PAID TO THE POSSIBLE SEXUALLY D DIMORPHIC RESPONSES WHICH COULD LEAD TO SEX SELECTIVE NEURO PROTECTION. THE OTHER CONCLUSION I'VE ALREADY MENTIONED -- THE OTHER CONCLUSIONS I'VE ALREADY MENTIONED. THANK YOU VERY MUCH. [APPLAUSE] ANY QUESTIONS, COMMENTS? >> WE'LL TAKE ONE QUESTION AND MOVE ON BECAUSE WE'RE RUNNING BEHIND QUITE A BIT. >> I NOTICED FOR YOUR STAGE 3, YOU WERE LOOKING AT OXYGEN CONSUMPTION. DID YOU LOOK AT MEMBRANE POTENTIAL? >> NO, NOT YET. >> ONE OF THE THINGS, ESPECIALLY WITH THE GHRELIN INFORMATION, IS ABOUT THE DIFFERENCE BETWEEN THE SYNTHESIS RATE, ONCE THE OXYGEN IS CUT OFF, YOU'RE GOING TO START HAVING -- YOU'RE GOING TO STOP SYNTHESIS AND HAVE TURN OVER, THE HARDER IT IS TO COME BACK FROM THE CHANGE IN EFFICIENCY, SO YOU'RE GOING TO HAVE TO WAIT FOR SYNTHESIS TO BEGIN, THAT'S GOING TO TAKE LONGER THAN THE LOSS YOU EXPERIENCED WHEN YOU GET TO 12 MINUTES, FOR INSTANCE, YOU WOULD HAVE HAD A HUGE TURNOVER. AND BY THE TIME YOU COULD REBUILD YOUR SYNTHESIS YOU WOULD HAVE A PROBLEM. IT WOULD BE INTERESTING TO SEE WHAT WAS HAPPENING AT THE MEMBRANE POTENTIAL. >> THAT WOULD BE EXCEEDINGLY DIFFICULT IN THE CANINE CARDIAC ARREST MODEL. YOU COULD POTENTIALLY DO IT ON THE SURFACE OF THE CORTEX. THE CORTEX AT THE TIME WE'RE USING IS RELATIVELY INSENSITIVE TO INJURY COMPARED TO THE HIPPOCAMPUS, AND THE MITOCHONDRIAL CHANGES IN THE CORTEX ARE INSENSITIVE, LESS THAN IN THE HIPPOCAMPUS. SO IF WE WANTED ANYTHING, WE NEEDED SOME KIND OF A FIBEROPTIC PROBE TO GO DOWN INTO THE BRAIN LOOKING AT THE MEMBRANE POTENTIAL IN THE HIPPOCAMPUS. THAT WOULD BE DIFFICULT. IN VITRO MODELING COULD DO THAT. >> THANKS, GARY. [APPLAUSE] THE NEXT SPEAKER WAS SELECTED AMONG THE POSTER SUBMISSIONS TO GIVING A TALK, SIDDESH ARAS FROM WAYNE STATE UNIVERSITY. FORCE. >> GOOD MORNING. I THANK THE ORGANIZERS. I'LL GIVE YOU A BRIEF TALK, WITH THE FOCUS OF OUR LAB RESEARCH. I'LL TELL YOU HOW WE GOT INTERESTED IN THIS PROTEIN CHCHD2 WHILE STUDYING THE TRANSCRIPTIONAL REGULATION OF ONE OF THE COX SUBUNIT ISOFORM, 2, AND I WILL DESCRIBE THE ROLE OF CHCHD2. THIS IS VERY INTERESTING BECAUSE IT'S ONE PROTEIN FUNCTIONING IN TWO CELL ORGANISMS, DIFFERENTLY. IN THE NUCLEUS IT'S A TRANSCRIPTION FACTOR, IN THE MITOCHONDRIA IT'S A REGULATOR. I'LL SHOW YOU DATA SUGGESTING CHCHD2 TO BE A MODIFIER GENE. THE TAKE-HOME MESSAGE IS THIS. UNDER NORMAL CONDITIONS IT'S LOCALIZED TO THE MITOCHONDRIA NECESSARY FOR FUNCTION. WHEN A CELL IS STRESSED, PREDOMINANT LOCALIZATION TO THE NUCLEUS, WHERE IT FUNCTIONS AS A TRANSCRIPTION FACTOR. SO THIS IS COX, MADE OF 13 SUBUNITS OF WHICH SUBUNIT 4 SHOWN IN RED MARKED BY THE ARROW IS OF PARTICULAR INTEREST. IT IS THE LARGEST NUCLEAR ENCODED SUBUNIT, AND FUNCTIONS ON BINDING NUCLEOTIDES. AS ISOFORM MAKES IT INTERESTING. A QUICK BACKGROUND ON THE FUNCTION OF SIGNIFICANCE OF COX 4 ISO FORM 2. IT'S MORE ACTIVE THAN COX THAT DOES NOT HAVE IT. IF YOU COMPARE LUNG AND LIVER COX, LUNG COX WHICH LAST ISOFORM 2 IS MORE ACTIVE THAN LIVER, WHICH DOES NOT. MICE HAVE A KNOCKOUT OF THIS ISOFORM ARE SIGNIFICANTLY REDUCED FOR COX ACTIVITY AS MEASURED BY THE NUMBER, AND ALSO SIGNIFICANT REDUCTION IN THE ATL LEVELS. LUNGS FROM THE KNOCKOUT MICE DISPLAY SIGNS OF PULMONARY INFLAMMATION, HALLMARKS OF INFLAMMATION. MOVING ON, HOW DOES THIS COME TO OUR ATTENTION IN TRANSCRIPTION IS STIMULATED, ATTRIBUTED TO THE PRESENCE OF A SEQUENCE IN THE PROMOTER FOR THIS ISOFORM WHEN MUTATED ABOLISHES HYPOXIC RESPONSE. ONE OF THESE PROTEINS WAS CHCHD2, CONFIRMED ON A YEAST ONE HYBRID, ALONG WITH RBPJK AND CXXC5. OF THE THREE PROTEINS, THE LEVELS OF CHCHD2 WERE SIGNIFICANTLY HIGHER AT LOW OXYGEN, MAKING THIS THE PROTEIN OF CHOICE FOR FURTHER DISSECTION. THE MODEL WHICH WE'VE ALREADY PUBLISHED, I WON'T GO INTO DETAIL, THE INHIBIT FACTOR OCCUPIES THE OXYGEN RESPONSIVE ELEMENT, WHEN THE OXYGEN TENSION GOES DOWN THERE'S AN INCREASE IN CELLULAR LEVELS OF 2, REPLACING 5, TO ENHANCE TRANSCRIPTION. CELLS WITH A STABLE KNOCKDOWN SHOW REDUCED 2 REPORTER ACTIVITY, AND THIS IS A REVERSIBLE EFFECT. WHEN WILDTYPE IS TRANSSECRETARY RTRANSSECTED, ITIS RESCUED. CELLS SHOW AN INCREASE IN THE LEVELS OF THE ENDOGENOUS CHCHD2. WE TOOK A LOOK AT THE PROMOTER FOR CHCHD2 AND FOUND THE OXYGEN RESPONSIVE ELEMENT FROM THE COX 4 PROMOTER. A CHCHD2 REPORTER SHOWS INCREASED ACTIVITY AT LOW OXYGEN, AND THIS IS FURTHER INCREASED. NEXT, THE WHAT DOMAIN IS NECESSARY FOR TRANSCRIPTION, CHCHD2 IS 1751 AMINO ACID PROTEIN, WITH THE HELIX DOMAIN AT C TERMINUS, AND A DOMAIN OF UNKNOWN FUNCTION IN BETWEEN. WHEN THE MUTANTS WERE TESTED, M-5, WHICH HARBORS THE LESION OF 101 TO 113 WAS DEFECTIVE. TO SUMMARIZE, CHCHD2 BINDS, FUNCTIONS AS A TRANSCRIPTION FACTOR AND TRANSCRIPTIONAL DOMAIN MAPS TO 101-113. BUT CHCHD2 IS ALSO A MITOCHONDRIAL PROTEIN. WHAT'S GOING ON HERE? IT'S ACTUALLY A LOCALIZATION ISSUE. AT NORMAL OXYGEN, WHEN IT'S AT NORMAL, THIS IS THE EXPERIMENTAL NORMAL HERE FOR CELLS, MOST OF CHCHD2 IS IN THE MITOCHONDRIA, A SMALL FRACTION IN THE NUCLEUS, WHEN THE OXYGEN TENSION GOES DOWN, MOST OF IT IS NOW LOCALIZED IN THE NUCLEUS, A SMALL FRACTION REMAINS IN THE MITOCHONDRIA. A SIMILAR LOCALIZATION IS OBSERVED WHEN CELLS ARE STRESSED AS SHOWN HERE. CONTROL CELLS HAVE MORE CHCHD2 IN THE MITOCHONDRIA, THE STRESSED CELLS HAVE MORE CHCHD2 IN THE NUCLEUS. SO TO FURTHER DISSECT THE ROLE OF CHCHD2, WE USE A CELL LINE WITH A STABLE KNOCKDOWN OF THIS GROUPING, TESTING THE ABILITY TO AFFECT FUNCTIONING OF COX. AS YOU CAN SEE, THERE WAS A REDUCTION IN COX ACTIVITY AND ALSO REDUCTION IN THE MITOCHONDRIAL MEMBRANE POTENTIAL. THESE WERE MEASURED WITH A COLLABORATOR AT CAMBRIDGE. OXYGEN CONSUMPTION WAS MEASURED IN CELLS WHERE IN ADDITION TO REDUCTION IN OXYGEN CONSUMPTION SEEN WITH THE KNOCKDOWN CELLS, CHCHD2 SHOWED AN INCREASE IN OXYGEN CONSUMPTION, WHICH WAS AGAIN REPLICATED ON A SEA HORSE ASSAY. THESE THREE DIFFERENT ASSAYS POINT OUT TO ONE FACT. THAT CHCHD2 IS A CRITICAL REGULATOR. AND IT DOES SO BY INTERACTING WITH COX. IN CELLS, CHCHD2 CO-PURIFIES WITH COX AS SHOWN HERE AND HERE ON A STAINED HIGH PERCENTAGE GEL, THE CARBON GEL IN OUR LAB, WHERE THIS IS PURIFIED COX, SEPARATED INTO A SUBUNIT AND THIS IS RECOMBINANT CHCHD2. THE RED ARROW POINTS OUT THE CHCHD2 BAND IN PURIFIED COX AND WE HAVE DATA TO SUGGEST AND CONFIRM THIS IS CHCHD2. THE LEVELS OF CHCHD2 IN PURIFIED COX APPEAR TO BE -- WHEN IT'S ADDED TO COX IT INCREASES COX ACTIVITY. WE USED THE MUTANT THAT WE GENERATED AND TESTED MUTANTS FOR INTERACTION WITH COX, M-4 WHICH HARBORS AMINO ACID 86-100 WAS DEFECTIVE IN COX INTERACTION. ANOTHER FOCUS OF THE LAB IS TO STUDY MODIFICATION OF MITOCHONDRIAL PROTEIN REGULATING FUNCTION, AND SO WE PERFORMED A PRELIMINARY BIOINFORMATIC. WHAT I'M GOING TO TALK ABOUT TODAY IS PHOSPHORYLATION. CHCHD2 HAS TWO RESIDUES, POSITION 99 AND POSITION 116. TYROSINE AT 99 IS PHOSPHORYLATED. PHOSPHORYLATED CHCHD2 INCREASES COX ACTIVITY FURTHER, WHEN NONPHOSPHORYLATED RECOMBINANT D-2 IS ADDED THERE'S A SMALL INCREASE IN COX ACTIVITY. WHEN RECOMBINAN RECOMBINANT IS ADDED, THERE'S A MAXIMUM INCREASE. WE TESTED THIS IN A CELL CULTURE SYSTEM. CELLS WERE TESTED. THE MITOCHONDRIAL FRACTION WAS PREDOMINANPREDOMINANTLY PHOSPHORYLATED. FURTHER, CELLS WHICH EXPRESSED A MUTANT WHICH CANNOT GET PHOSPHORYLATED SHOWED A DEDUCTION, THIS WAS SPECIFICALLY IMPORTANT BECAUSE THIS MUTATION IS IN THE COX INTERACTION DOMAIN. TO SUMMARIZE THIS PART, CHCHD2 IS NECESSARY FOR OPTIMAL FUNCTIONING OF COX, INTERACTS WITH AND CO-PURIFIES WITH COX, MAPS TO 86-100, ABC KINASE PHOSPHORYLATES CHCHD2. IT'S A NUCLEAR ENCODED PROTEIN IN THE MITOCHONDRIAL. IT BELONGS TO A LAST OF TWIN PROTEINS WHICH ESSENTIALLY HAVE CYSTEINE RESIDUES SEPARATED BY 9 AMINO ACIDS, THE PROTEINS ARE IMPORTANT IN THE MITOCHONDRIAL INTERMEMBRANE SPACE WITH THE PATHWAY SYSTEM BETWEEN THE CYSTEINE RESIDUE AND IMPORTED SUBSTRATE. TO TEST THE CHCHD2 IS IMPORTANT BY THIS PATHWAY, WE FIRST TESTED IF CHCHD2 INTERACTS, AND IT DOES. MIA-40. TO FIND OUT WHAT KISS TEEN CYSTEINES ARE IMPORTANT WE TESTED THESE MUTANTS FOR INTERACTION WITH MIA-40. THE MUTANT SHOWED REDUCED BINDING WITH MIA-40 SO DID OTHER MUTANTS. THE MUTANTS ALSO SHOWED DEFECTIVE IMPORT INTO THE MITOCHONDRIA. AN INDEPENDENT CONFIRMATION WAS DONE BY USING A SMALL MOLECULE INHIBITOR OF THE MIA-40 PATHWAY, PREVENTING IMPORT OF A TRANSSECTED PROTEIN BUT NOT THAT OF A NONTRANSSECTED NONSPECIFIC PROTEIN, CONFIRMING CHCHD2 IS IMPORTED INTO THE MITOCHONDRIA VIA THE MIA-40 PATHWAY. WHAT ELSE DOES CHCHD2 DO? THERE WERE COMPLEX 1 SUBUNIT LEVELS REDUCED IN THE CHCHD2 KNOCKDOWN CELLS, WHICH IS WHAT A GROUP PUBLISHED EARLIER. CHCHD2 ALSO APPEARS TO REGULATE CELLULAR ROS. THE KNOCKDOWN CELLS HAVE A HIGHER LEVEL OF ENDOGENOUS ROS, NOT SURPRISING BECAUSE THE LEVEL OF ROS SCAVENGERS IS PRETTY LOW. THE MITOCHONDRIA FROM THE KNOCKDOWN CELLS DON'T LOOK HAPPY. THEY DON'T FORM NETWORKS. AND THIS TAKEN TOGETHER WITH A REDUCTION IN THE LEVELS SUGGESTS A FISSION PROFILE AND THEY GROW SLOWLY. COMING TO THIS -- WHY CHCHD2 IS IMPORTANT CLINICALLY? A MUTATION IN THE TRANSCRIPTION DOMAIN OF CHCHD2 HAS BEEN FOUND IN A FAMILY DIAGNOSED WITH SARCOMA DISEASE, THE TRAN DESCRIPTION DOMAIN IS HIGHLIGHTED IN BOLD, MUTATION IS AT THIS RESIDUE. CMT-1-A IS CAUSED DUE TO A DUPLICATION OF PMP-22 AFFECTING THE LOWER LIMBS WITH SIGNS OF GAIT IMBALANCE. THIS IS THE PEDIGREE AND SEQUENCING. THE TH MUTATIONS ARE SHADED IN BLACK. PATIENTS WITH A PMP-22 MUTATION WITH THIS WERE WORSE. WE DECIDED TO TEST THE PHENOTYPE OF THIS MUTATION. THE MUTANT WAS TRANSCRIPTIONALLY DEFECTIVE. THERE WAS LESS IN CELLS EXPRESSING THE MUTANT AND LOWER LEVELS. THERE IS AN IMBALANCE IN THE MITOCHONDRIAL REGULATORY SYSTEM WITH THIS MUTATION. SO THIS, IN A WAY, MAY ENHANCE THE ACCUMULATION OF THE DELITIRIOUS MUTATION. CHCHD2 IS A MITOCHONDRIAL REGULATOR, WHERE IT BINDS TO COX AND INCREASES ENZYME ACTIVITY. PHOSPHORYLATION ENHANCES ITS ACTIVITY. IT IS IMPORTED BY THE MIA-40/ERV-1 PATHWAY AND REQUIRES TERMINAL CYSTEINES AND REGULATE MITOCHONDRIA POTENTIAL AND LOCALIZES TO THE NUCLEUS UNDER CONDITIONS OF STRESS AND BINDS TO OXYGEN RESPONSE ELEMENT AND REGULATES TRANSCRIPTION FOR AT LEAST COX 4I2 AND ITSELF. CHCHD2 KNOCKDOWN DEVELOPS WHY PLEITROPIC EFFECTS, INCREASED ROD LEVELS. MY MENTOR COULDN'T BE HERE BECAUSE HE INJURED HIS ANKLE. THAT'S HOW HE COMMUTES INTO THE DEPARTMENT, AND MY COLLEAGUES WHO IDENTIFIED THE ISOFORM AND INTERESTED IN STRUCTURAL DETAIL OF CHCHD2, THE CLINICIAN, AND LAST BUT NOT LEAST, THANK YOU ALL. ANY QUESTIONS? [APPLAUSE] >> I HAVE A QUESTION ABOUT POTENTIAL SIGNALLING PATHWAYS THAT MAY REGULATE EITHER THE TRANSCRIPTIONAL REGULATION OF CHCHD2 IN THE NUCLEUS, FOR EXAMPLE AN INTERACTION BETWEEN CHCHD2 AND RBCJ. >> J-CAPPA. >> INVOLVED IN NOT SIGNALING, ONE PATHWAY. >> UH-HUH. >> FOR EXAMPLE, DOES THAT AFFECT -- >> SO WITH IT' RESPECT TO NOT SIGNALLING, THERE'S TWO SCHOOLS OF THOUGHT. IT GETS INTO THE NUCLEUS AND FUNCTIONS AS A TRANSCRIPTION FACTOR. THE OTHER SCHOOL SAYS RGB-CAPPA IS ALWAYS PRESENT, WE THINK IT INHIBITS. >> WHAT ABOUT KINASE? >> THE KINASE, YEAH -- >> IS THERE ANY ROLE OF CHCHD2 LIKE CHCHD4? COULD CHCHD2 ALSO FUNCTION AS PART OF THIS MIA-40 IMPORT MACHINERY? WHAT ARE YOUR THOUGHTS ABOUT THAT? >> WE HAVEN'T LOOKED AT THAT. WE REALLY HAVEN'T LOOKED AT THAT. >> THANK YOU. [APPLAUSE] >> THE NEXT SPEAKER IS MARK COOKSON FROM THE NATIONAL INSTITUTE ON AGING. >> THANK YOU, MARK. I'M PARTICULARLY PLEASED TO COME TO THE MITOCHONDRIAL INTEREST GROUP BECAUSE I'M NOT A MITOCHONDRIAL BIOLOGIST AND NEVER HAD AN INTENTION. WHAT MY GROUP DOES IS LOOK AT GENES FOR PARKINSON'S'S DISEASE, AND FAMILIES IN THE LITERATURE, WHAT WE TEND TO DO IS CLONE THE PROTEINS OUT, PUT THEM IN CELLS AND NOW ANIMAL MODELS, AND LOOK WHAT THEY DO. ONE THAT WE WORKED ON OVER THE YEARS IS DJ-1 HERE, AND WHEN WE PUT IT INTO CELLS WE FOUND IT WOULD GO TO MITOCHONDRIA. THIS IS WHAT REALLY GOT US INTERESTED IN THE MITOCHONDRIAL PATHWAY. THE QUESTION WE FIRST ASKED, WHAT DOES THIS PROTEIN DO? AND ONE OF THE THINGS WE KNEW ABOUT WAS WHAT WAS EXPRESSED. HERE IS A SERIES OF EXPERIMENTS, IN VITRO AND IN RECEIV VIVO, SPORE SPORADI C PARKINSON'S DISEASE. I'M SUMMARIZING HUGE AMOUNTS OF DATA IN A COUPLE SLIDES BUT THE MECHANISM IS A CYSTEINE OBSERVATION, THIS IS WORK FROM MY COLLEAGUE, MARK WILSON. THE PROTEIN IS A DYMERIC STRUCTURE WITH A CYSTEINE STRUCTURE THAT GETS OXIDIZED, NEARBY AMINO ACIDS. THIS IS NOT NOT THE SPECIFIC WE THINK ABOUT BUT VERY DEFINED AND CONTROLLED REACTION. WHAT WE DID WAS REMADE A SERIES OF MUTATIONS THAT SHOULD EITHER SUPPORT OR NOT SUPPORT THE FORMATION AND CYSTEINE 1-R 06 IS B106 ISABSENT BUT HAS A GREATER MUTATING RESPONSE. I SHOW YOU THESE, I WANT TO SHOW YOU WHAT HAPPENS WHEN YOU PUT THOSE VERSIONS INTO CELLS. NORMALLY, AS I SHOWED YOU EARLIER, UNDER CONDITIONS OF STRESS IT GOES TO THE MITOCHONDRIA, TWO STRESSORS, AND IN THOSE CASES THE MITOCHONDRIA LOOK INTACT WITH THE PRESENCE OF DJ-1. HOWEVER IF WE BLOCK THAT, WE SEE THE MITOCHONDRIA FRAGMENT. THEY HAVE MITOCHONDRIAL DAMAGE, THEY ARE THICKER. NOW, THAT'S ALL BASED ON A PROTEIN. WITH EXPERIMENTS, IT SHOWS THE LACK OF ENDOGENOUS DJ-1 MAKES THE MITOCHONDRIA IN THESE CELLS DYSFUNCTIONAL. THERE ARE TWO DYSFUNCTIONS THAT HAVE BEEN WELL REPRODUCED IN LITERATURE, ONE IS A LOSS OF MEMBRANE POTENTIAL WITH TRME THAT CAN BE RESCUED BY EXOGENOUS ANTIOXIDANT. YOU CAN SEE HERE THE DJ-1 DEFICIENT SELLS HAVE INCREASED TURNOVER AUTOPHAGY. AGAIN, THIS BLOCK B BY -- THIS IS BLOCKED BY ADDING ANTIOXIDANT. WE FOUND A PROTEIN INVOLVED IN MITOCHONDRIA FUNCTION MORE OR LESS BY ACCIDENT, TIED TO STRESS IN TERMS OF HOW IT RESPONDS, ALSO WE COULD BLOCK IT BY EXOGENOUS PRECURSOR. RICHARD TALKED EARLIER, I'VE STOLEN THIS FROM HIM. RICH TALKED ABOUT THIS EARLIER. THE SAME PHENOTYPE IN HUMANS RICHARD MENTIONED, YOU HAVE LOSS OF MEMORY POTENTIAL AND INCREASED MITOPHAGY. THEY ARE ESSENTIALLY IN THE SAME PATHWAY IN THE CELL. THE DATA IS PUBLISHED, BUT FROM LABS, IMPORTANTLY ALSO PEOPLE WORKING IN VIVO AND MODELS, THE SUMMARY OF ALL OF THIS WORK IS THAT DJ-1 IS NEITHER REQUIRED OR PINK1 DEPENDENT WHICH RICHARD TALKED ABOUT EARLIER, NOR IS IT AFFECTED BY THESE. AND YET THE TWO LOSS OF FUNCTIONAL COPY EACH OTHER IN HUMANS. IN THE BRIEF TIME I'M GOING TO SPEAK, HOW DO WE GET TO PARALLEL PATHWAYS THAT CONVERGE ON THE SAME PHENOTYPE IN PEOPLE? IN FACT, WHAT I'M GOING TO SHOW YOU IS TWO DIFFERENT EXPERIMENTS WE'VE DONE IN THE LAB, ONE ON PINK1 PARKIN, ONE ON DJ-1, THE IMPORTANT THING HERE IS WE'VE GOT SO TIRED OF TRYING TO GUESS WHAT THE PROTEIN DOES, WE WENT BACK TO UNBIASED APPROACHES, THOSE ARE THE STORIES I'LL TELL YOU. THE FIRST THING IS RICHARD, AGAIN, ELEGANTLY EXPLAINED HOW YOU CAN GET PARKIN TO GO TO MITOCHONDRIA. HE WAS KIND ENOUGH TO GIVE US A CELL LINE THAT EXPRESSED WHICH WE ENGINEERED TO MARK MITOCHONDRIA. WE PUT IT THROUGH A GENE SCREEN, TO RECOVER A GENE THAT WOULD BLOCK PARKIN. WE KNEW THAT FROM RICHARD'S WORK. AGAIN, I'M GOING TO GO THROUGH THIS QUICKLY. HERE IS THE SCREEN WE DID, EVERYONE CAN GET THIS TO WORK. THE BLOCK BY PINK1, AND THEN FINALLY RECOVERY OF THE ADDITIONAL ONE, WE HAVE TEN OR TWELVE THAT WOULD REPRODUCE BIG BLOCKS. THIS ENZYME CONVERTS GLUCOSE AND OTHER SUGARS INTO GLUCOSE WHICH FEEDS METABOLIC PATHWAYS, CLUES CLUES -- THIRDLY, HEXOKINASE IS INVOLVED IN SIGNALLING, WHICH IT WAS NICE TO HEAR MARIE HARDWICK'S TALK, PATHWAY THAT CAN BE MODULATED BY HEXOKINASE. IT WAS REVERSED, REPRODUCIBLE. WE VALIDATED THIS IN EVERY WAY WE COULD THINK, WE SHOWED THEY WERE BLOCKED. WE SHOWED IT WAS A REAL BLOCK, NOT A DELAY. WE SHOWED IT HAD THE EXPECTED EFFECT OF BLOCKING MITOPHAGY DOWNSTREAM. I'LL COME BACK TO THIS IN A MONEY. HEXOKINASE 1 AND 2, YOU'LL SEE WHY IT'S IMPORTANT LATER. BOTH DO THE SAME CONVERSION, GLUE CLOSE TO GLUCOSE 6. YOU CAN SUBSTITUTE ONE FOR THE OTHER. YOU CAN SUBSTITUTE HEXOKINAS 1. WE FOUND AGAIN BY SURPRISE AN INTERESTING PATHWAY THAT TIES THE MITOCHONDRIAL ENERGY ETHICS INTO RECRUITMENT OF PARKIN, AND PARKINSONISM. NOW, IN PARALLEL WE'VE BEEN LOOKING AT DJ-1 IN ANIMALS. WE MOVED AWAY FROM THE CELL LINE BECAUSE WE'RE FRUSTRATED THAT SOME PHENOTYPES WERE NOT AS FAVORABLE AS WE HOPED FOR. WE WENT TO AN IN VIVO MODEL. WE LOOKED AT THE BRAINS OF THESE ANIMALS USING TWO LARGE SCALE TECHNIQUES, ITRAQ AND THEN WE WERE ABLE, BECAUSE WE HAD MICE AS WELL, TO VALIDATE RESULTS IN A SECOND SERIES. SO WE COULD HAVE FOUND ANYTHING. WHAT WE FOUND IN THE ITRAQ EXPERIMENTS, THE GENOMIC KNOCKOUT, WE HAD OTHER STUFF THAT'S SIGNIFICANT, AND WE LOOK DOWN THE LEFT, WE COULD HAVE FOUND ANY PROTEIN BUT WE FOUND HEXOKINASE IN THE BRAINS IN THESE RATS, DEFICIENCY TO DJ-1, SIGNALED PINK1 PARKIN. WE FOUND THIS IS A TRANSCRIPTIONALLY DEPENDENT, AND WE HAVE SOME PRELIMINARY MECHANISTIC DATA THAT EXPLAINS WHY THAT IS. THE NICE THING ABOUT THIS MODEL IS THAT WE CAN ACTUALLY TAKE THIS IN THE SECOND SPECIES, AND WE SEE THE SAME EFFECT, SO IT'S NOT UNIQUE TO THE RAT, IT'S SEEN IN RATS AND MICE, THE MAGNITUDE OF CHANGE IS SIMILAR. AND SO BECAUSE WE'RE SHORT OF TIME, AS I EXPECTED WE MIGHT BE, I'M JUST GOING TO SUMMARIZE THIS, WHAT WE'RE THINKING ABOUT, DJ-1 AND HOW PATHWAYS FIT TOGETHER. FUNDAMENTALLY DJ-1 IS INVOLVED IN SOME WAY OR ANOTHER IN THE REGULATION OF REACTIVE OXYGEN SPECIES. WE BELIEVE THAT GENERATES HEXOKINASE AND INCREASES ACTIVITY. FIRST OF ALL, IT WAS SHIFT AWAY FROM PHOSPHORYLATION, PHOSPATE PATHWAYS, WE THINK HE HEXOKINA IS HERE TO MITIGATE AGAINST LOSS OF DJ-1. MAYBE ONE OF THE REASONS THE ANIMALS -- ALL OF THESE ARE BEING UNIFORMLY -- MAYBE THE REASON, THEY ARE ABLE TO COMPENSATE THROUGHOUT THEIR LIFETIME. WE'RE NOW TESTING THAT THERE ARE PREDICTABLE CHANGES IN BIOENERGETICS IN MITOCHONDRIA, INITIATING EXPERIMENTS TRYING TO LIMIT HEXOKINASE AND THOSE EXPERIMENTS WE'RE GETTING ON WITH. I WANT TO THANK THE GROUP OF PEOPLE, THIS IS MY LAB, AGAIN, SAME BUILDING AS RICHARD. PARTICULARLY I WANT TO THANK DAVE HOUSER, IN THE AUDIENCE, A PH DID STUDENT. THANK YOU. [APPLAUSE] >> IN THE 70s AND '80s, A COMBINATION OF UBIQUITY, THE FACT THAT YOU FIND THIS CAN BE ASSOCIATED WITH THE MEMBRANE AND MODULATE MITOPHAGY IS AN INTERESTING RESULT. DO YOU HAVE ANY OTHER COMMENTS ABOUT THAT? >> I MEAN, I THINK IT'S A VERY INTERESTING -- I MEAN, IN A SENSE THIS IS A SINGLE FUNCTION ENZYME, BUT IT HAS SO MANY OTHER EFFECTS, PRESUMABLY SHIFTING TO GLUCOSE TO GLUCOSE 6 PHOSPATE, CERTAINLY IN THE LITERA LITERATURE IT'S THOUGHT. AS YOU SAY, BACK TO THE 70s HYPOTHESIS -- >> IF YOU COULD SEE THE IMPACT ON THE OTHER PHENOMENON. >> YES, SURE, SO WE ARE LOOKING AT USING THE PEPTIDES AVAILABLE, WE HAVEN'T DONE THOSE EXPERIMENTS YET BUT WE WILL PREDICT IT WILL REVERSE MANY DEFECTS. >> VERY NICE, MARK. DO YOU KNOW OR WOULD YOU PREDICT HEXOKINASE TO WORK UPSTREAM OR DOWNSTREAM. >> IT WILL NOT RESCUE IN THE ABSENCE OF PINK1, MODERATELY DOWNSTREAM OF PINK1. >> TO PUT IT UP OUR DOWN WITH UBIQUITIN. >> WE HAVEN'T LOOKED AT THAT BUT WE COULD LOOK AT HOW IT CHANGES THAT. I WOULD PREDICT LESS, I COULD BE VERY WRONG ON THAT. >> A LOT OF LABS ARE MAKING THAT ANTIBODY RIGHT NOW. >> THIS IS A VERY IMPORTANT AREA, GLUCOSE METABOLISM. DO YOU SEE THERE IS ANY CHANGE, FOR EXAMPLE, DURING THE AGING PROCESS ALSO, WHERE INSULIN ALSO HAS A ROLE IN GLUCOSE TRANSPORT, WHERE IT THEN GETS METABOLIZED LIKE IN THE LIVER, WHICH IS INSULIN DEPENDENT BUT IT'S NOT FOR JUST TRANSPORT. >> SO I CAN TELL YOU EXPERIMENTS WE HAVE DONE, EXPERIMENTS WE'RE PLANNING. WE HAVE DONE WAS LOOKED AT THE AKT DEPENDENCE WHICH INSULIN WOULD WORK THROUGH AKT, ALWAYS, ALL OF THIS IS BLOCKED BY AKT. WE WOULD PREDICT INSULIN SIGNALING WOULD BE FAR UPSTREAM OF HEX OHKINASE. WE'RE PLANNING TO LOOK OUTSIDE THE BRAIN, THE MICE DON'T HAVE BRAIN DISEASE WITHOUT -- THE RATS MAY HAVE SOME BUT IT'S PRETTY SMALL. AND SO WE ARE CONSIDERING LOOKING OUTSIDE OF THE BRAIN, INCLUDING LIVER METABOLISM BUT WE HAVEN'T DONE THOSE EXPERIMENTS YET. >> THANK YOU. >> DO YOU HAVE THE ABILITY TO CHECK DJ-1 IS DOWNREGULATED? >> WE COULD DO THAT. WE HAVE NOT. WHAT I WAS SAYING, EVERY TISSUE WE EVER LOOKED AT, IT EXPRESSED A HUGE AMOUNT, IT'S VERY EASY TO DETECT. I'VE YET TO SEE A PLACE WHERE IT'S NOT EXPRESSED FOR CELL LINES, SO IT ISN'T VERY TRANSCRIPTIONALLY REGULATED, REGULATION IS PREDOMINANTLY POST TRANSLATIONAL LEVEL THROUGH SITTING OXIDATION, AND ONCE OXIDIZED TURNS OVER. IT'S A VERY OLD PROTEIN. >> THANKS, MARK. [APPLAUSE] >> AN ANNOUNCEMENT. >> IF ANY OF THE FELLOWS OUT THERE THAT ARE PLANNING ON THE FELLOWS LUNCH WITH DOUG WALLACE, AT NOON, WE'RE GOING TO PUT IT OFF UNTIL 12:15 TO GIVE US TIME TO GET BACK ON TRACK WITH THE AFTERNOON, AND JAY CHUNG IS GOING TO TALK FAST. JAY CHUNG IS THE LAB CHEAP FROM THE NATIONAL LUNG, HEART AND BLOOD INSTITUTE. >> MY GROUP IS INTERESTED IN UNDERSTANDING MECHANISMS INVOLVED IN OBESITY AND AGING, AND UTILIZING THE MECHANISMS TO DEVELOP NOVEL THERAPY, TRANSLATIONAL MEDICINE, SO I'M GOING TO TALK TODAY ABOUT INHIBITING PDE4 AS POSSIBLE THERAPEUTIC STRATEGY. AS YOU KNOW, ALMOST 70% OF AMERICANS OLDER THAN 20 YEARS OF AGE ARE EITHER OBESE OR OVERWEIGHT. NOW, WHEN WE THINK OF OBESITY, WE THINK ABOUT OVEREATING, CERTAINLY FOOD INTAKE IS A MAIN CONTRIBUTOR, BUT THAT'S NOT THE WHOLE STORY. AVERAGE AMERICAN GAINS 30 POUNDS FROM AGE 20 TO AGE 50, ABOUT A POUND A YEAR. AND A POUND OF FAT IS 3500 CALORIES, 3500 CALORIES DIVIDEDDED BY 365 IS ABOUT 10 CALORIES A DAY. THAT'S ONE LIFESAVER RING THAT WE LEAVE UNBURNED EACH DAY, AND IF YOU THINK ABOUT 2700 CALORIES, THE AVERAGE AMERICAN MALE EATS, IN TERMS OF FEEDBACK, IT'S PRETTY GOOD, BUT EVEN A LITTLE BIT ACCUMULATES AFTER MANY YEARS. SO THERE'S ANOTHER ISSUE THAT IS PROBLEMATIC, THERE'S AGING DEPENDENT CHANGE IN FAT DISTRIBUTION. WHEN CHILDREN GAIN WEIGHT, THEY GAIN FAT UNIFORMLY. YOU SEE BUTTOCKS, BELLY, ARMS AND LEGS. BUT UNFORTUNATELY THOSE OF US IN MIDDLE AGE, WHEN WE GAIN FAT WE GAIN FAT PRIMARILY IN THE ABDOMINAL REGION, EVEN PEOPLE WHO ARE CONSIDERING TO BE SKINNY WHEN THEY WERE YOUNG, WE GAIN PREDOMINANTLY FAT IN THE BE ABDOMINAL REGION LINKED TO HYPERTENSION, HEART DISEASE AND STROKES. 70% OF HEALTH CARE COST GOES INTO DISEASES ASSOCIATED WERE THIS. THE ABDOMINAL FAT RELEASES CYTOKIINES, THAT'S THE DIFFERENCE BETWEEN SUBCUTANEOUS FAT AND ABDOMINAL FAT. THE ABDOMINAL FAT IS THE FAT UNDER THIS RED STRIPE, THE RECTUS ABDOMINUS MUSCLE, THE SIX-PACK MUSCLE, UNDER THAT, NOT ACCESSIBLE TO LIPOSUCTION. THAT MAY MAKE YOU LOOK ATTRACTIVE, IT DOESN'T DO MUCH FOR YOUR METABOLIC PROBLEM. SO THERE'S THE ISSUE THAT'S RELEVANT TO SUBCUTANEOUS FAT IN TERMS OF MORTALITY. THIS IS AN IOWA WOMEN'S HEALTH STUDY LOOKING AT OLDER WOMEN AND MORTALITY, ACCORDING TO BMI, DIVIDED INTO QUINTILES OR WAIST TO HIP RATIO. YOU CAN SEE PEOPLE WHO ARE -- WHO HAVE THE HIGHEST MORTALITY ARE NOT OBESE PEOPLE, IT'S THE SKINNIEST PEOPLE WITH ALL THEIR FAT IN THE WAIST. PEOPLE WITH THE LOWEST MORTALITY ARE THE PEOPLE HERE, WHO HAVE MODEST AMOUNT OF SUBCUTANEOUS FAT BUT SMALL WAIST TO HIP RATIO. SO THEY SAY IN HOLLYWOOD, THAT YOU CAN'T BE TOO RICH OR TOO THIN. WELL, I DON'T KNOW ABOUT BEING TOO RICH BUT YOU CAN BE TOO THIN. AND WHAT YOU CANNOT HAVE ENOUGH OF THOUGH IS SMALL WAIST AND SMALL WAIST TO HIP RATIO. THIS IS A MORTALITY LOOKING AT BMI ADJUSTED, WHAT YOU SEE IS THE LOWER THE WAIST CIRCUMFERENCE AND LOWER WAIST TO HIP RATIO THE BETTER IN TERMS OF MORTALITY. NORMALLY WHAT YOUR BMI IS. WHAT IS ABOUT WAIST TO THIGH OR HIP TO THIGH RATIO? SO STUDIES HAVE SHOWN IF YOU LOOK AT THE SKELETAL MUSCLE, PEOPLE WITH HIGH WAIST TO THIGH RATIO HAVE LOW MITOCHONDRIAL DENSITY IN THE SKELETAL MUSCLE. PEOPLE WITH SMALL WAISTS TEND TO HAVE HIGHER MITOCHONDRIAL DENSITY IN SKELETAL MUSCLE. MITOCHONDRIAL LOSS IS ASSOCIATED WITH AGING AND METABOLIC DISEASE. PEOPLE WHO ARE OBESE TEND TO HAVE LESS MITOCHONDRIA, 20%. PEOPLE WHO ARE TYPE 2 DIABETICS HAVE 40% LESS. LEAN, HEALTHY OFFSPRING OF DIABETIC, DESTINED TO DEVELOP TYPE 2 DIABETES, START OUT LIFE WITH 30% LESS MITOCHONDRIA. AND UNFORTUNATELY, HEALTHY ELDERLY PEOPLE HAVE 40% LESS MITOCHONDRIA. SO SKELETAL MUSCLE MAKES UP 40% OF BODY MASS, THE MAIN SITE OF YOUR GLUCOSE UPTAKE AND METABOLISM, AS I MENTIONED YOU LOSE MITOCHONDRIA IN SKELETAL MUSCLE AS WE AGE. ONE OF THE CONSEQUENCES OF THAT IS LIPIDS WHICH ARE OXIDIZED BY MITOCHONDRIA TEND TO ACCUMULATE, AND WHEN YOU DO THAT, LIPID CAUSES INSULIN RESISTANCE, AND OTHERS HAVE SHOWN THAT THE WAY TO REDUCE LIPID ACCUMULATION AND INCREASE MITOCHONDRIAL IS CALORIE RESTRICTION, AEROBIC EXERCISE OR TREATMENT WITH RESVERATROL. RESVERATROL IS A COMPOUND THAT IS FOUND IN RED WINE AS WELL AS OTHER PLANT-DERIVED FOOD AND HAS A NUMBER OF BENEFICIAL EFFECTS. IT BLOCKS GLUCOSE INTOLERANCE, MITOCHONDRIAL DYSFUNCTION, DECREASES INFLAMMATION, DECREASES BETA AMELOID. HOW DOES THIS SIMPLE COMPOUND DO ALL THESE THINGS? AND THAT'S WHERE WE GOT INVOLVED. IF YOU TREAT DIABETIC INDIVIDUALS WITH RESVERATROL YOU IMPROVE GLUCOSE TOLERANCE, LOWER THE GLUCOSE AFTER GLUCOSE FEEDING, SO A NUMBER OF PEOPLE HAVE PROMOTED RESVERATROL AS A DRUG BUT THAT HAS A LOT OF PROBLEMS. FIRST OF ALL, RESVERATROL HAS A PROMISCUOUS TARGET PROFILE, IT BINDS TO PROTEINS AND CAN TOXICITY. YOU NEED A COUPLE GRAMS A DAY, 500 BOTTLES OF RED WINE A DAY, POOR BIOAVAILABILITY. WE WANTED TO FIND OUT EXACTLY WHAT IS THE TARGET OF RESVERATROL. SO ONE OF THE DOWNSTREAM TARGETS, ALTHOUGH NOT DIRECT TARGET, TURNS OUT TO BE AMPK, SO WHEN CONSUMED BY ENERGY EXPENDITURE, AMPK IS REMARKABLY SIMILAR PHENOTYPE AS RESVERATROL, STIMULATE LAYING OF MITOCHONDRIAL BIOGENESIS, INCREASE THE METABOLIC RATE AND LOLLS OF FAT, INCREASED PHYSICAL ENDURANCE IMPROVED GLUCOSE TOLERANCE AND INSULIN SENSITIVITY. RESVERATROL INDIRECTLY ACTIVATES AMTK, SHOWING IF YOU KNOCK OUT AMTK -- AMPK, THE RESVERATROL DISAPPEARS. WHAT IS THE TARGET? IF YOU LOOK AT MICRO DATA, YOU LOOK AT THE ADIPOSE VERSUS RESVERATROL TREATED, VERSUS CALORIE RESTRICTED, THE GENE EXPRESSION PROFILES ARE SIMILAR. WHAT HAPPENS WHEN YOU CALORIE RESTRICT ANIMALS OR EXERCISE ANIMALS? TWO HORMONES ARE RELEASED, AND THEY ACTIVATE. WE CONSIDER THE POSSIBILITY MAYBE RESVERATROL IS TICKLING THE PATHWAY. IF YOU BLOCK THE SIGNALLING WITH MDL, AMPK ACTIVATION THAT WAS INDUCED BY RESVERATROL DISAPPEARS, SUGGESTING THE AMP ACTIVATION IS A CRITICAL PLAYER IN RESVERATROL ACTION. NOW, RESVERATROL CAN INCREASE CYCLIC AMP TWO WAYS, ACTIVATING OR BLOCKING. WORK SHOWS THAT IT'S THE LATTER, IF YOU LOOK AT THE PHOSPHO-DIESTERASE ACTIVITY, 5 AND 2 ARE NOT AFFECTED, BUT 3 ARE INHIBITED BY RESVERATROL. NOW, SO ONE OF THE PROBLEMS WITH USING PDE, YOU CAN'T WILLY-NILLY INCREASE IN THE BODY. IT HAS IMPORTANCE IN THE TISSUES. YOU DON'T WANT TO DO THAT NONSPECIFICALLY. WHAT'S MORE COMPLEX, IS THAT THERE ARE 11 FAMILY MEMBERS, EACH FAMILY HAS A NUMBER OF GENES, EACH GENE HAS A NUMBER OF DIFFERENT ISOFORMS, SO THERE ARE 21 GENES, SO IN SKELETAL MUSCLE FORTUNATELY PDE 4 IS A DOMINANT PDE. WE USE A DRUG WHICH SPECIFICALLY INHIBITS PDE4 TO SEE WHETHER IT CAN REPRODUCE RESVERATROL'S EFFECT WITHOUT ALL THE BAGGAGE THAT COMES WITH RESVERATROL. WHAT WE FIND IS ROLIPRAM ACTIVATES, AND IF YOU LOOK AT MITOCHONDRIAL CONTACT, TREATED MICE HAVE SIGNIFICANT INCREASE IN MITOCHONDRIAL CONTENT, AND IF YOU RUN THE MICE ON A TREADMILL UNTIL THEY GET EXHAUSTED, WHAT YOU FIND IS IT'S SIGNIFICANTLY IMPROVED THEIR PHYSICAL STAMINA. IF YOU LOOK AT THE EXPRESSION LEVELS OF MITOCHONDRIAL GENES, ROLIPRAM AND WHITE ADIPOSE TISSUE, YOU FIND A SIGNIFICANT INDUCTION BY ROLIPRAM, CONTROLLING DOWNSTREAM MITOCHONDRIAL GENES, INCLUDING UNCOUPLING PROTEINS, AND SAME WITH ADIPOSE TISSUE, YOU NOTICE ALL THE UNCOUPLING GENES ARE INDUCED BY ROLIPRAM. YOU FIND THESE INDUCTIONS ALSO REQUIRE AMPK. IF YOU FREED OF FEED MICE ROLIPRAM YOU SEE NO INDUCTION IF YOU DON'T HAVE AMPK. SO WHAT HAPPENS WHEN YOU TREAT ANIMALS FOR A LONG TIME WITH ROLIPRAM ON HIGH FAT DIETS? WHAT YOU FIND IS WEIGHT GAIN AND HIGH FAT DIET IS DIMINISHED, MICE DON'T ACCUMULATE FAT AS EASILY AS CONTROL ANIMALS. AND THERE'S LESS FAT CONTENT WHEN YOU LOOK AT TREATED ANIMALS, EVEN THOUGH FOOD INTAKE IS THE SAME. CONSISTENT WITH THE NOTION THAT THE UNCOUPLING PROTEIN LEVELS GO UP, THE METABOLIC RATE GOES UP, IF YOU LOOK AT THE BODY TEMPERATURE WHEN ANIMALS ARE FASTEST, BOTH ROLIPRAM AND RESVERATROL TREATED ANIMALS HAVE SLIGHTLY BUT INCREASED BODY TEMPERATURE. SO WE'VE DONE METABOLIC MEASUREMENTS AS WELL, AND THEY DO HAVE HIGHER OXYGEN CONSUMPTION RATES. SO WE WANTED TO SEE WHETHER INCREASING MITOCHONDRIAL CONTENT BY ROLIPRAM WAS AMPK DEPENDENT, AND IT IS. AND IF YOU DO TREADMILL RUNNING, WHAT YOU FIND IS THAT ROLIPRAM ABILITY TO INDUCE INCREASED PHYSICAL STAMINA IS BLUNTED IN THE ABSENCE OF AMPK. BUT MORE IMPORTANTLY, DOES THIS HAVE ANY EFFECT ON TYPE 2 DIABETES? SO IF YOU LOOK AT GLUCOSE INTOLERANCE, YOU HAVE WILDTYPE MICE IN BLACK CIRCLE AND TRIANGLE, ROLIPRAM IMPROVED THEIR GLUCOSE TOLERANCE. BUT IN OPEN SYMBOLS, THE AMPK KNOCKOUT MICE DO NOT RESPOND IN TERMS OF GLUCOSE TOLERANCE. SO WHAT WE THINK IS HAPPENING IS RESVERATROL AT LEAST IN SKELETAL MUSCLE BLOCKS PDE4, AND ACTIVATES THE DOWNSTREAM PATHWAY WHICH CULMINATES IN THE ACTIVATION OF AMPK WITHOUT ENERGY STARVING THE ANIMAL. AND THERE BY CONFERRING BENEFITS OF AMPK ACTIVATION LIKE EXERCISE AND CALORIE RESTRICTION WITHOUT HAVING TO DO THOSE THINGS. WE THINK PDE4 INHIBITOR MAY BE A DIABETIC THERAPEUTIC. IT'S INTERESTING THAT RESVERATROL AND A COMPOUND RECENTLY APPROVED FOR COPD, A PDE4 INHIBITOR, IF YOU LOOK AT THE STRUCTURE THERE'S UNCANNY RESEMBLANCE. HERE YOU HAVE PHARMACEUTICAL COMPANY MADE PDE4 INHIBITOR. FORTUNATELY, ROFLUMILAST IS 30,000-FOLD MORE POTENT THAN RESVERATROL, AND MY GROUP IS STARTING A CLINICAL TRIAL FOR PRE-DIABETIC INDIVIDUALS. SO DUE TO TIME I'M GOING TO CONCLUDE, RESVERATROL ACTIVATES AMPK, AND ROLIPRAM REPRODUCES BENEFITS. WE'RE DOING THE CLINICAL TRIAL RIGHT NOW. I'LL STOP THERE AND TAKE QUESTIONS. [APPLAUSE] THANK YOU. >> ALL RIGHT. THANK YOU FOR THE NICE TALK. THERE IS A LINK BETWEEN TYPE 2 DIABETES. DO YOU THINK PDE4 INHIBITORS MIGHT BE ALSO RELEVANT TO ALZHEIMER'S DISEASE TREATMENT? >> YEAH, THAT'S VERY CORRECT. INDIVIDUALS WITH ABDOMINAL OBESITY OR DIABETES AT MIDDLE AGE HAVE TWICE OR THREE TIMES THE RISK OF ALZHEIMER'S DISEASE 40 YEARS LATER. IT'S BEEN SHOWN IF YOU INJECT PDE4 INTO THE BRAIN, IT'S PROTECTIVE AGAINST AMELYOID TOXICITY AND MEMORY DEFICIENCITY. DON'T KNOW. I THINK THE PATHWAY THAT I TALKED ABOUT HERE MAY BE INVOLVED. IF AMPK ACTIVATION INCREASES AUTOPHAGY, MITTOPHGY, IT MAY BE RELEVANT FOR NEURODEGENERATIVE DISEASES AS WELL. >> SO THE -- WHEN THE AMPK PATHWAY IS ACTIVATED, AS YOU KNOW THE RESULTS ARE BECAUSE THERE IS ENERGY SHORTAGE. THERE IS AN INCREASE ALSO, STIMULATED AMPK SIGNALLING. MY QUESTION IS DO YOU EXPECT OR HAVE YOU SEEN ANY DIFFERENCE IN DIABETES WHEN YOU HAVE HYPERGLYCEMIA AND THE EFFECT OF THE PDE4 INHIBITOR? >> WHEN YOU HAVE -- >> HYPER GU HYPOGLYCEMIA. >> IT DOES BRING DOWN THE GLOW COAST IN PRE-DIABETIC ANIMALS. THESE ANIMALS ARE NOT SUPER HIGH HYPERGLYCEMIC BUT HAVE INSULIN RESISTANCE AND ELEVATED GLUCOSE. I DON'T KNOW IF I'M ANSWERING YOUR QUESTION. >> YES. WELL, I WAS TRYING TO SEE WHETHER THERE WAS SOME INTERACTION BETWEEN THIS MECHANISM AND GLUCOSE UTILIZATION. >> GLUCOSE UTILIZATION? YES, I THINK WE HAVEN'T LOOKED AT STUDIES CAN DIRECTLY LOOK AT GLUCOSE UPTAKE IN THE SKELETAL MUSCLE, BUT AMPK IS DIRECTLY INVOLVED IN GLUCOSE UPTAKE, IN SKELETAL MUSCLE, I WOULD EXPECT THAT WOULD BE THE CASE. >> YES, I WAS WONDERING IN LOOKING AT THE MICE THAT WERE TREATED WITH ROLIPRAM, WAS THERE A DIFFERENCE IN LIFE EXPECTANCY? >> SO THAT KIND OF STUDY COSTS A LOT OF MONEY BECAUSE YOU HAVE TO FEED THESE DRUGS FOR THE LIFETIME. WE HAVEN'T REALLY DONE THAT. BUT THAT'S SOMETHING THAT WE'RE INTERESTED IN. >> YOU TALKED A LOT ABOUT OBESITY, MOSTLY IN ADULTS. CHILDHOOD OBESITY IS ALSO A HUGE EPIDEMIC. IS THERE A LINK BETWEEN CHILDHOOD OBESITY AND MITOCHONDRIA? >> THERE HASN'T BEEN AS MUCH STUDY. MOST OF THESE STUDIES WERE DONE A WHILE BACK. AND THE APPRECIATION FOR CHILDHOOD OBESITY IS A RECENT THING. I'M SURE IT WILL BE DONE BUT IT REALLY IS DEFICIENT RIGHT NOW. >> THANKS, JAY. THANK YOU, EVERYBODY, FOR THE MORNING SESSION. [APPLAUSE] AND STEVE HAS A QUICK ANNOUNCEMENT BEFORE LUNCH. >> ALL RIGHT. SO I WANT TO THANK -- SINCE MOST EVERYBODY IN THE ROOM -- I WANTED TO THANK THE OFFICE OF DIETARY SUPPLEMENTS, UNDIAGNOSED DISEASE PROGRAM AND NIA FOR HELPING TO SUPPORT AND FUND THE VIDEO CASTING AND ARCHIVING AND POSTER SESSION, AND THANK THE RESEARCH ALLIANCE FOR SUPPLYING REFRESHMENTS. AND IN THE INTEREST OF CALORIC RESTRICTION, WE'RE GOING TO CUT LUNCH SHORT, YOU HAVE TO BE BACK HERE AT 1:15, WE'LL GET BACK ON TRACK, ON SCHEDULE, OKAY? THANK YOU VERY MUCH. I WANT TO INTRODUCE AGAIN THE EMCEE FOR THE MEETING TRACEY RAOUAL T, WHO WILL INTRODUCE OUR SPEAKERS. >> OUR FIRST SPEAKER WILL BE A ATIF. BEFORE COMING TO PITTSBURGH, HE WORKED AT THE INTERNATIONAL CENTER OF GENETIC ENGINEERING AS A JUNIOR RESEARCH FELLOW. IN HIS CURRENT PROJECT IN THE LAB, MITOCHONDRIAL MUTATION TO LOOK AT MITOCHONDRIAL PATH GENESIS AND DESIGN STRATEGIES TO MODULATE MITOCHONDRIAL PROTEIN EXPRESSIONS INVI VIVO. TODAY EL TALK ABOUT HIS DATA WHERE HE HAS TARGETED TWO IN INDODGEUS MITOCHONDRIAL PROTEINS PROTEINS. SO WELCOME, ATIF. >> THANK YOU VERY MUCH. GOOD AFTERNOON, EVERYONE. I'D LIKE TO START BY FIRST THANK THANKING THE ORGANIZERS FOR THIS WONDERFUL OPPORTUNITY TO PRESENT OUR WORK. TODAY I AM GOING TO DISCUSS ABOUT THE STRATEGY THAT WE USE TO MODULATE ENDODGENUS MITOCHONDRIAL EXPRESSIONS IN VIV VIVO BY MODULATEING SMALL RNAS INTO THE MITOCHONDRIA. MITOCHONDRIAL DISORDERS OCCUR IN THE PRESENCE OF COMPLEX CLINICAL SYMPTOMS, INCLUDING REDUCTION AND LONGEST, LOCOMOTOR DYS DYSFUNCTION AND MITOCHONDRIAL DYSFUNCTION. THESE DISORDERS CAN BE CAUSED BY THE MITOCHONDRIAL DNA OR THE NUCLEAR DNA. CONTRIBUTED SUBSTANTIALLY TO OUR UNDERSTANDING OF MITOCHONDRIAL AND MUCH HAS BEEN LEARNED BY USING MODEL ORGANISMS IN CASES WHERE NUCLEAR DNA IS INVOLVED. UNFORTUNATELY, THERE IS NO KNOWN FAPHARMACOLOGY GENE THERAPY AS A CURE FOR THESE DEVASTATING DISORDERS. OUR LAB HAS PREVIOUSLY IDENTIFIED AND CHARACTERIZED A MODEL OF MITOCHONDRIAL MUTATIONS 861. 08 80 P 6 IS A SUBUNIT OF THE COMPLEX. THIS ATP 61 MUTATION IS A MIXED MUTATION THAT LEADS TO A SUB SUBSTITUTION OF LICE ENE BY GLUTAM ATE, AS SHOWN IN THIS PARTIAL STRUCTURE. THE FLIES AFFECT BID THIS MUTE MUTATION SHOWS REDUCED LONGEST. THEY HAVE LOCOMOTOR DYSFUNCTION. AND SHOW MITOCHONDRIAL HISTOLOGY HISTOLOGY, AS SEEN IN THIS MONO MONOGRAM. IN ADDITION, THEY ALSO HAVE MULTIPLY REDUCED INHIBIT AASE ACTIVITY. SUGGESTING THAT THIS ATP 6 MODEL IS AN EXCELLENT MODEL TO VALID EIGH VALIDATE -- SO MITOCHONDRIA IS WELL-KNOWN TO IMPORT MACRO MOLL MOLECULES. THEY HAVE RECENTLY GAINED SIGNIFICANT INTEREST, AND WE HAVE A FAIR AMOUNT OF WORKING KNOWLEDGE OF THE MECHANISMS INVOLVED IN MITOCHONDRIAL RNA IM IMPORTS, SOME OF WHICH ARE SMOUN HERE. THIS PROCESS IS THOUGHT TO BE REGULATED, AND CONSERVED IN CERTAIN SPECIES. SOME ARE COMPLEX AND THE GENE PHASES. GENE PHASES HAVE RECENTLY BEEN SHOWN TO AUGMENT THE IMPORT OF R RNACK INTO THE MITOCHONDRIA. ON THE EXTREME RIGHT HA-HAND SIDE IS AN RNA COMPLEX THAT ARE KNOWN TO IMPORT C RNA INTO THE RIBE RIBOSOME MITOCHONDRIA. SO THE EXISTENCE OF THESE IN INDODENDOGENOUS IMPACT -- -- OPENS UP A POSSIBILITY TO MODULATE PRO PROTEIN EXPRESSIONS BY TARGETING RNA INTO THE MITOCHONDRIA. WE DESIGNED A UNIQUE SET OF VECT VECTORS, WHICH WE CALL AS MP TO MODERATE MITOCHONDRIAL PROTEIN IN VIVO. IT STANDS FOR MITOCHONDRIAL TARGETED EXPRESSION SYSTEMS AND THE MAP IS SHOWN ON THE LEFT HAND SIDE LIST. THIS IS A VECTOR AND HAS A SITE, NON-CODING LITER SEQUENCE, FOLLOWED BY A PASSENGER AND ENDING IN AN R NAP DETERMINATION DETERMINATION, WHICH IS A SEQUENCE. I'D LIKE TO BRING YOUR ATTENTION TO THIS NON-CODING SEQUENCE. THIS IS AN RNA STRUCTURE, WHICH IS KNOWN TO BE IMPORTED INTO THE MITOCHONDRIA. IN OUR STUDY, WE USED THREE DIFFERENT SEQUENCES. THIS NC L IS 120 NUCLEOTIDES NUCLEOTIDES-LONG NON-CODING SEQUENCE, WHICH WE HAVE AND OTHERS HAVE IDENTIFIED TO BE MOST ABUNDANT LY LOCALIZED INTO THE FLY MITOCHONDRIA. MR P ARE MUCH SHORTER, AS COMPARED TO FIBER AND HAS BEEN SHOWN TO BE IMPORTED INTO THE MITOCHONDRIA. SO WE WERE ABLE TO DEVELOP A HIGHLY SELECTIVE ANTIBODY AGAINST A FLY ATP AND THIS ENABLES US TO ASK A VERY SIMPLE QUESTION -- DOES THE MUTANT ATP 61 GETS TRANSLATED AND IS STABLE IN THE FLY MITOCHONDRIA? WE PERFORMED A BLOT AND WE WERE VERY INTERESTED TO OBSERVE THAT THE ATP 6 LEVEL OF THE MUTANT FLY WAS VERY SIMILAR, AS COMPARED TO THE Y-TYPE ATP 6 FLY FLY. NOW THIS EXPERIMENT HAS VERY SERIOUS IMPLICATIONS. WE KNOW THAT MITOCHONDRIAL COMPLEXES ARE ASSEMBLED FROM SIBLING INDIVIDUAL SUBUNITS AND THE FACT THAT THE MUTANT PROTEIN IS BEING STABLEY TRANSLATED AND IS FOUND STABLEY IN THE MITOCHONDRIA, COULD BE A FORMED FORMIDABLE CHALLENGE. A VAST MAJORITY OF THE MITOCHONDRIAL MUTATIONS ARE APPROXIMATELY 300, THE MAJORITY OF WHICH ARE T RCHLS NNA MUTATIONS AND WE ENVISION THAT THE SAME CHALLENGE WILL BE PRESENTED IN MUTATIONS. SO WE USED A SELECTOR IS AS A TOOL TO ENGINEER AN RNA, WHICH WE CALL AS TRANSLATIONAL INHIBIT INHIBITOR. THIS TLI, ONCE IMPORT ED INED INTO THE MITOCHONDRIA, HIRYDROGIZES WITH M (M)RNA AND DOCKING OF THE SMALL SUBUNIT OF THE MICROSOME AND TRANSLATION OF THE TARGETED PRO PROTEIN. SO THE PROPOSED MECHANISM OF ACTION IS KNOCK DOWN OF THE PRO PROTEIN BY INHIBITING THE TRANS TRANSLATION OF THE (M)RNA. SINCE WE KNEW HOW THE ATP 61 MUTATION LOOK LIKE, WE CHOSE IT AS THE FIRST TARGET OF TLI. FOR THIS, WE DESIGNED TWO ATP 6 TLI, A, 1, AND B. THEY DIFFER ONLY BY ONE BASE AND THERE IS A COMPLEMENT AARY RELATIONSHIP WITH THREE NUKE NUCLEOTIDES INB, AS COMPARED TO ATP 6 A. SHOWN HERE AS THE LONGEST ASSAY OF THE FLY EXPRESSION THE IN INHIBITOR IN RED, AS COMPARED TO THE CONTROLLED SLIDES FLIES. SIMILARLY, THIS IS THE FLY WITH ATP 6 BTLI AND WE OBSERVED 75% KNOCKDOWN WITH A SIGNIFICANCE, AS COMPARED TO THE Y FLY. AGAIN, I'D LIKE TO THIELT THE FLIES SHOWED REDUCED LONGEST. WE THEN PERFORMED A LOCOMOTOR A ASSAY ASKING THE QUESTION WHETHER THESE FLIES THAT EXPRESS EXPRESSED T LMENTI ATP 6 SHOWED ANALOGOUS BEHAVIOR OF FENPHENOTYPE AS THE ATP 61 FLY DOES. SHOWN HERE ARE THE LOCOMOTOR DIS DYSFUNCTION ON DAY ONE, VERSUS DAY 50 IN FLIES WITH ATP 6 A AND TLI ATP 6B. IN BOTH CASES WE OBSERVED THAT THE FLIES DID NOT SHOW LOCOMOTOR DYSFUNCTION, WHEREAS THERE WAS DYSFUNCTION IN THE FLIES ACCEPTING THE TLI, SUGGESTING THE FLIES EXPECTING TLI DEMONSTRATE LOCOMOTOR DIS DYSFUNCTION, WHICH IS SIMILAR TO THE ATP 61 MUTANT SGLIECHLTZ WE THEN INVESTIGATED THE TARGET PRO PROTEIN IN THIS CASE THE ATP 6 PROTEIN FOR THE LEVEL. SHOWN HERE IS A BLOCK AND ATP 6 HERE. THIS IS THE WILD TYPE, AND THE PRENON-CODING SEQUENCE WITH THE TRANSLATIONAL INHIBITORS TO TARGET ATP 6. AND WE OBSERVED SIGNIFICANT RE REDUCTION IN THE EXPRESSION OF THE PROTEIN LEVELS, AS COMPARED TO THE Y WILD TYPE CONTROLS. THESE TRANSLATION INHIBITORS ARE EXACTLY THE SAME. THE ONLY DIFFERENCE IS THE SEQUENCING AND MR P, FIBERS AND RNA. SO WE WERE EXCITED, AS THIS KNOCKDOWN LEVEL WAS ANALOGOUS TO SOME OF THE KNACK-OF-KNOCKDOWN LEVELS INDEPENDENT OF RNA AMERIMEC MECHANISMS BY OTHER GROUPS. IN OUR CASE, WE SHOW THIS KNOCK KNOCKDOWN IN THE MITOCHONDRIA. SO THE NEXT TARGET OBVIOUS QUESTION WAS DO TLIS MODULATE MITOCHONDRIAL PROTEIN IN A RNA DEGREDATIOE DEGREADATION? WE QUANTITY AIATED THE LEVELS IN THE RNA AND THE FLIES EXPECTING TLI ATP 6B AND WE DID NOT FIND ANY SIGNIFICANT CHANGE IN THE LEVEL OF (M)RNA, SUGGESTING THAT THE TRANSLATION -- IT AFFECTS TRANSLATION AND REDUCE THE TARGET PROTEIN LEVELS, WHICH IS INDEPENDENT OF ALTERING RNA LEVELS. WE ALSO LOOKED AT SOME NON- NON-SPECIFIC TARGETS OF MITOCHONDRIAL RNA AND CHOSE COX COX-2 INHIBIT (M)RNA IN THE FLIES WITH ATP 6 A SCAAND SCOMBCHLS WE DID NOT FIND ANY SIGNIFICANT CHANGE IN EITHER CASES. B. WE ALSO WANT TED TO INVESTIGATE THE NON-TARGET MITOCHONDRIAL PRO PROTEIN EXPRESSION LEVELS. AND AGAIN, WE CHOSE COX-2 INHIBIT AT THE PROTEIN LEVEL, WHICH SHOWED THAT THERE WAS NO SIGNIFICANT CHANGE IN EITHER OF THE NON-CODING GENEING SEQUENCE EXPECTING TLI ATP 6, SUGGESTING THAT THE TLI SHOWED ANY SPECIFIC TARGETING IF THERE WAS NO PRO PROTEIN TARGET IING, WE HAVE OBSERVED CHANGES IN EITHER THE M (M)RNA LEVELS OR THE PROTEIN LEVELS OF NON-TARGET PROTEINS. WE THEN ASKED A QUESTION WHETHER WE CAN REENGINEER THE VECTOR AND USE IT FOR THE MAMMAL IIAN. SO WE PLACED THE INITIATED SEQUENCE IN FLIES. AND FOR THE MAMMAL IIAN SYSTEM WE CHOSE THE TLI AGAINST COX-2 INHIBIT. SHOWN HERE IS THE BLOCK AGAINST THE ANTI-COX-2 INHIBIT LEVEL. THE FIRST SHOWS THE CONTROL AND THE REST SHOWS THE REDUCTION OF THE KNOCKDOWN COX-2 INHIBIT LEVELS IN M RCHRP AND FIBER. SHOWN HERE IS THE -- THIS SHOWS THAT THE MORE ROBUST DECREASE IN THE PROTEIN LEVELS, COMPARED TO THE M RCHRP, SUGGESTING THAT THE -- IT COULD BE MODIFIED TO TARGET PROTEINS IN THE MAMMAL IIAN SYSTEM AS WELL. SO WE ASKED ANOTHER IMPORTANT QUESTION WHETHER ADDING THIS EX EXTRA TLI WITH THE FIBER AFFECTS THE IMPORTABILITY OF THIS RNA INTO THE NOT MITOCHONDRIA. SO BY A RADIO TAG AND PERFORMED A MITOCHONDRIAL IMPORT ASSAY, WHICH HAS BEEN PREVIOUSLY ESTABLISHED. WE OBSERVED ROBUST IMPORT OF 5 R RNAS JUST BY ITSELF. JUST TO NOTE THAT IT HAS PREVIOUSLY WILL NOT SHOWN THAT THE IMPORT OF FIBERS ARE DEPENDENT ON THE POTENTIAL. SO AS A CONTROL, WE USED ATPS AND THIS KNOCKDOWN IS BASICALLY COMPLETING AGGREGATED THE IMPORT OF FIBER (M)RNA INTO THE MITOCHONDRIA. WE THEN ASKED THE SAME QUESTION OF IMPORTABILITY OF THE CRIM ER ERICK R ITNAN AND -- RNA AND OBSERVED SIMILAR ROBUST IMPORT OF THE TLI WITH COX-2 INHIBIT INTO THE MITOCHONDRIA, WHICH WAS AGAIN ABROGATEED INTO. HAVING Y AND ALSO THAT ANY SEQUENCE, ANY TLI SEQUENCE CAN ALSO BE A ATTACHED TO THIS PARTICULAR FIB FIBER. SO THIS IS THE LAUGHST SLIDE OF MY DATA, AND THIS WAS ONE OF THE MOST IMPORTANT CONTROLS THAT WE DESIGNED. AND THIS WAS -- THE EXPERIMENT WAS DESIGNED TO ASK THE SEQUENCE SPEC FIIFICITY SELECTIVITY QUESTION QUESTION. IN THIS CASE, WE USED THE FIBBER AND THE TLI COX-2 INHIBIT AND USED AN AUTOMATED SOFTWARE TO RE RESEFSMBLE THE SEQUENCE -- ESTABLISH THE SEQUENCE WHICH WE CALL TLI. THIS IS THE DATA OF THE BLOCK THAT SHOWS THAT THE COX-2 INHIBIT WAS UNABLE TO KNOCK DOWN THE PROTEINS IN THIS CASE THE COX-2 INHIBIT, AS COMPARED TO THE TLI COX-2 INHIBIT, WHICH SHOWED A ROBUST KNOCKDOWN, AS COMPARED TO THE CONTROLLED VOESHTH. THIS SUGGESTS THAT THE TLI SHOWED SEQUENCE SPEC FIIFICITY. VECTOR. SO IN SUMMARY, WE HAVE DEMONSTRATED THE UTILITY OF TWO NEW VECTORS, ONE IN THE MAMMAL IAN CELLS THAT CAN TARGET RNA INTO THE MITOCHONDRIA IN VIVO. THESE SHOWED ROBUST KNOCKDOWNS OF SPECIFIC TARGET PROTEINS. THEY ARE PROPOSED TO WORK IN A NON-CAT LALYTIC MECHANISM, WHICH ON THE BASIS OF OUR DATA, SHOWS INDEPENDENT DEGREDATION. WE HAVE ALSO SHOWN THAT ADDING T TLI SEQUENCES DO NOT IMPACT THE IMPORTABILITY OF THIS KIN AASE RNA RNA. OUR DATA SUGGESTS THAT WE COULD POTENTIALLY TARGET GENES IN ADDITION TO THE ATP 6 AND COX-2 INHIBIT, THAT I SHOWED YOU HERE. HOWEVER, THIS VECTOR HAS CERTAINLY LIMITATIONS. ONE, THAT IT CANNOT BE USED AGAINST PROTEINS, BECAUSE THEY ARE SFLALZ FOR THE DETERMINATION DETERMINATION. IN ADDITION, THERE ARE SOME VARIABILITIES IN THESE INHIBIT INHIBITORS, WHICH WE BELIEVE COULD BE VIEWED TO THE SEQUENCE SELECTIVITY AND CURRENTLY THEY ARE WORKING ON FURTHER OPTIMIZE OPTIMIZATION OF THE VECTORS. WE ARE ALSO CURRENTLY TESTING WHETHER THIS VECTOR CAN BE USED TO TARGET LONGER RNA SEQUENCE S IN VIVO, WHICH COULD POTENTIALLY BE A WIDER STRATEGY. WITH THAT, I WOULD LIKE TO ACKNOWLEDGE MY MENTOR, DR. MIKE MICHAEL SAL LIDINO AND OTHER MEMBERS. DR. SOLATTO AND AN UNDERGRANT -- UNDER-- UNDERGRADUATE SDWRUVENLTS I WOULD ALSO LIKE TO THANK MY PROGRAM, THE SCHOOL OF MEDICINE, UNIVERSITY OF PITS PITTSBURGH, AND THE PITTSBURGH INSTITUTE FOR NEWER LODUROLOGICAL DISORDERS, WHERE OUR LAB IS PHYSICAL LY LOCATED. LAST BUT NOT LEAST, I WOULD LIKE TO THANK THE ORGANIZERS, THANK YOU VERY MUCH. THANKS FOR YOUR ATTENTION. APPLAU [APPLAUSE] >> THANKS FOR YOUR TALK. IT LOOKS IN ONE OF YOUR EARLIER DRAFTS SDWLASHGS THE MR P TARGET WAS ACTUALLY THE MOST EFFICIENT. BUT IT LOOKS LIKE YOU SPENT THE MOST TIME WITH THE R (M)RNA. DO YOU THINK WHEN YOU SPECULATE ON WHICH OF THESE THREE METHODS IS MOST EFFICIENT AND ARE YOU INTERESTED IN PURSUING TRYING TO SORT OUT WHICH ONES IS WORTH TARGET? >> RIGHT. SO THE FIRST SET OF DATA WHERE M MR P IS BETTER IN THE FLY. IN THE SECOND SET, WHICH WE FURTHER USED FOR ALL OF THE IM IMPORT ASSAY AND SAMPLING IS IN THE MAMMAL IIAN SYSTEM. SO WE DO SEE SOME KIND OF VARIATION, AND AS I MENTIONED, THIS COULD BE A SPREAD TO THE SELECTIVITY. WE HAVEN'T LOOKED INTO DETAILS AS TO THE IMPORTS, SPECIFICALLY COMPARING THESE DNA SEQUENCES, BUT WE DO, BASED ON THE KNOCK KNOCKDOWN LEVELS, WE DO OBSERVE THESE VARIATIONS. >> ONE THING THAT HAS CONFUSED ME ABOUT READING SOME OF THE PAPERS OUT THERE ABOUT THE RNA IS -- SO IT'S A BIGGER PIECE AND YOU HAVE TO EXPLAIN THINGS DIFFERENTLY FLAU[INAUDIBLE] WHICH CAN EACH, IF I'M REMEMBERING CORRECTLY, INDEPENDENT LY BE IM IMPORT ED INED INTO THE MITOCHONDRIA. HAVE YOU PLAYED AROUND THE ALL WITH TRYING TO SEPARATE THE FUNCTION OF THOSE TWO? >> YEAH, WE ARE IN THE PROCESS OF CHARACTERIZING THE NCS AS WELL, SO YES. THE ANSWER IS YES. >> THANK YOU. >> THANK YOU VERY MUCH. APPLAU [APPLAUSE] >> I'D LIKE TO THANK ATIF FOR THAT INTERESTING TALK AND INTRODUCE DR. MIGUEL AOU. AND HE HIS COLLABORATORS HAVE BEEN STUDYING THE ROLE OF MITOCHONDRIA AND METABOLISM IN HEART FUNCTION AND DIABETES. WITH A MAIN FOCUS IN THE DR. AOU AOU'S IS HOW IT'S AFFECTED BY DIABETES IN THE HEART. SO MICHAEL -- MIG >> THANK YOU VERY MUCH. THANKS TO THE ORGANIZERS FOR THIS KIND OF PRESENTATION. IT'S EXCITING. AND THE TOP IC TODAY IS ABOUT MITOCHONDRIAL ADAPTIVE MECH MECHANISMS THROUGH OVERCOMING HEART DYSFUNCTIONS IN DIABETES. AND -- SORRY. IT STARTS WITH A QUESTION, WHICH IS WHY KEEPING A RELIABLE AND COMPROMISED ENERGIES OF FLY SUPPORTING THE HEART, PARTICULARLY IN DIABETES. AND A BASIC FIPHYSIOLODGGICAL FACT I WILL STRAITS THE IMPORTANCE OF THIS QUESTION. A HUMAN MALE LIKE ME, 150 POUNDS POUNDS, WILL CONSUME 430 ELILITERS OF OXYGEN PER DAY, AND THAT ACTIVITY CAN INCREASE FIVE TO TEN TIMES, DEPENDING ON PHYSICAL ACTIVITY. OF ALL THAT OXYGEN, MORE THAN 90 90% WOULD BE CHANNELED INTO THE CHAIN, WHERE -- IS REVIEWED AND MAKING THE MITOCHONDRIA SUSCEPT SUSCEPTIBLE TO OXIDATIVE DAMAGE. ADDITIONAL LY, THE HEART IS TEHE ORGANI, FROM THE HUMAN BODY, THAT CONSUMES ON A SPECIFIC BASIS, THE HIGHEST AMOUNT OF OXYGEN WITH RESPECT TO THE WHOLE BODY, WHICH, ON THE OTHER HAND, THE HEART MAY EXHIBIT AN AORTIC MET METABOLISM. SO IT'S HIGHLY DEPENDENT ON THE MITOCHONDRIA FOR OBTAINING THE ENERGY. SO WE HAVE TWO THOSE ASPECTS -- ENERGETIC AND RELOCK, WHICH ARE EXTREMELY IMPORTANT. AND WE WILL SEE THAT THOSE -- THIS IS WHAT I CALL THE CRUCIAL ENERGY REDOX LINK IS AFFECTED IN MITOCHONDRIA AND IN DIALITYIC MITOCHONDRIA. AND ALSO, WEST EXTRA THREAT OF HIGH FRIBILLATEING LEVELS OF THE TWO MAIN FUELS OF THE HEART, WHICH RADIORFAT AND GLUC OOSE. SO WHEN TAKING ALL OF THESE INTO ACCOUNT, IT IS ALMOST LODGICAL THAT SOMEHOW THERE SHOULD BE A ADAPTIVE MECHANISMS, PROTECTIVE MECHANISMS OF MITOCHONDRIAIAL FUNCTION IN DIABETES, AND WE WILL TALK ABOUT THAT. MITOCHONDRIAL. SO I WILL START ADDRESSING THE CRUCIAL ENERGY REDOX LINK IN THE FRAMEWORK OF THE REDOX OPTIMIZED HYPOTHESIS AND IN THE CONTEXT OF THE FUNDAMENTAL RELATIONSHIPS BETWEEN MITOCHONDRIAL RES RESPIRATION AND ROSS. JUST AS A REMINDER, CONSIDER THIS BASIC ENERGY REDOX PROCESS PROCESSING MITOCHONDRIA, PRODUCED IN THE CHAIN AND THERE ARE A WHOLE ARRAY OF ANTI-OXIDANT SYSTEMS THAT WILL MODULATE IN THE REFLEX FROM THE MITOCHONDRIA WILL BE THE NET RESULT OF THESE TWO PROCESSES. BUT THE MAIN DRIVING FORCE BEHIND THE ROSS INFLECTION FROM THE MITOCHONDRIA IS THE REDOX ENVIRONMENT AND THE REDOX POSTU POSTULATE THAT WE INTRODUCED IN 2010. THIS HYPOTHESIS SAID THAT THE ROSS LEVELS IN RED HERE, WILL A ATTAIN AT THE FUNCTION OF THE RE REDOX ENVIRONMENT A MINUIMUM OF INTERMEDIATE VALUES OF REDOX ENVIRONMENT. AND THAT IT -- ROTH LEVELS LIN CEASE TOWARDS THE MORE REDUCED OR THE MORE OXIDIZED EXTREMES, BUT BY COMPLETELY DIFFERENT MECH MECHANISMS. ON THE RIGHT, WE WILL HAVE LOW E ELECTRON FLOW. AND SO THE ELECTRON CARRIERS WILL BE HIGHLY REDUCED, SO THE PROBABILITY OF GIVING ELECTRONS TO OXYGEN WILL INCREASE, DESPITE HIGH ANTI-OXIDANT DEFENSE LEVELS IN GREEN HERE. ON THE, OTHER IT WILL BE DUE TO OVERWHELMING ANTI-OXIDANT DEFENSE DEFENSES. SO IN THE MINUIMUM, WE HAVE A VISION IN WHICH ROTH LEVELS ARE LOW, ALTHOUGH NON-VISIBLE AND IT WILL BE XIBL WITH FIPHYSIOLODGGICAL ROTH SIGNAL LY. AND IT IS A VERY IMPORTANT POSTU POSTULATE THAT THAT MINUIMUM OF ROTH WILL BE ATTAINED WHEN MITOCHONDRIAL RESPIRATION IS AT THE MAXIMUM. AND THAT IMPORTANT POSTULATE WAS DEMONSTRATED AND TESTED IN A PAPER PUBLISHED THIS YEAR, IN WHICH WE JUST USED ISOLATED MITOCHONDRIA WITH NORMAL SUB SUBSTRATES THROUGH ATP AND WE IN INCREASED THE RES PRPIRATORY RATE WITH ATP AND LOOKED AT ROTH E EMISSIONS. AND AS A FUNCTION OF THE REDOX ENVIRONMENT AND WHAT WE SEE IS THAT WHEN THE RESPIRATIONS ARE AN ENERGETIC MAXIMUM, THE ROTH E EMISSION GOES A MINUIMUM. WE ADDITIONAL LY TESTED THE MITOCHONDRIA STRESS OXIDATIVE STRESS, BECAUSE AS WE KNOW IN AGING, AS WELL AS IN DISEASE, AND PARTICULARLY IN D'AIURETICS, AS WE WILL SEE, MITOCHONDRIA FUNCTION IN A MORE OXIDATIVE ENVIRONMENT. SO WHICH IS THEN COMPROMISELING THE REGENERATION OF GSH AND ADD ADDING EXTRA HIRYDROGEN PROJECTION PROJECTIONIDE. AND WHAT WE SAW IS THAT THE RES RESPIRATION DECREASED AND ROTH E EMISSION ALSO INCREASED. BUT THEY FOLLOW ESSENTIALLY THE SAME KIND OF RELATIONSHIP. AND TO GO TO THE MORE EXTREME OX OXIDATIVE ENVIRONMENT, WE WILL NEED AN EXTRA ENCOUPLEING IN ORDER TO SEE AN INCREASE, BUT NOT IN ROTH LEVELS. BUT THAT WILL BE MORE IN PATH AH PATHOFIPHYSIOLODGGICAL EREGIME. SO WHAT WE SEE THEN FROM THIS IS THAT WHAT I CALL THE CRUCIAL ENERGY REDOX LINK IS THAT WHEN YOU INCREASE ADP TO THE MITOCHONDRIA, WHICH WILL STIM STIMULATE AND INCREASE IN ENERGY DEMAND, ROTH SHOULD DECREASE. AND THAT, -- WE SEE THAT THIS IS SOMEHOW DAMAGED. AND WE TESTED THIS IN DIABETIC MITOCHONDRIA FROM ONE OF THE AN ANIMAL MODELS THAT WE WORKED WITH, IS DIABETIC BEGGUINEA PIGS IN INDUCED BY TYPE 1 DIABETES. WHEN WE ISOLATE MITOCHONDRIA FROM CONTROL ANIMALS, DIABETIC OR DIABETIC TREATED WITH INSULIN AND LOOKED AT HIRYDROGEN PER OXIDE EMISSIONS AS A FUNCTION OF ADP CONCENTRATIONS LOOKING AT MITOCHONDRIA IN CYAN HERE. THERE IS A DECREASE IN ROTH EMISSION IN ALL GROUPS. WHEN WE STRESSED THE MITOCHONDRIA, WE STILL SEE A DE DECREASE AS A FUNCTION OF ADP CONCENTRATION WITH THE EXCEPTION OF THE DIABETIC GROUP, IN WHICH WE OBSERVED AN INCREASE, RATHER THAN A DECREASE, IN ROTH AE AEMISSION, WHICH MAKES THE MITOCHONDRIA ON ONE HAND. BECAUSE THAT ENERGY, REDOX LINK IMPORD A SOURCE OF OXIDATIVE STRESS IN DIABETES. SO I SAID AT THE BEGINNING THAT IN DIABETES, ALSO THE HEART AND THE CARDIAC REDOX ENVIRONMENT IS SUBJECTED TO THE THREAT OF HYPER HYPERGLYCOEMIA AND HYPEREPIDEMIO HYPEREPIDEMIOIA. AND WORKING WITH TWO DIFFERENT ANIMAL MODELS WITH DIABETES, THE MOUSE LEPSIN RECEPTOR TRANSGENIC MOUSE AND THE SOUPER DIABETIC FAT FATTY RAT. WE DO -- DID THIS CRUCIAL OBSERVATION. IN HYPERGLYCOEMIA, WHEN WE LOOKED AT THE CONTRACT AISLE ACTIVITY IN -- CONTRACT I'LL ACTIVITY IN THE PER FUSE HEART AND RAT HEART TRAVEHICKIA AND ALSO IN CONTRACT ACTIVITY, WE SEE THAT IT IMPROVES CONTRACTILE ACTIVITY, WITH THE OPPOSITE HAPPENING IN THE CONTROLLED WILD TYPE. SO THIS LED US TO THINK THAT SOMEHOW THE DIABETES HEART IS DIFFERENT AND HANDLES IN A DIFFERENT WAY WITH RESPECT TO NORMAL HEART. IN THIS CASE THE LIPIDS AND ALSO THE IN THE CONTEXT OF HYPERGLYCO HYPERGLYCOEMIA, WHICH IS THE WHOLE MARKER OF DIABETES. SO THIS LEADS ME TO THE FIRST PART OF MY -- OF THE ADAPTIVE MECHANISM THAT CONCERNS LIP ID-E LIPID-ELICITED IMPROVEMENT IN ACTIVITY IN TYPE-2 DIABETES DIABETES. HERE, WORKING WITH TYPE-2 DIABETES DIABETES, MICE, THE DB D BCHB MOUSE AND DOING TWO PHOTO IMAGEING OF ISOLATED CARD YA MIOMYO CARDIOMYOCITES FROM WILD TYPES AND FROM DIABETIC IN BLACK HERE. WE LOOKED AT THE REDOX OF THOSE CARD IOMIOCYTES USING SIMULTANEOUS LY THROUGH DIFFERENT OXIDATIVE STRESS AND ALSO NADH AND DETECTING REDUCED GLUC OOSE. AND WHEN WE EXPOSED THE CARD YIO MIOCYTES TO INCREASING CONS CONCENTRATION OF HIGH GLUC OOSE HERE, AND A HIGH GLUCOSE IN THE PLAQUE BETA STIMULATION, STIMULATING METABOLIC STRESS AND ALSO HIGHER ENERGETIC DEMANDS BY ISOPRO TERENOL IN THIS CASE. AND WHEN WE DO THAT, WE OBSERVED THAT THE POOL IS INCREASING LY OX OXIDIZED AS THE METABOLIC STRESS AND THE ENERGETIC DEMAND IN INCREASES.^ AND ALSO, THE OX OXIDATIVE STRESS IN THE DIABETES -- DIABETIC INCREASES. BUT WHEN WE ADD PAL MITT AATE, THEN THERE IS AN INCREASE BOTH IN THE WILD TYPE AND THE DIABETES SIGNIFICANT AND THE DRAMATIC DECREASE IS IN THE OX OXIDATIVE STRESS. AND SO WE -- BY OTHER KIND OF EXPERIMENTS, WE SHOWED THAT THAT IMPROVEMENT OF THE REDOX ENVIRONMENT PERFORMED BY THE ACTION OF PER MUTATE CORRELATED POSITIVELY WITH A RESCUE OF THE CONTRACTILE ACTIVITY OF THE DIABETES CARD IO CITE. AND BY INDEPENDENT EXPERIMENTS, INCLUDING THE REDOX ENVIRONMENT EXPERIMENTS INDEPENDENT FROM THE PRESENCE OF FARMUT TATE, INCLUDING THE ENVIRONMENT WITH GRUDOSCIENCE, WE ALSO OBSERVED THE SAME. WE IMPROVED REDOX IN THE SAME WAY THAT PAR MUTATE DOES AND CONTRACT AISLE ACTIVITY, MEANING THAT THE ENVIRONMENT IS INTERMED INTERMEDIAR Y AND THE DEFECT -- DIRECT EFFECT OF THE CAUSE OF RESCUE IN CONTRACTILE ACTIVITY. SO WE WANTED TO UNDERSTAND WHERE THE EFFECT OF PAL MUTATE. AND FOR THAT, WE WENT TO THE NEXT LEVEL, TO THE WHOLE HAEART AND MONITORED IN THIS CASE FOR THE LEFT VEN CLICK LE FUNCTION THAT DEVELOP PRESSURE IN THIS CASE. WHEN WILD TYPE AND DIABETIC HEARTS WERE SUBJECT ED ED TO THEIR NORMAL GLUC OOSE CONCENTRATION LEVELS AND ALSO PAR MUTATE LEVELS. SO AT BASELINE, WHEN THERE IS JUST GLUC OOSE, THERE IS NOT MANY DIFFERENCES IN CONTRACTILE ACTIVITIES. WHEN WE HAD BETA ENERGETIC STIMULATION, IT IS MUCH HIGHER IN THE WILD TYPE THAN IN THE DB, WHICH IS QUITE BLUNTED. AND PAR MUTATE RECOVERS -- PRESERVES THE CONTRACTILE FUNCTION WHEN PRESENT ALSO IN THE PRESENCE OF HYPOFLIES EM IMIA. AND WHEREAS IN THE WILD TYPES, THERE IS AN ASLIGHT DECREASE, ALTHOUGH NOT SIGNIFICANT. NOW, IF WE WANT TO UNDERSTAND BETTER THE MECHANISMS OF THE EFFECTS OF PAL MUTATE, WE TOOK HEARTS FROM EACH ONE OF THESE TREATMENTS, FROM THE TWO GROUPS AND ANALYZED BY METABOLIC. AND WHAT WE OBSERVED IS THAT MET BOLL ITES FROM THE MAIN GLUCOSE DEGREDATION PATHWAYS IN THE HEART, LIKE SULF AASE AND GLYCOCOL SIS AND POLLIOS, SHOWED AC ACCUMULATION OF MET BOLL IITES HERE IN BLACK, IN THE DIABETES. SO IN ORDER TO TAKE THIS TO A MORE QUANTITATIVE LEVEL, WE TOOK THE MET BOLLITE AND WE APPLIED A NOVEL PROCEDURE TO OBTAIN, FROM WHICH THE MET BOLLITE FLIES EMERGE AND THIS IS THE KIND OF RESULT THAT WE OBTAINED. HERE IS THE WHOLE GLUC OOSE FLAXUM OF THE HEART IN WHICH THE FLAX PHYLAXIS ARE SPREAD. IN THIS CASE THE WILD TYPE IN BLUE AND THE DIABETIC IN RED. TWO OF THE MAIN RESULTS FROM THIS FLAXUM ANALYSIS WAS THAT THEOSOLVEIC PATH WWAY INCREASED ABOUT 50% WITH FLAX BUT THAT PATH WWAY. AND WHEN WE LOOKED AT GUY COLYCOLYSIS GLYCOLYSIS, IT WAS NOT AFFECTED EVEN BY THE PRESENCE OF THE PAL MUTATE, AND IT WAS ALWAYS HIGH. SO THAT EXPLAINED WHY PAL MUTATE IS INCREASING OR IMPROVING THE REDOX ENVIRONMENT, BECAUSE THE PENTOSOLVEIC PATH WWAY, AS YOU WELL KNOW, IS A MAIN PRODUCER OF NAD BRPH, WHICH IS RESPONSIBLE FOR GSH GLUTOSCIENCE REGENERATION IN THE CIYTOPLASM. AND THIS EXPLAINS THE IM IMPROVEMENT THAT WE SAW BY PAR MUTATE. SO NOW LET ME GO TO THE THIRD PART OF MY TALK, WHICH IS ABOUT DIRECT CONTACT MITOCHONDRIAL DROPLETS DRIVEN BY HIGH-ENERGY SGLAVENLTD SO ALL THIS IS GOOD IN THE SHORT TERM, BUT WE NEED TO UNDERSTAND WHAT HAPPENS IN THE LONG TERM. AND WAY WE DID THAT IS WE SUBJECTED THE MICE AND WILD TYPES TO AN EXERCISE DIET PROGRAM FOR FIVE WEEKS, IN WHICH WE SUBSTITUTED CERTAIN LIPIDS, KEEPING THE SAME FAT PERCENTAGE IN THE DIET BY PAR MUTATE. AND THEN AFTER THE FIVE WEEKS, WHEN WE LOOK AT THE FIPHYSIOLOGY OF THE DIABETIC, THEY EXHIBITED A EXCHANN EXCHANGE RATIO OF 0.7, PLANING THAT THEY WERE DOING THROUGHOUT THAT PERIOD OF TRAINING A FAT OXIDATION. ALSO, THEY LOST 29% OF THEIR WEIGHT WITHOUT CHANGING FOOD CONSUMPTION. AND THEY CHANGED -- EXCHANGED THE PERCENTAGE BODY FAT INTO LEAN MUSCLE IN THESE PERCENTAGES PERCENTAGES. AND ALSO, THE INVI VIVO HEART PERFORMANCE OBSERVED BY THE THIRD WEEK IMPROVED. EJECTION FRACTION AND DECREASED LV AND DIAL STOLLIC DIMENSION. AND SO THESE ANIMALS THAT ARE IN THE TREADMILL, ITS HEART RATE COULD HARDLY KEEP WITH LOW IN INCLINATION AND LOWEST SPEED ON THE TREADMILL ENDED UP AFTER FIVE WEEKS RUNNING AT THE SAME LEVEL AS THE WILD TYPE. SO WHEN WE LOOKED INTO THE ULTRA ULTRASTRUCTURE OF THE HEART OF THOSE ANIMALS, THE DIABETIC EXERCISE SHOWED A LOT OF LIP IID DROPLETS INTERLOCKED WITH THE MITOCHONDRIA AND NO LIP ID DROPLETS IN THE WILD TYPES. BUT THE INTERESTING FEN APHENOMENON WAS THIS ONE, IN WHICH THE EXERCISE GROUP IN THE DIABETIC EXHIBITED A TWO-FOLD INCREASE IN THESE FIGURES, IN WHICH LIP ID DROPLETS APPEAR SURROUNDED WITH MITOCHONDRIA. THERE SEEMS TO BE SOMEHOW RE RECRUITED AROUND THE LIP IID DROP DROPLETS. THAT WHEN THEY CAME IN CLOSE CONTACT, THEY ALWAYS APPEAR THIS KIND OF HOLLOW, WHICH HAS BEEN INTERPRETED AS THE ACCUMULATION OF STLEE GLYCEROLLINGS IN THE PRIME MINISTERIPHERY BECAUSE THEY ARE MORE CHOLESTEROL ESTHERS THAN ARE IN THE CORES OF THE LIP ID DROPLETS. SO WE CALL THIS FEN APHENOMENON D DIRECT CONTACT DROPLETS THAT INVOLVES A DROPLET OF THE MITOCHONDRIA AND ALSO A DIRECT CONTACT. ANOTHER INTERESTING OBSERVATION IS THAT THE DIABETIC, THE NUMBER OF MITOCHONDRIA DECREASED SIGNIFICANTLY IN THE EXERCISE GROUP. BUT THE SIZE OF THE MITOCHONDRIA WAS BIGGER IN THE DIABETIC. AND THAT SUGGESTED TO US THAT THERE MAY BE SOME KIND OF FUSION NEN FEN APHENOMENON GOING ON IN THIS PROCESS. SO NOW WHAT IS THE FIPHYSIOLOGICAL SIGNIFICANCE OF THIS FEN APHENOMENON? DOES IT HAVE SOMETHING TO DO WITH THE FUNCTION? IS IT BENEFICIAL?{}WELL, THIS LEADS ME TO THE LAST PART OF MY TALK, ABOUT MITOCHONDRIA ENERGY ENERGETIC CHANGES IN THE DINE DYNAMICS OF LIP ID STORAGE AND UT UTILIZATION. SO WE WANTED TO TAKE THESE OBSERVATIONS TO THE CELLULAR LEVEL, WE STAINED LIP ID DROPLETS WITH A FLUORESCENT ANALOGUE CALLED BODIFFY AND THE LIP IID DROPALITIES PEAR HERE AS BRIGHT GREEN SPOTS. AND IN A PERFECTLY POLARIZED MITOCHONDRIA, WHICH IS A SENSOR OF MITOCHONDRIAL MEMBRANE POTENTIAL. AND WE SHOWED ALSO A MEASURE A DIRECT MITOCHONDRIAL DRIP LIP ID DROPLETS THE COLONIZATION OF THE RED SIGNAL OF CM RCHR M AND BODIFF Y THAT APPEAR AS YELLOW DOTS HERE. THE LIP ID DROPLETS ARE OUTSIDE THE MITOCHONDRIA ARE COMPLETELY GREEN. SO MEANING THAT THERE IS -- THAT WE CAN DEMONSTRATE THAT THESE LOCALIZATION THERE MAY BE SOME DIRECT CONTACT. BUT NOW -- OKAY, BUT WHAT DOES IT MEAN FOR THE CELL TO HAVE -- THOSE LIP ID DROPLETS ACCUMULATED ACCUMULATED? WELL HERE IS A VERY GOOD REASON FOR THE CELL TO KEEP THAT CON CONFINED, THE LIPIDS. WHEN WE ISOLATED MITOCHONDRIA AND LOOKED AT THE RESPONSE OF HIRYDROGEN PER OXIDE EMISSION AS A FUNCTION OF LIP ID PRECURSORS OF REDOX DATION IN THE PRESENCE OF EL CARNATE AND THEN IN OXIDATION CONDITION, WE OBSERVED THAT THE ROTH EMISSION WAS LOW UNTIL 10 MICROMOLAR AND AFTER THERE IS A SEVERALFOLD INCREASE IN THE ROTH EMISSION. THERE IS ROTH OVERFLOW FROM THE MITOCHONDRIA. AND WHEN WE LOOKED AT THE RES RESPIRATION, TOGETHER WITH ROTH EMISSIONS, WE OBSERVED THAT, THE LIP ID CONCENTRATION IS AT OR BELOW THE THRESHOLD, DESPITE HIGH LEVELS OF RESPIRATION, THE ROTH EMISSION IS LOW. BUT AFTER THE THRESHOLD, THERE IS A HUGE INCREASE THAT IS EXPLAINED BY REDOX OXIDATION. A GOOD REASON WOULD BE THAT BY KEEPING CONFINED IN LIP ID DROPLETS, THE CELL WILL BE ABLE TO AVOID AN INCREASE IN THE IN INTRACELLULAR PREFATTY ACID CONCENTRATIONS, THAT IS IN ROTH -- THUS LIMITING THE ROTH OVER OVERFLOW AND DAMAGING THE ENVIRONMENT. NOW, IN THAT SIGNAL, THE SIGNAL FOR THOSE LIP ID DROPLETS TO AC ACCUMULATE COMES COME FROM THE MITOCHONDRIA. IN OTHER WORDS, THE MITOCHONDRIA PLAYS A CERTAIN ROLE IN THIS. AND THIS SIMPLE EXPERIMENT SHOWS THAT THIS MIGHT BE THE CASE. IF YOU COUPLE THE MITOCHONDRIA IN A CELL THAT OTHERWISE DONE SHOW ANY LIP I HAD DROPLETS AND THEN YOU PER FUSE, YOU WASH OUT AFTER FIVE MINUTES ONLY AND THEN YOU WASH OUT IN THE PRESENCE OF PAR MUTATE, YOU WILL SHOW THAT WITHIN FIVE TO TEN MINUTES THERE IS A HUGE ACCUMULATION OF LIP ID DROPLETS IN THE CELL. AND THIS MEANS THAT, WHEN MITOCHONDRIAL ENERGETICS ARE SOMEHOW COMPROMISED OR DAMAGEED, THAT WILL BE A SIGNAL FOR THE CELL TO ACCUMULATE A LIP ID DROP DROPLET. SO HERE IS OUR WORKING HYPOTHESIS OF METABOLIC CHANNEL CHANNELING OF LIP ID OXIDATION AT THE CONTACT SITE IN VC MLD. SO THE LIP ID DROPLETS IS SURROUNDED BY THE MITOCHONDRIA, WILL BE INTERACTING OR WILL BE OXIDIZEING THE THREE LIST ERROLS FROM THE LIP ID DROPLETS. REMEMBER THAT I SAID THAT THIS HOLLOW IS A LOCALIZATION AND RELEASING THE FATTY ACIDS AND THIS WILL BE METABOLICICALLY CHANNELED WITH REOXIDATION, A AVOIDING THE DELUSION OR THE EL ELEVATION OF THESE FATTY ACIDS INTO THE CIYTOPLASMIC BULK. AND IN THIS WAY, TWO THINGS WILL BE DONE AT THE SAME TIME THAT ARE BENEFICIAL FOR THE CELL. ON THE ONE HAND, FROM AN ENERGY ENERGETIC POINT OF VIEW, BY MET METABOLICICALLY CHANNELING THE FATTY ACIDS THROUGH REOXIDATION IN THE MITOCHONDRIAL MATRICX, THAT WILL BE A MORE EFFICIENT WAY TO UTILIZE THE LIPIDS. AND ON THE OTHER HAND, THE FACT THAT THIS WILL AVOID THE PRE-FAT PRE-FATTY ACID ELEVATION OF THE CONCENTRATION IN THE CIYTOPLASM WILL RULE OUT THE POSSIBILITY OF TRIGGERING ROTH OVERFLOW FROM THE MITOCHONDRIA. AND IN THAT WAY, DAMAGING THE RE REDOX ENVIRONMENT OF THE CELL. SO, I CONCLUDE SAYING THAT THE MITOCHONDRIAL REDOX ENERGETIC IS -- LINK IS DAMAGED IN DIABETES AND THAT THAT FEN APHENOMENON UNDER UNDERLIES THE IMPAIRMENT IN TYPE TYPE-2 DIABETES DIABETES, LEADING TO AN OX DIZED INTRACELL INTRACELLULAR REDOX ENVIRONMENT. ON THE OTHER HAND, HYPERFLIES EM IA TRIGGERS A REDOX IMBALANCE, SHOOTING GLUCOSE TO THE PATH PATHWAYS, THAT CONSUME NAD BRPH AND PAR MUTATE ISN'T ABLE TO RE REMOBILE THE GLUCOSE FLAX SUM BY ACTIVATION OF THE PATHWAYS, IN INCREASING NAD BRPH AND GSH, BECAUSE IN ROTH HAS THAT'S COUNTED IN THE EFFECT OF HYPER HYPERGLYCOEMIA. AND THAT IN THE LONG TERM, ON THE HIGH ENERGETIC DEMAND FOR THE CONDITIONS, MITOCHONDRIA IN INCREASED DIRECT CONTACT WITH LIP ID DROPLETS AND INCREASE STORAGE UTILIZATION. LET ME FINISH, THANK YOUING MY COLLEAGUES. SEONIA CAR TESSA, WITH WHOM WE DEVELOPED THE INITIAL STAGES OF THIS PROJECT. TO THE CONSTANT SUPPORT AND ADVICE BY BRIAN ARUK. AND TO THE GROUP OF TALENTED AND HARD-WORKING GROUP -- PEOPLE. BRIAN STANLEY AND VIVA. THANK YOU. APPLAU [APPLAUSE] >> HI. YOU'RE RIGHT, I ENJOYED THAT. IN THE STUDIES WHERE YOU WERE GIVING THEM PULL MUTT AATE AND YOU WERE FEEDING THEM A HIGH-PULL MUTATE DIET, THE OTHER ANIMALS WERE GETTING THE SAME AMOUNT OF FAT AND WHAT KIND OF FAT WERE THEY GIVEN? >> WELL, IT'S THE DIET IS WITH NATURAL INGREDIENTS. AND YOU WILL -- WHEN I SAY THAT YOU SUBSTITUTE ONE FAT FOR THE OTHER IS IN THE PAL MUTATE RICH DIET THERE WILL BE PALM OIL INSTEAD OF >> SABINA: OIL. NO HIGH FAT. WE SUBSTITUTE CERTAIN LIPIDS BY PAR MUTATE. >> THANK YOU. >> MIGUEL, I HAVE A QUESTION FOR YOU. WHAT IS THE INTRAMATRICKX CONS CONCENTRATION OF GSA IN NON- NON-KINASE? >> IT IS ABOUT 1 TO 2. AND MITOCHONDRIA DOES NOT INTRODUCE PRODUCE. THEY JUST IMPORT IT FROM THE CIYTOPLASM, WHICH IN CARD YA MAIOMYO CARDIOMYOCITES, IS ABOUT 3 MILLI MILLIMORALS. >> OKAY, -- MOLEORS MOLARS. >> OKAY, GREAT. >> TOWARDS THE END -- INAUDIBLE [INAUDIBLE] >> WELL, IN THE MITOCHONDRIA WHAT HAPPENS IS I DIDN'T SHOW THE DATA. I WANTED TO SHOW ANOTHER VIEW. BUT WHAT HAPPENS, FOR INSTANCE, IF YOU REFERRED TO THE EFFECTS OF LIP ID PRECURSORS, WHAT HAPPENS IS THAT THE LIPIDS, WHEN THEY GO OVER THRESHOLD IN OTHER THINGS THAT THEY DO -- WELL, THEY DO SEVERAL THINGS. THEY HAVE A FUEL EFFECT ON THE MITOCHONDRIA AND THAT'S WHAT HAPPENS. THERE IS OXIDATION OF THE REDOX WHEN IT GOES OVER THE THRESHOLD. THERE IS ALSO LIPIDS -- ALSO AND THAT EFFECT IS INDEPENDENT OF OX OXIDATION. FOR INSTANCE, PAL MUTATE CO8 ACTS ON 1. AND THE EFFECT OF THE OTHER NINE NINE. SO WHEN YOU ARE BRO THE THRESHOLD -- BELOW THE THRESHOLD THRESHOLD, ANY OF THOSE EFFECTS HAPPENS. THE OTHER WAY AROUND. INSTEAD OF B -- ABOVE THE THRESHOLD, EMISSION DECREATIONS. >> -- DECREASES. >> TO WHAT EXTENT DO YOU THINK ISOLATING MITOCHONDRIA IS MEASURE -- AFFECTS WHAT YOU ARE MEASURING? >> CAN YOU REPEAT THE QUESTION? >> WHEN YOU ISOLATE MITOCHONDRIA AND YOU PERFORM THESE EXPERIMENTS, WHAT EXTENT DO YOU THINK THAT INFLUENCES WHAT YOU ARE MEASURING VERSUS IF YOU WERE TO DO -- >> OKAY. WELL, OF COURSE WE OBSERVED -- WHAT WE OBSERVED IS I SHOWED DATA FOR IMAGEING, FWHI WE SEE THAT THE DIABETIC WILD TYPES, WHEN THEY ARE EXPOSED TO HIGH ENERGETIC DEMAND, THEY WILL HAVE A MORE OXIDIZED -- AND ALSO, THAT WILL PRODUCE THE TYPES OF MITOCHONDRIA NOW WHEN YOU SUBJECT THEM TO THE ENERGY, WHEN YOU INCREASE ADP AND YOU INCREASE THE ENERGETIC DEMAND THAT THEY WILL RELEASE MORE ROTH ROTH. THE MECHANISM DISH DIDN'T TALK ABOUT THE MECHANISM, BUT WE HAVE BEEN STUDYING SEVERAL THINGS THAT ARE GOING ON IN THOSE MITOCHONDRIA. AMONG THEM, FOR INSTANCE, IT DEPENDS ON THE SYSTEM. FOR INSTANCE, WITH ROTH, UPMOD UPMODULATES ALL THE -- BUT THE MOUSE HAS -- INAUDIB[INAUDIBLE] OF COURSE, THE -- THE MITOCHONDRIA IS THE SORT OF HYPE THAOTHETICAL WAY OF ADDRESSING WHAT COULD BE HAPPENING IN THE CARD IOMIOCYTE. BUT IN THE MOUSE WHEN WE INK INCUBATEED THE CARD YA MIOMYOCITES, WE SAW AN IMPROVEMENT, AND WE SAW A DECREASE ALSO IN ROTH E EMISSIONS. AND WHEN WE DID THAT -- WHEN WE DID THAT INCUBATION IN ISOLATING MITOCHONDRIA FROM DIABETIC AN ANIMALS, WE SAW THAT THE GLUCO GLUCOKINANS IMPROVED THE REDOX ACTIVITY IN THE MITOCHONDRIA. SO THERE IS AN INTERPLAY BETWEEN THE TWO THINGS, THAT YOU CAN SOMEHOW -- INAUDIB[INAUDIBLE] FROM WHAT HAPPENS. >> OKAY, I THINK WE'D BETTER MOVE ON. THANK YOU, MIGUEL. OUR NEXT SPEAKER -- OH, YES. APPLAU [APPLAUSE] OUR NEXT SPEAKER IS WILLIAM MARS MARSTON LINEHAM, WHO IS CHIEF OF NEWER LODUROLOGICAL SURGERY AT THE NCI. HE'S HAD A LONG STANDING INTEREST IN IDENTIFICATION OF GENETIC BASIS OF THE CANCER AND KIDNEY AND HE AND HIS COLLEAGUES HAVE DEFINED THE BOTH METHODS FOR CLINICAL MANAGEMENT OF KID KIDNEY CANCER ASSOCIATED WITH HEREDITARY FORMS, INCLUDING CARC CARCINOMA, WHICH ARE CAUSED BY MUTATIONS OF THE HE ENZYMES HIGH DROGEN ANASE. WELCOME. >> THANK YOU. >> THANK YOU. >> THANK YOU VERY MUCH. WE' WE'RE RUNNING ABOUT 20 MINUTES LATE. SO I'VE BEEN ASKED -- I'M GOING TO SKIP OVER A FEW THINGS AND HOPE TO GET YOU ALL BACK A LITTLE BIT MORE ON TRACK. AS TRACEY MENTIONED, I'M A NEWER LODGEIC SURGEON HERE AT THE NCI. FOR 32 YEARS OR SO I'VE BEEN WORKING ON THE CANCER OF THE KID KIDNEY. KIDNEY CANCER. WHEN WE STARTED, WE THOUGHT IT WAS A SINGLE DISEASE. IT'S NOT A SINGLE DISEASE. IT'S MADE UP OF A NUMBER OF DIFFERENT TYPES OF CANCERS THAT HAPPEN TO OCCUR IN THE KIDNEY. THEY HAVE DIFFERENT HISTOLOGIES, DIFFERENT CLINICAL COURSES, RESPOND DEFENSIVELY THERAPY. AND AS I'LL SHOW YOU BRIEFLY A LITTLE BIT ARE CAUSED BY DIFFERENT GENES. WE KNOW OF THE 13 GENES, THIS IS A METABOLIC DISEASE. EACH OF THESE GENES AFFECT THE CELL'S ABILITY TO DETECT OXYGEN, IRON, GLUTRANSOR ENERGY. WHAT I AM GOING TO DO IS TALK WITH YOU BRIEFLY ABOUT THREE DIFFERENT TYPES OF KIDNEY CANCER CANCERS, IN WHICH WE KNOW THE GENES FOR THESE DISEASES. AND THESE GENES ARE ASSOCIATED WITH WITH ALTERED MITOCHONDRIAL FUNCTION. I'LL TALK FIRST ABOUT D SHELTH L, WHICH IS THE MOST COMMON TYPE OF KIDNEY CANCER. WE'LL ALSO TALK ABOUT WHAT TYPES OF -- TWO TYPES OF HERDDRY -- HEREDITARY CANCER THAT'S RIGHT CAUSED BY MUTATION. PER MUTASE AND HIREYDROJEN ATNATE. SO WE'LL START WITH D SHELTH L, HERD HEREDITARY KIDNEY CANCER SYNDROME IN WHICH A -- TUMORS ARE IN A NUMBER OF ORGANIS, INCLUDING THE KIDNEY. AS THEY GET BILATERAL, THESE ARE ALWAYS WHAT WE CALL CLEAR CELL KIDNEY CANCER AND WE SPECULATE THESE PATIENTS GET UP TO 600 TUMORS PER KIDNEY. SO WE MANAGE THESE PATIENTS BY WHAT'S CALLED ACTIVE SURVEILLANCE. WHEN THE LARGEST TUMOR GETS TO BE 3 CENTIMETERS, WE RECOMMEND SURGERY. WITH PATIENTS WHO -- WITH SMALLER TUMORS, WE DO ACTIVE SURVEILLANCE. WE DON'T OPERATE ON THEM. AND IN 28 YEARS, WE'VE NOT HAD ONE PATIENT DEVELOP MET ASTATIC DISEASE. I AM GOING TO SHOW YOU THAT THIS IS MASSIVELY DIFFERENT IN THE CANCERS THAT ARE CAUSED BY RECYCLED ENZYME MUTATION, VERY DIFFERENT FEPHENOTYPE. SO THE THEME FOR THIS IS CHROME CHROMOSOME 1. IT'S A D SHELTH L GENE, WHICH WE FOUND IN 1993. WE DETECTED MUTATION IN 170 FAMILIES, BASICALLY 100%. ASSOCIATE WITH -- WHEN WE LOOK AT CLEAR CELL KIDNEY CANCER, WE FIND MUTATION IN THIS GENE IN 90 90% OF BOTH. D SHELTH L IS BASICALLY THE GENE THAT IS HEREDITARY AND THE SPRATORADIC FORM OF CLEAR CELL KIDNEY CANCER CANCER. NOW D SHELTH L, THE PRODUCT GENE FORMS A COMPLEX WHICH TARGETS HYPE TOX IA UBIQUITIDATION. TARGET AND DEGRADE IT. ALL IN PATIENTS WHERE THERE ARE MUTATIONS IN D SHELTIH L, REGARDLESS OF WHETHER IT'S HIYPOXY, YOU CAN'T DEGRADE IT. IT OVERACCUMULATES. SO FOR THE PAST 21 YEARS, WE'VE SEEN THE APPROVAL OF 7 DRUGS NOW TARGETING OUR FIRST KIDNEY CANCER GENE PATH WWAY AND WE WERE THRILLED ABOUT IT BUT WE'RE STILL NOT CURING THESE PATIENTS WITH THIS THERAPY. THE QUESTION IS WHAT'S GOING ON HERE? WHY AREN'T WE CURING? SO WE AND OTHERS LOOKED -- THIS WAS FROM THE RECENTLY PUBLISHED P COMMERCIALICGA STUDY AND LOOKED AT KIDNE Y CANCERS IN SIGNIFICANTLY MUTATED GENES. OTHER THAN THESE, THE MOST COMMONLY MUTATED GENES ARE THESE THREE HERE THAT ARE CHROMATIN REMODELING GENES. NOW WHEN CHARLES SANITY ON, AN IN INCREDIBLE PUBLICATION IN THE NEW ENGLAND JOURNAL TWO YEARS AGO AND JUST RECENTLY IN "NATURE GENETICS," LOOKED AT VERY LARGE KIDNEY TUMORS AND FOUND SIGNIFICANT GENOMIC HERT HETEROGENEITY IN THESE TUMORS AND ALSO FOUND LATE THESE MUTE MUTATIONS THAT I JUST TALKED TO YOU ABOUT, THESE CHROMATIN RE REMODELING MUTATIONS. SO THIS, WE FOUND IN PCPA AND CHARLES SANITY ON FOUND IN THEIR STUDIES THAT THESE GENES ARE MUTATED IN LARGE TUMORS THAT ARE MIGRATED P HIGH-STAGE, LOW VIEFL -- SURVIVAL. SO THE BAD TUMORS. SMALL ONES BEHAVE WELL, AS I'VE TOLD YOU. LARGE ONES SPREAD AND ARE LETHAL LETHAL. NOW, THE OTHER THING WE SHOWED AFTER THE PAPER WAS THE FOLLOWING. THAT WHEN WE DID AN INTEGRATEIVE ANALYSIS LOOKING AT TUMORS THAT ARE BAD TUMORS, THAT ARE HIGH- HIGH-GRADE, LOW SURVIVAL, HIGH- HIGH-STAGE, DECREASE CYCLE FUNCTION, INCREASE GLY COCOLYSIS, WHAT'S CALLED REDUCTIVE CARB OXYL ATION, DEPENDENT CARB OXYL ATIOATION TO MAKE LIPIDS. IN OTHER WORDS, THIS IS A MODEL VERY SIMILAR TO THE MODEL I AM GOING TO SHOW YOU IN A MINUTE WITH THE TCA CYCLE MUTATION. SO THAT D SHELTH L CLEAR CELL, AGGRESSIVE TUMORS UNDERIGO A MET METABOLIC SHIFT, AROEROBIC GLY COL SIS AND DECREASE OXIDATIVE. NEXT I AM GOING TO SHOW YOU THIS TUMOR. THIS IS ANOTHER HEREDITARY CANCER SYNDROME CALLED HERDDRY CELL CANCER, HLRCC. THESE PATIENTS ARE AT RISK TO DEVELOP MUTTERINS AND VERY AGGRESSIVE FORMS OF KIDNEY CANCERS. THIS IS A HEADTRY CANCER SYNDROME. WE MICHELLE?{}LY DESCRIBED IN' 9 95 AND NOW GOES BY THE TERM HLRC HLRCC. THESE ARE ACTUALLY TUMORS IN THE ERECTOR PYOLIC. WHEN IT GETS COLD, THIS MUSCLE CONTRACTS AND THIS IS WHAT YOU GET. WHAT'S CALLED A LYOMIOMA. WE SEE THE EXACT SAME THING IN THE UTERUS. 92% OF THESE FAMILIES HAVE IT. AND THIS IS WHAT GOT US INTO THIS. THIS FIRST PATIENT WAS IN MAY OF 1989. I TOOK OUT THAT KIDOMY. SHE WAS A 15-YEAR-OLD AND CAME UP WITH HER MOM. I TOOK THAT OUT. SHE WENT ON TO DIE NINE MONTHS LATER OF MET ASTATIC DISEASE. HER MOM DIED AFTER THAT AND IT TOOK US 18 YEARS TO FIGURE OUT WHAT SHE HAD. SO THIS IS WHEN I SAW HER, WE SAW HERE -- HER. AND TWENTY TIME WE SAW HER AND SAW HER FIRST KCOUSIN 18 YEARS LATER, HER MOTHER DIED, HER MOM DIED AND UNCLE DIED, GRANDMOTHER AND GREAT AUNT DIED OF MET ASTAT METASTATIC KIDNEY CANCER AND SUB SUBSEQUENTLY WE LOST HER FIRST KCOUSIN. FOR YEARS WE HAD ABSOLUTELY NO THERAPY FOR THIS DISEASE. SECOND PATIENT I SAW, LARGE TUMOR. WE TOOK THIS OUTSIDE AND HE DIED 17 MONTHS LATER. THE PATIENT HAS SKIN BUMPS. HE CAME TO US HAVEWA VERY SMALL TUMOR. HAD AS A KRIS IN HIS LEFT KIDNEY AND INSIDE THAT CRAFT WAS A SMALL TUMOR. AND HE PRESENTED -- HE ALREADY HAD A LARGE NODE THAT WAS POSITIVE FOR MET ASTATIC CANCER. IN OTHER WORDS, THIS CANCER SPREAD WHEN IT'S SMALL. BLEW OUR MINDS. YOU DON'T SEE THAT IN KIDNEY CANCER. YOU DO IN THIS KIND OF KIDNEY CANCER. A VERY AGGRESSIVE TYPE OF TYPE-2 DIABETES PAP LAILLARY KIDNEY CANCER CANCERS. YOU GET A RANGE OF ACTIVE ACTIVE-LOOKING NUCLEI WITH A NUCLEAR LAR HALO. WE SEE IT IN 77-YEAR-OLDS. SO IT'S A PATIENT THAT CAME TO US WHO WAS 24-YEAR-OLD WHO CAME TO US WITH THIS. WE COULDN'T TELL IF IT WAS TUMOR OR NOT. MRI AND COULDN'T CALL IT. SHE CAME BACK NINE MONTHS LATER AND WE DID THIS. SAW THIS MASS IN HER LEFT KIDNEY KIDNEY. WHEN WE DID AN MRI, WE CALL THIS THE DOUBLE BUMP SIGN HERE AND WE SEPARATED -- OPERATED. SO WE'D BECOME INCREDIBLY NERVOUS ABOUT THESE TUMORS BECAUSE WE KNOW THAT THEY CAN IN INFILTRATE. SO WE WENT WIDE ON THIS SURGERY ON THIS YOUNG WOMAN. INSTEAD OF DOING THE KIND OF OPERATION YOU DO ON A PATIENT, WHERE WE CAN WATCH THEM AND DO ACTIVE SURVEILLANCE FOR YEARS UNTIL THEY REACH A CERTAIN SIZE AND OPERATE, HERE, YOU CAN'T DO THAT. THESE ARE SMALL AND WE TOOK OUT THE WHOLE LOWER HALF OF HER KID KIDNEY. SO SHE HAD A CYST, WHICH LOOKED ON IMAGEING LIKE A TUMOR INSIDE THE CYST. OSO OUR PATHOLOGIST SAID YOU'VE GOT TO LOOK AT THIS. THIS IS A 24-YEAR-OLD. AND SHE SAID YOU'VE GOT TO TAKE A LOOK AT THIS. SHE SAID THERE IS YOUR CYST AND INSIDE THAT YOU'VE GOT A VERY AGGRESSIVE TUMOR. AND SHE SAID LOOK AT THIS. THAT TUMOR HAS INFILTRATED UP INTO YOUR NORMAL KIDNEY. MARIA, DON'T TELL ME I'VE GOT A POSITIVE MARGIN HERE. SHE SAID MONTH. YOU'RE FINE. I SAID WE COULDN'T SEE THAT FROM THE IMAGEING? I SAID WE COULDN'T SEE IT. YOU'RE A SURGEON. SO WE NOW CAN HAVE COME TO UNDERSTAND THIS AS A VERY AGGRESSIVE CANCER AND VERY IN INFRILL -- VERY INFILTRATEIVE. AND IT COMES UP VERY QUICKLY. THIS IS IMAGING AND SHE SHOWED UP AT NORMAL IMAGEING AND A FEW YEARS LATER SHOWED UP WITH THIS BIG TUMOR, THESE NODES, THIS CANCER HERE AT 10 TO 59 LYMPH NODES WERE POSITIVE AND ARE NOW TREATING HER FOR MET ASTATIC DISEASE. HERE WE MANAGE THESE TWO VERY DIFFERENTLY. WE IMAGE THEM EVERY SINGLE YEAR AS LOANING AS THEY'RE ALIVE. AND WE DO VERY CAREFUL SURGERY. THIS GENE IS ON CHROMOSOME ONE. IT'S THE GENE FOR -- ANOTHER GENE I'LL SHOW YOU IN JUST A MINUTE. SO WE DETECT MUTATIONS IN THESE PATIENTS. WE HAVE FOUND MUTATION IN 179 FAMILIES. SO THIS IS A CLASSIC, WHAT YOU CALL TUMOR SUPPRESSOR GENE, WHERE YOU HAVE THE MUTATION. SO WE MAKE A -- THE 24-YEAR-OLD I SHOWED YOU, DO IT IN THE LAB AND PUT IT IN THE MOUSE AND THAT'S WHAT WE SEE, A TUMOR. OKAY. SO WE MAKE ONE CHANGE TO THOSE CELLS. WE PUT A NORMAL COPY OF HIRYDROIT IS A BACK IN, BOOM. WE SEE THE DEVELOPMENT OF A SMALL TUMOR OR NO TUMOR. AMAZING. SO WE WONDERED HOW ON EARTH COULD KREBS CYCLE CAUSE KIDNEY CANCER? SO OVER THE YEARS WE AND OTHERS HAVE SHOWN THAT, WHEN HUMMEROUS HIRYDROIT IS A IN IS KNOCKED DOWN, IT AFFECTS FOPHOSPHORYLATION. AND WHAT HAPPENS IN THE CELLS IS A VERY AGGRESSIVE CANCER. SO IT FLIPS TO AROEROBIC GLY COL JISHGS USING A WHOLE LOT OF GLUCOSE. SO YOU CAN MEASURE THAT. AND THESE CELLS TAKE UP VERY LITTLE OXYGEN, ABOUT 20% NORMAL COMPARED TO THESE RESTORED AND H HH 2 CELLS. THEY SWITCHED TO AROEROBIC FLY COL SIS AND THEY ARE USING JUST GLUCOSE. THEY ARE VERY DEPENDENT ON GLUCOSE. YOU PUT THEM IN THERE IN A LOW- LOW-GLUCOSE ENVIRONMENT AND THEY DIE. NOW, I AM GOING TO SKIP OVER THIS BECAUSE WE'RE SHORT OF TIME. SO WE CAN USE THAT TO IMAGE THESE PATIENTS. AND THIS IS A PET SCAN, WHICH IN OTHER TYPES OF KIDNEY CANCER CLEAR CELLS, IS NOT USUALLY VERY POSITIVE. THIS DETECTS GLUCOSE TRANSPORT. SO WE DO A PET SCAN AND WE CAN PICK UP EVERY ONE OF THESE TUMORS. IT'S AMAZING WITH THIS. THIS WAS INITIAL LY CALLED NORMAL ON A CT SCAN HERE IN THE LIVER. WE WERE STILL WORRIED ABOUT THIS TO REGULATE -- PET SCAN. HE HAD A BIG TUMOR IN THE LIVER HERE. WE COULDN'T TELL IF TESTIMONIES IT WAS TUMOROUS OR NOT. ON THE PET SCAN WE SURE COULD. THE TUMOR IN THERE. THIS PAISHT HERE I SHOWED YOU WITH A SMALL TUMOR OF THE SKIN, CUTANEOUS. IT BARELY EVEN PICKED UP, AS OUR LADIES, WHEN WE LOOK AT THESE UTERUS. SO WHAT HAPPENS HERE IS THE CYCLE GETS REPROGRAMMED STEWITO GO COUNTERCLOCK WIWISE IN A WAY AND YOU -- THEY BECOME DEPENDENT ON GLUT AMINE ON CARB OOXYL ATIOATION WHETHER IT GETS AGGRESSIVE. WE ALSO SHOWED AND THEN MORE RECENTLY WITH TRACEY, THAT WHEN IT GOES UP, IT THEN -- WE TALKED A MINUTE AGO ABOUT D SHELTH L TARGET TARGETING DEGENERATION. IT'S HARD TO BASICALLY -- WHAT HAPPENS IS IT ACCUMULATES. AND IT DERIVES TRANSCRIPTION GENES LIKE GROWTH FACTOR AND THE GLUCOSE TRANSPORTERS. WELL, FOR THIS VERY AGGRESSIVE CANCER CELL, WHICH IS VERY DEPENDENT ON GLY COCOLYSIS, THIS IS PERFECT FOR THIS CELL, THE PERFECT STORM FOR THIS CELL. SO WE DEVELOPED A THERAPEUTIC A APPROACH TARGETING THIS PATH WWAY. SO CONDUCTED THE TARGET WITH A GLUCOSE TRANSPORT AND GLY COCOLYSIS GLYCOLYSIS. AND RECALL THAT WE LOST EVERY ONE OF THESE TO ADVANCED DEVELOPED DISEASE. THIS WAS A PRIVATE TRIAL. WE NOW HAVE A FORMAL PHASE 3 TRIAL. AND WE ARE SEEING VERY DRAMATIC RESPONSES IN THESE PATIENTS. THIS IS ONE OF THE FIRST PATIENTS WE SAW. AND HE HAD A VERY NICE RESPONSE FOR 18 MONTHS. THIS WOMAN CAME TO US, 42 42-YEAR-OLD WOMAN FROM UP NORTH. BOTH HER SISTERS HAD DIED OF MET METASTATIC KIDNEY CANCER, AS DID HER FATHER. SHE CAME TO US WITH ADVANCED DISEASE. WE PUT HER ON THIS THERAPY, THIS PATH WWAY, AND AT 3 MONTHS SHE WAS -- WE HAVE NOT YET SEEN HER. WE SAW HER LAST SPRING SEVEN YEARS OAT -- OUT. WE CANNOT DETECT DISEASE. SO IN THE INTEREST OF TIME, I AM GOING SKIP OVER THIS BECAUSE YOU HAVE ANOTHER THING COMING ON. JUST VERY BRIEFLY, THERE IS ANOTHER KREBS CYCLE ENZYME CANCER THAT IS VERY SIMILAR TO H HLRCC. IT'S HIRYDROJEN ATNATE. THESE PARITIES GET KIDNEY CANCER CANCER, AND WE CAN SEE THESE FAMILIES WITH KIDNEY CANCER ARE JUST KIDNEY CANCER. THIS IS WHAT YOU SEE IN THESE PATIENTS, YOUNG PEOPLE. THESE TUMORS ALSO ARE VERY ACTIVE. THEY CAN SPREAD. THIS IS 1.8 CENTIMETER TUMOR N THIS LADY IT SPREAD TO HER LIVER LIVER. THAT LADY DIVED THIS DISEASE, 33 33-YEAR-OLD. SAME THING. WE TOOK THIS OUT AND SHE WENT ON TO DIE. BUT WE MADE A CELL LINE FROM THAT TUMOR. AND YOU SEE A VERY SIMILAR PICTURE HERE. THEY SWITCH TO AROEROBIC GLY COLL GLYCOLYSIS. HERE, THIS IS COMPLEX 2. SO HERE, WE DETECT ALMOST NO OX OXYGEN UPTAKE. AND THEY ARE DOING A WHOLE LOT OF FLY COGLYCOLYSIS. ALSO, THESE ARE ALL CHARACTER CHARACTERIZED BY REDUCTIVE CARB OX IL ATILATION. AND A SIMILAR STORY WHERE IT IS TREATED. ALSO AFFECTS THE HIGH DREDOG NASE. AND I MIGHT ALSO ADD, JUST ADD A LITTLE CLOSURE, THIS TUMOR, THIS PATIENT AND THIS FAMILY AND THIS VERY TUMOR IS RIGHT SMACK IN THE MIDDLE -- WE TALKED ABOUT THE IY RCH IYR REGION OF SV 8B. THIS DOESN'T HAVE THAT THERE ARE WHERE YOU WOULD PREDICT. THAT WOULD AFFECT THE HSD 20 AND THE COCHAP PER ONONE BINDING. SO IT GIVES A LITTLE -- OUTCOME OF THAT. SO WHAT I'VE SHOWN HERE IS KID KIDNEY CANCERS AND A NUMBER OF CANCERS AND THAT IN CLEAR CELL KIDNEY CANCER, IT'S AGGRESSIVE, WHEN IT'S LARGE, HIGH-GRADE, HIGH-STAGE, LOW-SURVIVAL, THE TUMORS UNDERGO A METABOLIC SHIFT AROEROBIC GLY COLCOLYSIS AND DECREASE FOPHOSPHORYLATION. IN THE TUMORS THAT ARE CAUSED BY THE HUMAN ER ATATE HIGH DREDGOG NASE IN PATIENTS, THOSE WERE MORE AGGRESSIVE. THOSE ARE BORN WITH DECREASED FOPHOSPHORYLATION FLY COLGLYCOLYSIS. AND THIS IS AN ASPECT OF CANCER THAT WAS DESCRIBED IN 1924 BY THE GREAT BERG OF THE BLUEBERG EFFECT AND IT IS TRULY AN EXTREMELY AGGRESSIVE FEPHENOTYPE FOR THESE CANCERS. AND IT IS OUR HOPE THAT BY UNDERSTANDING THE IMPAIRED MITOCHONDRIAL FUNCTION AND KID KIDNEY CANCERS WILL HELP PROVIDE THE FOUNDATION FOR THE DEVELOPMENT OF EFFECT YIIVE FORMS OF THERAPY FOR PATIENTS WITH THESE CANCERS. I WANT TO ACKNOWLEDGE MY COLLEAGUES IN OUR LABORATORIES. OUR COLLEAGUES IN THE OGOLOGY GRANTS, MEDICINE PROGRAMS. COLLEGE OF LONG TERM -- MARIA MORINO, WHO DID IMAGE. AND PETER CRICE AT SOUTHWESTERN AND THE UNIVERSITY OF KEN TUTUCKY. THANK YOU VERY MUCH. APPLAU [APPLAUSE] >> VERY INTERESTING TALK. I HAVE A QUESTION FOR YOU. >> WHY DON'T WE TAKE QUESTIONS AFTERWARDS. WE CAN CHAT. I KNOW YOU HAVE TO GO. YOU'VE GOT THE TALK AT 3:00, SO WHY DON'T YOU AND I CHAT? >> OKAY. >> THANK YOU ALL VERY MUCH. APPLAU [APPLAUSE] >> THANK YOU ALL VERY MUCH FOR COMING TO THE -- TO THIS SIM POSEIUM. AND -- SIM POYMPOSIUM. WE'RE ABOUT 10 MINUTES LATE JOB, WE'VE GOT TO GET OUT OF HERE SO THAT DOUG CAN GET UP FOR HIS WEDNESDAY AFTERNOON LECTURE. THE POSTER SESSIONS ARE OPEN. I THINK THERE ARE SOME REFRESHMENTS THERE AND SHOW BACK UP HERE AT 3:00 FOR THE WEDNESDAY AFTERNOON LECTURE. THANK YOU ALL VERY MUCH. SEE YOU NEXT TIME. >> GOOD AFTERNOON, EVERYONE. WELCOME BACK TO THOSE WHO HAVE BEEN HERE FOR MUCH OF THE DAY, A WONDERFUL SYMPOSIUM ON MITOCHONDRIAL BIOLOGY AND DISEASE. WELCOME TO THOSE WHO HAVEN'T BEEN HERE ALL DAY & HAVE COME FOR THE WEDNESDAY AFTERNOON LECTURE BY DR. DOUGLAS WALLACE A MITOCHONDRIAL ETIOLOGY OF METABOLIC AND DEGENERATIVE DISEASES, CANCER AND AGING. I'VE HAD THE PLEASURE OF KNOWING DOUG SINCE WE'VE SHARED INTERESTS IN THE FIELD OF HUMAN GENETICS. HE GOT HIS UNDERGRAD DEGREE AT CORNELL, TWO YEARS IN THE PUBLIC HEALTH SERVICE, HERE? NO, I WAS HOPING YOU WERE IN THE NIH GANG OF THE YELLOW BERETS, BUT WENT ON TO GET HIS Ph.D. AT YALE. AFTER THAT, HE SPENT SEVEN YEARS AT STANFORD, I GUESS HE'S ONE OF THOSE PEOPLE WHO GETS RESTLESS, ENDED UP AT EMORY WHERE HE WAS FOR A SIGNIFICANT PERIOD OF TIME IN A PRODUCTIVE PERIOD OF TIME, ALMOST 19 YEARS, THEN MOVED TO UNIVERSITY OF CALIFORNIA, IRVINE, AND MOST RECENTLY FOUR YEARS AGO HAS MOVED TO THE UNIVERSITY OF PENNSYLVANIA AND CHILDREN'S HOSPITAL OF PHILADELPHIA WHERE HE HAS PERHAPS ONE OF THE LONGEST TITLES I'VE SEEN IN A WHILE, MICHAEL AND CHARLES BARNETT ENDOWED CHAIR IN PEDIATRIC MITOCHONDRIAL DISEASE. HE HAS DEFINED THE FIELD OF HUMAN MITOCHONDRIAL GENETICS, MITOCHONDRIAL DNA, BACK THERE A FEW YEARS AGO DEMONSTRATING THE VERY FIRST EXAMPLE OF A DISEASE THAT IS INHERITED BECAUSE OF MUTATIONS IN MITOCHONDRIAL DNA AND THEREFORE INHERITED THROUGH THE MATERNAL LINE. A LONG LIST OF OTHER ACHIEVEMENTS FOR WHICH HE'S BEEN RECOGNIZED BY ELECTION TO THE NATIONAL ACADEMY OF SCIENCES, INSTITUTE OF MEDICINE, AND MOST RECENTLY RECEIVED THE HIGHEST PRIZE GIVEN IN GENETICS, THE GRUBER FOUNDATION GENETICS PRIZE IN 2012. WE'RE FORTUNATE TO HAVE HIM HERE TO TALK TO US THIS AFTERNOON. PLEASE JOIN ME IN WELCOMING DR. DOUG WALLACE. [APPLAUSE] >> I WANT TO REALLY THANK YOU, FRANCIS, FOR BEING HERE, FOR THE GRACIOUS INTRODUCTION. THANK YOU VERY MUCH, STEVE, FOR ALL THE EFFORT YOU PUT IN HAVING THE FABULOUS SPEAKERS WE HEARD TODAY, FOR ALL OF YOU FOR BEING HERE THIS AFTERNOON. WE'RE AT AS FRANCIS MENTIONED TO ME A UNIQUE TIME. THERE'S NO TIME IN WESTERN MEDICINE, BIOMEDICAL SCIENCE WITH MORE TOOLS AND MORE OPPORTUNITIES TO MAKE REAL IMPACT ON THE HEALTH OF OUR SOCIETY, AT THE SAME TIME WE'RE LOOKING AT CONTINUAL DECLINE IN THE SUPPORT FOR OUR SCIENCES, WITH PARTICULAR ANXIETY AND CONCERN FOR THE FUTURE OF OUR YOUNG SCIENTISTS WHO OF COURSE ARE THE FUTURE FOR THIS FIELD. AND WE HAVE TO ASK OURSELVES AT THIS POINT WHY IS IT THAT OUR SOCIETY IS TURNING AWAY FROM THE CONCEPT THAT BIOMEDICAL RESEARCH COULD ACTUALLY BE A MAJOR ASSET TO THEM, WHY ARE THEY THEN PULLING BACK FROM THEIR SUPPORT? AND I THINK THE REASON FOR THAT IS THAT UNFORTUNATELY, THERE IS AN INVERSE CORRELATION BETWEEN THE FREQUENCY OF THE DISEASES THAT IMPACT OUR SOCIETY, AND OUR ABILITY TO DIAGNOSE THEM EFFECTIVELY AND TREAT THEM. AND THIS MEANS THAT FOR THE VAST MAJORITY OF THE AMERICAN CITIZENS AND ALSO FOR THOSE THROUGHOUT THE WORLD THERE'S A GROWING DISSOLUTIONMENT WITH WHAT MEDICINE CAN DO, SINCE THESE ARE THE VERY PROBLEMS THAT THEY ARE CONFRONTED WITH. SO HAVING THIS PROBLEM IN FRONT OF US WHICH I THINK WE MUST ADMIT IS TRUE WE HAVE TO ASK OURSELVES THEN WHY IS IT WITH ALL THE EFFORT THAT YOU ALL ARE PUTTING IN AND WITH ALL THE SUPPORT THAT GOVERNMENTS AND PRIVATE INDUSTRY HAVE PROVIDED THROUGHOUT THE WORLD, WHY HAVEN'T WE BEEN ABLE TO DO BETTER? WHY HAVEN'T WE CURED A SINGLE CASE OF ALZHEIMER'S DISEASE? WHY IS DIABETES INCREASING? WHY IS OBESITY AN EPIDEMIC, ET CETERA, ET CETERA. AND THERE WAS A PHILOSOPHER OF SCIENCE, TIMUS KUHN WHO PROPOSED A NUMBER YEARS AGO WHEN SCIENTIFIC EFFORT IS INVESTED MORE AND MORE HEAVILY IN TRYING TO SOLVE SOME PROBLEMS, AND YET PROGRESS SEEMS TO NOT BE MOVING FORWARD AT THE RATE YOU WOULD EXPECT, IT MAY BE TIME TO ASK, WHAT ARE THE BASIC PREMISES ON WHICH THE EVERY DID HE EVER DID HE ENDEAVO RS ARE ADDRESSED AND ARE THEY THE RIGHT ONES AND WHAT ARE THE PROBLEMS WE'RE CONCERNED ABOUT? THERE ARE ALL THE NEURO PSYCHIATRIC DISEASES, BEING IN PEDIATRICS, I WENT TO LOOK AT PEDIATRIC SEDUCES, ONE OF THE MOST OBVIOUS IS AUTISM, NOW ASSUMED TO BE ONE IN 88 BOYS BEING AFFECTED, OR ALZHEIMER'S, PARKINSON'S, MIGRAINE, DEPRESSION, SCHIZOPHRENIA, OBSESSIVE COMPULSIVE, MYALGIA, CHRONIC DISEASE, VISCERAL DISEASES, GASTROINTESTINAL, OR METABOLIC, TYPE 2 DIABETES, OBESITY, HYPERTENSION, INFLAMMATORY DISEASE, LUPUS, CANCER AND AGING, ALL OF THESE ARE IN FACT THE MAJOR CONCERNS WE HAVE AND YET THEY ARE THE ONES THAT WE SEEM TO BE LEAST ABLE TO ADDRESS. IF KUHN IS RIGHT THAT MAYBE THERE'S SOMETHING WRONG WITH HOW WE APPROACH, WE SHOULD ASK WHAT ARE THE BASIC ASSUMPTION? I'D LIKE TO PROPOSE WESTERN MEDICAL PHILOSOPHY HAS BEEN BASED ON TWO BASIC IDEAS, ONE THAT GOES BACK TO A MAN NAMED VISALIUS 500 YEARS AGO IN IT EL, THE FIRSITALY,THE FIRST TO DEFINE THE ANAT OMY OF THE HUMAN BODY, A BREAKTHROUGH, AND ALL FUTURE PHYSICIANS AND FUTURE INVESTIGATORS THEN BEGAN TO SPECIALIZE IN DIFFERENT PARTS OF THE BODY. SO NOW TODAY WE HAVE OPHTHALMOLOGISTS, NEPHROLOGISTS, ALL OR BEGAN-SPECIFIC SPECIALTIES. IT LED TO AN UNSPOKEN COROLLARY, IF YOU HAVE A HEADACHE, YOU GET REFERRED TO THE NEUROLOGIST BECAUSE THE ASSUMPTION IS THERE'S SOMETHING WRONG WITH YOUR HEAD. THAT IS THE IDEA IS IF YOU HAVE A SPECIFIC TISSUE-SPECIFIC SYMPTOM THIS MUST BE DUE TO A TISSUE-SPECIFIC DEFECT. THE OTHER IDEA GOES BACK TO GREGOR MENDEL, OBSERVED A SUBSET FOLLOWED A PATTERN WHERE IT SEEMED LIKE THE ADULT PLANT HAD TWO COPIES OF SOMETHING, EACH SEX CELL GOT ONE OF THOSE COPIES, AND THEN OFFSPRING GOT TWO COPIES, WE CALL THAT THE LAWS OF MENDELIAN INHERITANCE. THE TURN OF THE CENTURY, STUDYING THE ANATOMY OF THE FRUIT FLY, ALMOST EVERY ONE OF THEIR ANATOMICAL PROBLEMS WAS INHERITED ACCORDING TO THE PATTERN AND SOON LEARNED THE CHROMOSOMES FOLLOWED THOSE SAME IDEAS AND THAT LED TO WHAT WE CALL THE MENDELIAN LAWS OF INHERITANCE. THE IDEA THERE'S A LAW OR LAWS OF INHERITANCE HAS AN UNSTATED COROLLARY, IF SOMETHING IS TRANSMITTED IN A FAMILY ACCORDING TO THE LAWS OF MENDEL, GENETIC. IF NOT, IT'S ENVIRONMENTAL. THE TWO IDEAS HELD US IN GOOD STEAD, WE MADE PROGRESS, BUT MAYBE THEY EXHAUSTIONED SOM EXHAUSTED THEI R CAPACITY TO SERVE US BECAUSE ALL OF THE MENDELIAN GENES ON CHROMOSOMES ARE WHERE THE ANATOMICAL GENES ARE, THERE WAS AN INTERN CONSIS CONSISTENCY. LIFE IS NOT JUST ABOUT ANATOMY, IT'S ABOUT BEING ANIMATED. IN FACT, YOU'RE THE MOST ANIMATED THING IN OUR KNOWN ENVIRONMENT. AND NEWTON INDICATED, AGAIN HALF A MILLENIA AGO, ANY KIND OF MALTER WAS INANIMATE UNLESS IT WAS ACTED ON BY ENERGY. YOU'RE HIGHLY ANIMATED, ONE OF THE MOST IMPORTANT THINGS ABOUT YOU IS THE FLOW OF ENERGY. SO THEREFORE WE CAN REALIZE LIFE IS REALLY INTERACTION OF ANATOMY, ENERGY AND INFORMATION FOR ANATOMY AND INFORMATION FOR ENERGY. ONCE WE REALIZE ENERGY IS SOMETHING WE SHOULD ALSO BE LOOKING AT, THAT ENWE CAN REALIZE THAT DIFFERENT ORGANS RELY ON ENERGY TO DIFFERENT EXTENTS. SO YOU COULD THEN IMAGINE A SITUATION WITH A PARTIAL ENERGETIC DEFECT AND IT WOULD SPECIFICALLY AFFECT THOSE ORGANS THAT HAD THE HIGHEST ENERGY DEMAND. AND THAT IN FACT TURNS OUT TO BE THE BRAIN, HEART, MUSCLE, RENAL AND ENDOCRINE SYSTEMS, AND SOME ARE NOT INHERIT AND NOT ACCORDING TO THE LAWS OF MENDEL AND THEREFORE THIS WHOLE ASPECT OF INHERITANCE HAD BEEN OVERLOOKED FROM A CLINICAL POINT OF VIEW. SO NOW WE REALLY HAVE BASICALLY FOUR SETS OF PARADIGMS, WHEN WE ADD THESE TOGETHER I THINK, I HOPE TO BE ABLE TO CONVINCE YOU WE'LL HAVE A MORE COHERENT VIEW OF THE DISEASE PROCESS. SO WHERE DID THIS DICHOTOMY OF ENERGY AND ANATOMY COME FROM? IN A SYMBIOSIS TWO BILLION YEARS AGO BETWEEN A BACTERIA AND AN OXIDATIVE BACTERIA, THE ALPHA PROTEAL MATERIAL THAT CAME TOGETHER TO FORM A RELATIONSHIP, THAT ULTIMATELY BECAME TIGHT ENOUGH THAT THEY MERGED TOGETHER. THE GENOME WHICH WAS THE SAME SIZE AS THE ORIGINAL BACTERIA BEGAN TO ACCUMULATE GENES BECAUSE BACTERIA EXCHANGE GENES. IT'S WHAT THEY DO. THERE WAS A GENE FLOW FROM THE OXIDATIVE BACTERIA TO THE METHIANOGEJ NUCLEUS. WHY WOULD THERE BE A GENE CELL? TURNS OUT THAT IT TAKES ABOUT ONE BACTERIA SIZE CELL'S ENERGY TO REPLICATE ITS DNA, TRANSCRIBE INTO RNA AND TRANSLATE INTO PROTEIN. THE REASON BACTERIA DON'T HAVE BIGGER GENOMES IS THEY CAN'T MAKE ENOUGH ENERGY. ENERGY IS LIMITING THROUGH THE BIOENERGETIC PROCESS. THROUGH THE BIOSYNTHETIC PROCESS. IF ALL THE BACTERIA WOULD USE UP ALL THE ENERGY, THERE WOULD BE NO ADVANCES. BUT IF IN FACT A GENE FROM THE THE BACTERIUM WAS TRANSFERRED TO THE NUCLEUS, INSTEAD OF HAVING TO HAVE A THOUSAND COPIES, ONE IN EVERY BACTERIUM, YOU COULD HAVE ONLY TWO. NOW THOSE TWO IN THE NUCLEUS COULD BE REPLICATED, TRANSCRIBED, TRANSLATED WITH A THOUSAND-FOLD SAVINGS OF ENERGY. BY THIS TRANSFER THEN OF GENES FROM THE MITOCHONDRIA TO THE NUCLEUS, THERE WAS A TREMENDOUS ENERGY SAVINGS. AND THAT ENERGY SAVINGS THEN CREATED ENOUGH RAW ENERGY TO ALLOW THE NUCLEUS TO ACCUMULATE 25,000 GENES AND THUS ALLOW MULTI-CELLURITY TO OCCUR FOR ARMS, LEGS, QUESTIONS. WE HAVE TWO ORGANISMS THAT HAVE UNDERGONE A SPECIALIZATION, SPECIALIZED IN ANATOMY AND IN ENERGY. HOW DOES THIS ENERGY FLOW THROUGH OUR CELLS? FOR US THAT LIVE ON THE SURFACE OF THE EARTH, HIGH ENERGY PHOTONS IMPINGE AND TAKES THE ENERGY FROM THE SUNLIGHT AND SPLITS WATER INTO HYDROGEN AND OXYGEN, RELEASES OXYGEN IN THE ATMOSPHERE AND THEN THE PLANT THEN USES THAT HYDROGEN CONDENSED WITH CARDON T CARBON TO GIVE YOU SUGAR. WE GET THE ENERGY OF SUNLIGHT IN THE STARCH IN THE PLANTS, WHY WE CLEARED THE MIDWEST. WE EAT THE STARCH, IT GOES INTO OUR CELLS, SPLIT INTO TWO, THE MITOCHONDRIA STRIPS THE HYDROGEN TO GIVE YOU THE WATER BACK AND RELEASE ENERGY IN CHEMICAL FORM AS ATP, KINETIC FORM AS HEAT. THE IMPORTANT POINT, ALL ENERGY FLOW IS NOT THROUGH THE NUCLEAR CYTOCOLIC ORGANISM BUT BACTERIA. UNTIL WE UNDERSTAND THE FLOW, WE CAN'T UNDERSTAND THE PATHOPHYSIOLOGY OF COMPLEX DISEASE. SO THIS IS MITOCHONDRIAL BIOCHEMISTRY LESSON FROM HELL. I NEVER HAD A MEDICAL STUDENT THAT LIKED IT BUT IN FACT I LOVE IT AND THEREFORE YOU'LL BE TORTURED. WE HAVE AN OUTER MEMBRANE, INNER MEMBRANE SPACE, MATRIX, THIS IS THE MITOCHONDRIA, GLUCOSE HAS GONE THROUGH GLYCOLYSIS, THE CYCLE STRIPS THE HYDROGEN OFF THE CARBON AND PUTS THEM INTO THIS CARRIER, NAD, TO GIVE YOU THE REDUCED FORM, NADH. WE'RE GOING TO BURN THOSE WITH OXYGEN YOU'RE BREATHING, ELECTRONS FLOW DOWN THE WIRE, REDUCING TO A MOLECULE OF WATER. THE PRISO PROBLEM WITH ELECTRON FLOW, IT'S DIFFICULT TO STORE THE ENERGY BECAUSE IT'S KINETIC. HOW DID MOTHER NATURE SOLVE THIS PROBLEM? SHE SOLVED THAT BY CREATING A CAPACITOR, IT PUMPS POSITIVE CHARGES TO THE INSULATOR INTO THE MEMBRANE SPACE TO GIVE YOU POSITIVE ON THE OUTSIDE, ALKALINE AND NEGATIVE ON THE INSIDE, THE GREAT INSIGHT OF PETER MITCHELL. THIS STORED ENERGY CAN THEN BE USED FOR LOTS OF PURPOSES, ONE OF WHICH CAN BE USED TO ADP INTO ATP WHICH CAN BE EXCHANGED, GOES THROUGH THE VOLTAGE CHANNEL AND THE ATP CAN DO WORK. WE'VE COUPLED OXIDATION WITH PHOSPHORYLATION. NOW EVERYBODY IN THIS ROOM HAS A DIFFERENT EFFICIENCY AT OX-PHOS. SOME ARE EFFICIENT AND CONVERTING HYDROGEN INTO ATP, TIGHTLY COUPLED. EVERY CALORIE YOU EAT IS ONE UNIT OF HEAT, THE MOST EFFICIENT PEOPLE EAT THE LESS LEAF CALORIES FOR MAXIMUM AMOUNT OF WORK AND GENERATE LEAST AMOUNT OF HEAT. OTHER PEOPLE ARE LESS EFFICIENT, AND THESE PEOPLE ARE LOOSELY COUPLED, AND THEREFORE TO MAKE THE SAME AMOUNT OF ATP THEY HAVE TO EAT MORE CALORIES. BY BURNING MORE CALORIES THEY ARE MAKING PROPORTIONALLY MORE HEAT PER UNIT WORK. THIS COUPLING EFFICIENCY AS I'M GOING TO ARGUE IS A VERY, VERY IMPORTANT COMPONENT OF THE VARIATION OF PEOPLE IN THE ROOM AND IN THE WORLD. NOW, THIS SYSTEM IS LIKE ANY FURNACE, IT HAS INCOMPLETE OXIDATION, COMBUSTION, THAT'S PERSONIFIED BY THE ELECTRONS AND COMPLEX ONE AND THREE, BEING ABLE TO DONATE DIRECTLY TO O 2 TO GIVE YOU AN UNPAIRED ELECTRON WHICH WANTS TO OXIDIZE LIPIDS, PROTEINS AND DNA, TWO WILL BE DISMUTATED INTO HYDROGEN PEROXIDE, YOU CAN GET ANOTHER ELECTRON TO GIVE YOU A RADICAL WHICH AN UNPAIRED ELECTRON, OXIDIZING YOUR MITOCHONDRIA AND YOUR CELLS, THE OXYGEN RADICALS. BUT YOU'LL ALSO SEE THEY ARE CRITICAL IN SIGNALING. THE MITOCHONDRIA BEING A CAPACITOR PICKS UP POSITIVE CHARGE REGULATING CALCIUM AND PERMEABILITY TRANSITION PORE, SELF DESTRUCT SYSTEM, NORMALLY A CLOSED DOOR, MAINTAINING THE MEMBRANE PR POTENTIAL BUT WHEN IT DECLINES, CALCIUM OVERLOAD BECOMES HIGH, OXIDATIVE STRESS IS HIGH, IT GOES FROM CLOSED DOOR TO OPEN CHANNEL, SHORTS THE CIRCUIT, MEMBRANE POTENTIAL, THE STRENGTH OF THE MATRIX CAUSES FLUIDS TO FLOW IN, INNER MEMBRANE SWELLS, BAX AND BAT FORM A NEGATIVE A CHANNEL, RESTORING PROTEIN, DEGRADING THE NUCLEUS AND CYTOSOL WHEN MITOCHONDRIAL ENERGETICS IS IMPAIRED. THE MITOCHONDRIA GENERATION URGES ENERGY, REGULATES REDOX, AT HIGH LEVELS IS DAMAGING, REGULATES CALCIUM, CELL DEATH, REGULATES THINGS LIKE ATP. BASICALLY THEN WHAT WE CAN THINK OF THE CELL AS TWO LIFE FORMS, NUCLEAR CNTOSOLIC LIFE FORMS, GENES IN THE NUCLEUS, AND ABOUT ONE TO TWO THOUSAND OF THE NUCLEAR ENCODED PROTEINS WERE ORIGINALLY FROM THE MITOCHONDRIAL DNA AND ARE NOW MADE ON THE CYTOSOL AND TRANSPORTED TO MAKE THE ANATOMY OF THE MITOCHONDRIA. THESE ARE TRANSLATED ON MITOCHONDRIAL-SPECIFIC RIBOSOMES, SENSITIVE, AND THESE 13 PROTEINS ARE SEVEN OF THE 45 PROTEINS OF COMPLEX 1, ONE OF THE 11 PROTEINS OF COMPLEX 3, 3 OF THE 13 PROTEINS OF 4, TWO OF THE 17 OF COMPLEX FIVE. YOU MIGHT ASK, IF YOU PUT 2000 GENES IN THE NUCLEUS WHY KEEP 13? THE ANSWER, I BELIEVE, COMES FROM THE FACT THAT EVERY ONE OF THE PROTEINS IS COMPLEX 1, 3, 4 AND 5. AND WHAT DO 1, 3, 4 AND 5 HAVE IN COMMON? THE MEMBRANE POTENTIAL. SO THESE PROTEINS THEN ARE THE WIRING DIAGRAM OF THE POWER PLANT. IF ANY ONE OF THESE COMPLEXES BECAME LEAKY FOR PROTON, IT WOULD SHORT THE CAPACITOR, ONCE THAT COLLAPSES YOU LOSE YOUR POTENTIAL FOR ENERGY, STOP BREATHING, BECOME INERT, WE CALL THAT DEAD. THIS THEN IS CRITICAL THIS SYSTEM BE MAINTAINED, AS ALL OF THESE ENZYMES HAVE TO CO-EVOLVE AND BE BALANCED TOGETHER. SO HOW DOES THAT OCCUR? WELL, THE PROBLEM THEN IS YOU CAN'T HAVE RECOMBINATION, BECAUSE IF WE MIXED AND MATCH MITOCHONDRIAL DNA FROM ANY TWO OF YOU, YOU HAVE SLIGHTLY DIFFERENT CIRCUIT DIAGRAMS, WE GET A MISMATCHED CIRCUIT THAT WOULD SHORT. SO IN FACT WE CANNOT ALLOW RECOMBINATION. THAT'S WHAT THE NUCLEUS DOES. SO MOTHER NATURE HAD TO SOLVE THIS PROBLEM. BEING A WOMAN, SHE SOLVED THE IN A LOGICAL SAY WEIGHING THE MITOCHONDRIAL DNA WOULD BE INHERITED ONLY FROM THE MOTHER, SO IT'S INHERITED TO HER CHILDREN, DAUGHTERS TO THEIR CHILDREN, A MALE'S MITOCHONDRIAL ARE SELECTIVELY THROWN OUT AND DESTROYED. MEN HAVE BEEN THROWN OUT FOR TWO BILLION YEARS, DON'T FEEL BAD. [ LAUGHTER ] EACH CELL THEN HAS THOUSANDS OF THESE MITOCHONDRIAL DNA'S AND THEY ARE CONSTANTLY REPLICATING INSIDE YOUR CELL. SO THEY ARE REPRESENT INDICATING, INCREASING IN NUMBER. THEY ARE BEATING EATEN UP, CREATING MUTEAND AND NORMAL BACTERIA. RED IS MUTANT, BLUE IS NORMAL. IF THE CELL DIVIDED DOWN THIS WAY, BOTH CELLS WOULD HAVE MUTE QUANTITY ANMUTANT AND NORMAL. POINT IS MITOCHONDRIAL DNA GENOTYPES SEGREGATE DURING MITOSIS. A HETEROPLASTIC SKY GOA ZYGOTE WILL GIVE RISE TO TWINS WITH DIFFERENT PHENOTYPES, OR IF HETEROPASS PLASTIC, SOME WOULD BE PRIMARILY MUTANT, AND THE MORE THERE WERE, THE LESS ENERGY, THE EQUIVALENT OF METROPOLITAN BROWNOUT. ORGAN SYSTEMS WOULD MALFUNCTION, THAT TURNS OUT TO BE THE BRAIN. WE HAVE A HIGH MUTATION RATE. WE'LL TALK ABOUT WHY THAT IS BUT THE MUTAGEN IS PROBABLY ROS. DID DID TRANSFER RNA'S PUNCTUATE GENES, AND COMPLEX ONE, COMPLEX FOUR, SIX AND EIGHT FOR COMPLEX FIVE WITH A CONTROL REGION THAT REGULATION TRANSCRIPTION AND REPLICATION. SO IT HAS A VERY HIGH MUTATION RATE. ONE OF THE THINGS THAT WE GET IS MA TERNALLY INHERITED DISEASE. THIS MUTATION, A NUCLEOTIDE POSITION, IF YOU INHERIT THAT FROM YOUR MOTHER YOU'RE FINE UNTIL MID-LIFE AND YOU'LL GO DEAF. THIS GIVES YOU DIABETES, A HIGH PERCENTAGE GIVES YOU STROKE, EVEN HIGHER LEVEL WILL KILL YOU AS AN INFANT. MUTATION IN THE TRNA GENE AT 8344 GIVES YOU A KIND OF EPILEPSY, THERE ARE HUNDREDS OF THESE PROTEIN SYNTHESIS MATERNALLY INHERITED GENES. WITH ONE YOU'LL BE FINE UNTIL MID-LIFE AND LOSE VISION IN ONE EYE. UP HERE, THE SAME MUTATION, 14484 GIVES YOU THE SAME PHENOTYPE, 14459, WHEN THE MUTATION AT 8993, 70% GIVES YOU RETINAIZED PIGMENTATION, SOME OF THE SUBUNIT 1 GENES IN PROSTATE CANCER. WE HEARD SEVERAL NICE LECTURES IN THAT AREA SO I DROPPED THEM FOR TIME'S SAKE. THE MITOCHONDRIAL GENOME INVOLVES THOUSANDS OF ON ONE TO TWO THOUSAND NUCLEAR GENES, 35 MITOCHONDRIAL GENES, MA TERNALLY INHERITED DISEASES. EVERYBODY IN THE ROOM HAS A DIFFERENT SEQUENCE. SOME ARE COMMON, THIS ONE IS FOUND IN 3/4 OF SUBSAHARAN AFRICANS, THIS AROSE IN ASIA, CROSSING THE BEHRING LAND BRIDGE, AND THESE I'M GOING TO ARGUE CHANGED THE MITOCHONDRIAL PHYSIOLOGY TO ALLOW PEOPLE TO ADAPT TO DIFFERENT ENVIRONMENTS. OKAY. SO ONCE WE BEGIN TO THINK ENERGETICALLY, GETTING CLOSE TO THE INTRODUCTION, BE PATIENT, ONCE WE BEGIN TO THINK ENERGETICALLY, THEN WE CAN REALIZE WE CAN THINK ABOUT ANATOMY FROM AN ENERGETIC POINT OF VIEW. HIGH ENERGY TISSUES LIKE THE BRAIN HAS THE HIGHEST ENERGY DEMAND, LOWEST RESEARCH, 2% OF THE BODY WEIGHT, USESE USES 20% OF OXYGEN. A 10% REDUCTION IN OXYGEN WILL GIVE YOU A BAD HEADACHE. WE HAVE RESULTS ON HEARTS, RENAL, ENDOCRINE, THEY CAN BE MORE TOLERANT. WE HAVE ENERGY STORAGE TISSUES, ADIPOSE STORES FAT. WE HAVE AN ENERGY HOMEOSTASIS TISSUE, THE LIVER, WHY DOES IT REGULATE BLOOD GLUCOSE? THAT'S YOUR CONNECTION WITH SUNLIGHT. AN ENERGY SENSITIVE TISSUE, PANCREATIC BETA CELL TELLS THE SWELLS TO SWITCH TO TOR GLYCOLYSIS, OR TURN ON CYCLIC AMP TO BURN FAT. ALL OF THIS LEADS TO THIS CONCEPT THAT IF WE NOW MOVE ANATOMY OUT OF THE CENTER OF OUR THINKING AND PUT IN ENERGETICS, ALL OF THE COMMON COMPLEX DISEASES COULD BE UNDERSTOOD FROM THE SAME PATHO PHYSIOLOGICAL MESSAGE. THIS IS THE MAJORITY OF THE ENERGY AND THE MOST SENSITIVE. SO WE COULD HAVE VARIATION IN NUCLEAR GENES, MUTATIONS OR POLY MORPHISMS, CHANGES IN THE EXPRESSION THAT WOULD HAVE CHANGED ENERGETICS. THIS IS HOW YOU PROCESS ALL YOUR CALORIES, AND THE OXYGEN THAT YOU'RE BREATHING. IT'S WHERE THE SOURCE OF ENERGY TO GROW, MATURE AND REPRODUCE ARE. IT'S ALSO THE MOST SENSITIVE TO TOXINS, SO IF YOU DON'T LIKE YOUR NEIGHBOR, YOU WANT HER TO STOP BOTHERING YOU, PUT A LITTLE CYANIDE IN YOUR TEA. SHE'LL STOP BOTHERING YOU. ALMOST ALL THE MAJOR TOXINS ARE DIRECT INHIBITORS EVER MITOCHONDRIA. WHY? THAT'S YOUR ACHILLES HEEL. IF YOU INHIBIT FUNCTION, YOU WILL ACCUMULATE SOMATIC MUTATION BECAUSE REPLICATION IS NOT GOING WELL GIVE YOU A DECLINE IN FUNCTION. AND THAT GIVES YOU THE DELAYED ONSET OF PROGRESSIVE COURSE WE SEE IN ALL COMPLEX DISEASES AND AGING AND WE BELIEVE THAT'S THE AGING CLOCK. WHAT WOULD BE THE INTERMEDIATATES, PARTIAL DEFECTS? THEY ARE GOING TO AFFECT THE BRAIN, HEART, MUSCLE AND RENAL, WITH THE COMPLEX DISEASES. IT WILLEIT WILL ALSO MESH PERTURB THE FLOW OF ENERGY IN YOUR SYSTEM. EVERY TIME A CELL BREAKS OPEN, IF THE MITOCHONDRIA ARE NOT DIGESTED, YOU'RE GOING TO RELEASE THOSE POLY PEPTIDES AND OTHER THINGS AND YOU'RE GOING TO AMASS A MASSIVE INFLAMMATION RESPONSE. THIS IS A VERY GROWING AREA RIGHT NOW, THE SO-CALLED DAMPS, THE INFLAMMATION SYSTEM SECONDARY TO MOST OF THE DEGENERATIVE DISEASES. FINALLY, HOW YOU MANAGE ENERGY IS CRITICAL TO WHETHER CANCER GROWS, IT'S NOT THAT ONE SYSTEM IS DEFECT ACTIVE, CANCER CELL HAS THE ABILITY TO SWITCH. THAT'S THE BACKGROUND. NOW WE'RE GOING TO HAVE EXAMPLES. THIS IS JUST THE FAMILY WHERE THIS PERSON WENT BLIND, RELATED TO THIS FEMALE, TO THIS PERSON WENT BLIND, THESE PEOPLE, TO THIS FEMALE, THESE MEN WENT BLIND AND THIS WOMAN, AND THEN TO THIS FEMALE, THIS BLIND PERSON AND IT'S A LATE ONSET DISEASE, YOUNG PEOPLE DIDN'T SHOW IT. THIS IS NEUROPATHY, THIS IS A MUTATION. SOME OF THE THINGS YOU CAN IMMEDIATELY SEE ABOUT THIS, NOT EVERYBODY ON THE MATERNAL LINE IS BLIND. PEOPLE THOUGHT THIS WAS AN EXCELLENEXCELLENT GENE ORIGINALLY, THERE'S A 4 TO 1 PREFERENCE IN MALES. WHY WOULD YOU HAVE THAT WITH MALE BIAS? MALE BIAS IS IN FACT A THEME. ONE OF THE INTERESTING THINGS IS THAT YOU ALSO SEE IT IN AUTISM, IN PARKINSON'S DISEASE, MANY OF THESE COMPLEX DISEASES SHOW THIS MALE BIAS WHICH WE THINK IS A SEX LIMITED FEATURE OF ENERGETICS. STILL, THAT ASKS THE QUESTION, WHY IS IT THERE'S VARIABILITY IN A MITOCHONDRIAL DNA DISEASE GENE? ONE OF THE MAJOR FACTORS IS THE BACKGROUND IN WHICH THAT MUTATION OCCURS. THE MITOCHONDRIAL DISEASE, EVERYBODY HERE HAS A DIFFERENT MITOCHONDRIAL DNA. A MUTATION WOULD HAVE DIFFERENT EFFECTINGS OEFFECTS ON YOU. HERE ARE THREE, THERE ARE NOW DOZENS. THIS ONE CAUSES A SEVERE COMPLEX ONE DEFECT, 1178, A MODERATE DEFECT, AND A MILD, YET THEY ALL GIVE THE SAME PHENOTYPE. WHY WOULD THAT BE? THE ANSWER IS THAT THE BACKGROUND IS IMPORTANT. THIS IS GROUP J FOUND IN 20% OF PEOPLE OF EUROPEAN BACKGROUND. THIS SEVERE MUTATION DOESN'T MATTER YOUR BACKGROUND, WHETHER IT'S J OR NOT. YOU'RE GOING TO GO BLIND. THIS MORE MODERATE ONE, ONE-THIRD OF THE PEOPLE ARE ALSO J, SO J HAD TO INCREASE THE PENETRANCE, AND FOR THIS MILD ONE ALMOST 3/4 ARE J. SO IN FACT J INCREASED THE PENETRANCE OF THE MILDER MUTATIONS. HERE IS ANOTHER MUTATION. THAT ALSO WILL INCREASE THE PENETRANCE OF THE MILDER MUTATIONS. SO WHAT ARE THESE LINEAGES? THESE LINEAGES ARE IN FACT CONTINENT AND REGIONAL SPECIFIC MITOCHONDRIAL DNA LINES. SO IF I SEQUENCE THE MITOCHONDRIAL DNA OF EVERYBODY IN THE ROOM, ANY TWO OF YOU, WITH NUCLEOTIDE SUBSTITUTIONS, WOULD BE PROPORTIONAL IF YOU SHARED A COMMON MOTHER, BY USING THAT LOGIC, AND SEQUENCING THE MITOCHONDRIAL DNA OF INDIGENOUS PEOPLE AROUND THE WORLD WE FOUND IN FACT MITOCHONDRIAL DNA INITIATED IN AFRICA ABOUT 150,000 YEARS AGO, GAVE RISE TO L-1 AND L-2, PI PYGMIES, SOUTHEAST ASIA, THE TROPICS, DOWN TO AUSTRALIA, BUT DID SOMETHING UNIQUE, MOVING NORTH TO THE TEMPERATE ZONE, AND H, J, TU, V AND M MOVED NORTH, AND 20,000 YEARS AGO THE AMERICAS WERE COLONIZED. ALL ALELES ARE FOUND THROUGHOUT THE WORLD. THIS WOMEN NEED TO BE ABLE TO RUN AWAY FROM LIONS, OTHERWISE THEY GET EATEN. HOW DO THEY RUN FROM LIONS? THEY HAVE TO BE EFFICIENT. UP HERE THE LIONS FROZE TO DEATH. HOW ARE THEY GOING TO SOLVE THAT PROBLEM? ACCUMULATING MITOCHONDRIAL DNA MUTATIONS THAT DECREASE COUPLING EFFICIENCY. NOW THEY HAVE TO EAT A HIGH FAT DIET FOR THE SAME AMOUNT OF ATP GENERATING MORE INTERNAL HEAT. THEY WILL HAVE TO KILL MARINE MAMMALS, EVERYBODY WILL LOOK DOWN ON THEM. THAT'S THE WAY IT IS. THE POINT IS THAT THESE MITOCHONDRIAL DNA LINEAGES BECAME ADAPTIVE AND THEY ALLOWED OUR ANCESTORS TO LIVE IN THESE DIFFERENT GEOGRAPHICAL REGIONS, THIS SHOWS OUT OF AFRICA, LINEAGE M WHICH STAYED IN THE TOPICS HAS SYNONYMOUS MUTATIONS, RANDOM MUTATIONS, BUT N WHICH MOVED TO THE TEMPERATE ZONE, HAS THREE. WHAT DOES THAT DO? DECREASED THE MEMBRANE POTENTIAL AND ALTERED CALCIUM METABOLISM MAKING IT LESS EFFICIENT. AN AMIN AMINO ACID, THIS VARIANT, THIS AMINO ACID IS CONSERVED DOWN. THESE MUTATIONS IN THE B GENE, THIS ONE IS CONSERVED IN ALL MESOZOANS ALL THE WAY TO E. COLI. WAIT A MINUTE. THE AMINO ACID CHANGES CONSERVED THROUGHOUT SPECIES EVOLUTION, POLY MORPHIC IN THIS ROOM, THAT'S UPSIDE DOWN FROM EVERYBODY WE'VE BEEN TALK ABOUT MOLECULAR EVOLUTION. HOW COULD THIS BE? REMEMBER, THE MITOCHONDRIAL DNA HAS INCREDIBLY HIGH MUTATION RATE. HOW WOULD IT WORK THEN? THE IDEA THAT WE HAVE IS THAT WHEN -- SO THIS IS THE MEAN ENVIRONMENT, THE MOST COMMON ENVIRONMENT. WE HAVE THEN A MITOCHONDRIAL LINEAGE THAT HAS BECOME ADAPTED TO AN EXTREME ENVIRONMENT, TWO STANDARD DEVIATIONS, IT THEN IS GOING TO ACQUIRE A MUTATION, SOME ALREADY FUNCTIONAL, CREATING LINEAGES THAT MOVE BACK TOWARD THE MODAL ENVIRONMENT, ULTIMATELY FIXING THESE FUNCTIONAL MUTATIONS WITH RANDOM VARIATION. THAT THEN WILL RADIATE AND THEN ACQUIRE THE FUNCTIONAL MUTATIONINGMUTATIONSAND CREATE A MODE ENOU GH TO CREATE A NEW SPECIES, YOU GO BACK THROUGH THE SYSTEM AGAIN. WHEN YOU TAKE THE AVERAGE OF THE SPECIES, THE ENVIRONMENT HAS SELECTED FOR CONVERGENT EVOLUTION ON THE SAME AMINO ACID CHANGES. AT EXTREME ENDS, WE HAVE THEN THE FUNCTIONAL VARIATIONS THAT WE SEE IN THE ARCTIC OR EXTREME AFRICA. I THINK THEN THIS IS THE DRIVING FORCE FOR ADAPTATION WITHIN SPECIES WHEREAS NUCLEAR VARIATION IS THE DRIVING FORCE FOR ADAPTATION BETWEEN SPECIES. THIS IS STUDIES IN TIBET. WE SEQUENCED MITOCHONDRIAL DNA FROMTY YETIAN TIBETANS AT MANY ALTITUDES. THERE'S LINEAGE M AND N. OUT OF AFRICA IS M AND N. WHAT WE FOUND IN TIBET IS THAT THIS MUTATION, T-339-4C AROSE THREE INDEPENDENT TIMES. IF WE ASK THE FREQUENCY OF THAT MUTATION VERSES ALTITUDE, WE SEE AS THE ALTITUDE INCREASES, SO THIS THIS. THERE IS AN ODDS RATIO, THIS OCCURS BY CHANCE OVER 25. SO WHAT THIS MEANS IS THIS MUTATION IS BEING SELECTED BY ALTITUDE. BUT WAIT A MINUTE. THAT'S THE SAME VARIANT WE SAID INCREASES PENETRANCE OF LABORS. HOW COULD IT BE BAD FOR LABORS, GOOD FOR ALTITUDE? OKAY. THIS IS ANOTHER VARIANT. WE'LL COME BACK TO THAT. THIS IS ANOTHER LINEAGE, LINEAGE FOR F-4, THIS IS JUST LOOKING AT OBESITY, ONE OF MANY, MANY STUDDIZE WE'VSTUDIES WE'VE BEEN DOING. IT HAS A SIGNIFICANT EFFECT, INCREASED BMI LEVELS, WE CAN DO THAT FOR DIABETES, CARDIOVASCULAR DISEASE, ON AND ON. THAT I VARIANT OCCURRED SEVEN TO SEVEN THOUSAND YEARS AGO IN EUROPE, IT'S ONLY ABOUT .4% OF EUROPEANS, BUT IF YOU NOW LOOK AT PEOPLE WITH ALZHEIMER'S AND PARKINSON'S DISEASE, 3% HAVE ALZHEIMER'S, 5% -- 3% OF ALZHEIMER'S HAVE THE MUTATION. IT PREDISPOSES TO LATE ONSET NEURODEGENERATIVE DISEASE. A MUTATION SPONTANEOUS, 3397, THIS IS A CODE, BUT THAT'S THE ADJACENT CODON TO THE MUTATION, THIS ONE GIVES YOU ADPD. HOW DO WE MAKE SENSE OF THIS? WHAT WE REALIZE SENTENCE PHENOTYPE DEPENDS ON CONTEXT. OUT OF AFRICA, WE HAVE L, AND THAT GIVES RISE TO N WITH THESE TWO FOUNDING MUTATIONS, AND THE TRNA GLUTAMIN IS PREDISPOSED, WE CAN GET THIS, THAT'S TOXIC AND GIVES YOU ADPD. HOWEVER, YOU CAN GET THE 3394 MUTATION, THAT IS AT A HIGHER FREQUENCY INCREASING YOUR RISK OF LABORS BUT ALSO OBESITY AND DIABETES. BUT OVER HERE, WHEN WE SKIPPED THESE MUTATIONS, IN THAT CONTEXT, HIGH ALTITUDE, THIS PARTICULAR MUTATION IS NOW COMMON AND IT'S ADAPTIVE TO ALTITUDE BECAUSE THIS IS ONLY BAD WHEN IT'S IN THE CONTEXT OF THESE. SO HOW CAN WE SHOW YOU THAT'S FUNCTIONALLY IMPORTANT? WE TAKE BLOOD PLATELETS, WE HAVE A CELL LINE THAT LACKS ITS OWN MITOCHONDRIA, WE SELECT FOR THESE SOMATIC SELF HYBRIDS, AND WE ASSAYS. F IS THE DIABETES, YOU CAN SEE WHEN WE HAVE THIS C MUTATION, WE GET A 15 TO 30% REDUCTION IN COMPLEX 1, SO IT'S FUNCTIONALLY IMPORTANT. LOOK AT THE RELATIONSHIP BETWEEN THE T ALELE FOR F AND B. B AND F NOT ONLY ARE RELAVENT TO EACH OTHER, RELAVENT TO THE NEW MUTATIONS. FINALLY IF WE LOOK AT THE LINEAGE, THE C IS AS GOOD AS THE T MUTATION ON B. BACKGROUND IS EVERYTHING. CON TEXT IS WHAT MATTERS WITH MITOCHONDRIAL ENERGETICS. WE CAN THEN USE THESE LINEAGES TO DO A LOT OF ASSOCIATION, AND THEY HAVE CORRELATED WITH RISK FOR ALZHEIMER'S, AIDS, OSTEOPOROSIS, AGING, CANCERS, ATHLETIC PERFORMANCE. OKAY. ALL RIGHT. SO HOW CAN I PROVE TO YOU IN FACT THESE ARE RELEVANT TO DISEASE? WHAT WE DID IS WE CREATED A MOUSE WHICH HAS A SPECIFIC MITOCHONDRIAL DNA POINT MUTATION, WE MADE THIS ORIGINALLY TO LOOK AT WHETHER WE COULD SHOW THAT THESE MUTATIONS IN FACT CAUSED THE DISEASE. IN ONE LINE, WITH A FRAME SHIFT, IN THIS THIS, WE TAKE THE FOR EXAMPLEMENTFRAGMENTAND TAKE THIS, PUT IT I NTO A FOSTER MOTHER, GOT CHIMERIC FEMALE AND BREAD THROUGH THE FEMALE LINE, THUS PICKING UP THE MITOCHONDRIAL DNA. OKAY. SO THIS SHOWS IN FACT THE MUTATION WHICH WAS INSERTION OF EXTRA C, THROWING IT OUT OF FRAME. THIS EMBRYONIC STEM CELL DELETED THE ADJACENT T AN AND REVERTED BACK TO WILDTYPE AND CARRY THE MUTATION IN THE CO-1. AFTER MANY TRIES WE GOT THIS PARTICULAR EMBRYONIC STEM CELL LINE AND THE FEMALE WITH THIS LINE HAD IN DIFFERENT ORGANS DIFFERENT PERCENTAGES OF THE MUTANT, REPLICATED SEGREGATION. WE CROSSED THIS FEMALE WITH A NUCLEAR MATCH CONTROL, SHE STARTED WITH 47%, HER FIRST AND SECOND LITTERS WERE 16%, THE THIRD LITTER WAS 6%, FOURTH, FIFTH AND SIXTH WERE 0%, THERE WAS A SELECTIVE LOSS OF THE FRAME SHIFT. WE TOOK THE 16% AND MATED THEM, SIX OR ZERO. WE SUPE SUPER OVULATED THEM. THIS IS A TRUNCATED DISTRIBUTION, IN THE OVARY THERE'S A SYSTEM THAT SELECTS AGAINST DILATARIOUS MUTATIONS. WHY WOULD THAT BE IMPORTANT? IF YOU HAVE AN EXQUISITELY HIGH MUTATION RATE, FOR GENES ABSOLUTELY CRITICAL FOR LIFE, YOU LET ALL THOSE MUTATIONS INTO THE POPULATION, THE POPULATION WOULD GO EXTINCT. SINCE MITOCHONDRIA CAN FUNCTION IN A SINGLE CELL, YOU CAN NOW HAVE SELECTIVE PRESSURE ON THE CELL BEFORE YOU EVER GET A ZYGOTE, ALLOWING US TO HAVE A VERY HIGH MUTATION RATE WITHOUT GENETIC LOAD. LET'S SAY WE SEGREGATE THE MUTATION AND THIS CREATES A REDUCTION, GIVES YOU DEGENERATING MUSCLE FIBERS, CARDIOMYOPATHY AND MUSCLE SYMPTOMS. LET'S TALK ABOUT ANOTHER DISEASE, A FAMILY REPORTED, THIS WOMAN HAD 50% OF A MUTATION THAT CHANGED THIS GENE, AND SHE HAD CEREBELLAR. ALL THEIR CHILDREN DIED. WE HAVE THE INHERITANCE OF THIS HETEROPLASMIC MUTATION. AN ARTICLE JUST CAME OUT THAT ARGUES DARWIN'S FAMILY, THE REASON HE WAS SICK, HE HAD A MUTATION, IF YOU TRACE HIS PEDIGREE, EVERYBODY ON THE MATERNAL PEDIGREE IS AFFECTED. THAT'S AN ASIDE. WHAT WE HAVE IS THIS RANDOM SEGREGATION OF HETEROPLASMY. HOW CAN WE SEE IF THIS IS THE CAUSAL MUTATION? WE SCREEN FOR MANY YEARS TO FIND A CELL LINE WITH EXACTLY THAT MUTATION. HERE IT IS. WE TAKE OUR FEMALE EMBRYONIC STEM CELL, PUT IN THE MITOCHONDRIAL DNA, THE FOSTER MOTHER, TRANSMIT THE MUTATION. WHAT DO WE GET? THIS IS LOOKING AT THE OPTIC NERVE, THIS IS THE CONTROL OPTIC NERVE WITH THE NORMAL FIBERS AND YOU CAN SEE IN THE MUTANT SWOLLEN FIBERS. IN FACT, THE SMALL CALIBER HIGH ENERGY FIBERS ARE PREFERENTIALLY AFFECTED. YOU CAN SEE THAT THE RED ARE THE NORMAL MICE, THE BLUE ARE THE MUTANT, AND YOU CAN SEE THERE'S A BIAS TOWARDS SMALL CALIBER FIBERS IN THE WILDTYPE VERSUS BLUE AND THAT GETS WORSE AS THEY AGE. IF WE THEN LOOK AT THESE MORE GENERALLY WE SEE DEMYELENNATION, WHAT YOU WOULD SEE IN MULTIPLE SCLEROSIS. IN FAMILIES, MALES GO BLIND, FEMALES GET MULTIPLE SCLEROSIS. THIS IS JUST LOOKING AT THIS CELL LINE. WE DECLINE IN REACT ACTIVITY IN THE BASAL GANGLIA, INCREASE IN ANXIETY, DEPRESSIVE EFFECT. WE SEE SEVERE EFFECTS ON MOTOR AND WE SEE A LEARNING DISABILITY. AND IF WE NOW DO AN MRI WE CAN SEE WE'RE GETTING SPECIFIC ALTERATIONS IN THE FIBER DENSITY. THIS MITOCHONDRIAL DNA POINT MUTATION NUCLEUS PERFECTLY NORMAL IS GIVING YOU CLINICAL NEUROLOGICAL PHENOTYPES OF WHAT WE WOULD CALL PARKINSON'S DISEASE. SO THAT MEANS THAT THIS MUST BE AN ENERGETIC DISEASE BECAUSE THAT'S THE ONLY CHANGE THAT HAS OCCURRED. OKAY. SO HOW CAN WE TELL MORE ABOUT THIS DISEASE? WELL, NOW THAT WE HAVE A MOUSE WE CAN ISOLATE AND STUDY THE MITOCHONDRIA IN SITU. WE FIND THIS COMPLEX 1 DEFECT REJUICEREDUCES ENERGETICS 30% HAS NO AFFECT ON ATP LEVEL BUT AFFECTS RESPIRATION AT REST BUT IT CAN GO TO NORMAL BUT THE REACT REACTIVE OXYGEN GOES TO HIGH LEVELS. NOW, LET'S AGE THESE CO 1 MICE TO TWO MONTHS. NOW WE SEE WE GET INSULIN RESISTANCE. HERE IS THE INSULIN LEVEL OF THE MUTANT AND WILDTYPE AND FOR THE CO-1 WE NOW HAVE A GLUCOSE INTOLERANCE. SO THIS POINT MUTATION THEN IS GENERATING ALL THE PHENOTYPES THAT WE SEE IN COMMON DISEASES. SO, OKAY, THOSE ARE PATHOGENIC MUTATIONS, BUT WHAT ABOUT NATURALLY OCCURRING VARIANTS LIKE YOU HAVE IN WE DECIDED TO TRY AND MIX TWO NATURALLY OCCURRING MITOCHONDRIAL DNA'S, WE MIXED NZB WITH 129, PUT ANYTIME THE ANIMAL, NOW WE HAVE THE FOUNDER FEMALE AND THAT'S THE NZB, 129, THIS IS HER DAUGHTER. WE MATED HER AND ALL OF THEY ARE OFFSPRING, MALES OR FEMALES, HAVE THE HETEROPLASMA AND DAUGHTERS TRANSI TRANSMITTED, MALES DO NOT. THESE ANIMALS HAVE BEEN BACK-CROSSED 20 GENERATIONINGS ON A COMMON INBRED BACKGROUND SO THERE'S NO NUCLEAR VARIATION WITHIN WHAT WE CAN CONTROL FOR. SO NOW WHAT WE DO IS TAKE THE HETEROPLASMIC ANIMAL AND ASK THE PHENOTYPE. IF WE LOOK AT ACTIVITY, WE FIND THE 129 AND NZB ARE ACTIVE AT NIGHT AS THEY SHOULD BE, BUT THE HETEROPLASMIC ARE HYPO ACTIVE, ASSOCIATED WITH FOOD -- DECREASED FOOD INTAKE. THE RESPIRATORY CONTROL RATIO IS SLIGHTLY LOWER BUT WHEN WE PUT THEM IN AN ENVIRONMENT, THEY BECOME HYPER-EXCITABLE. CAN THEY LEARN? HERE IS BLACK IS NZB, BLUE HAS DIFFERENT BOUTS OF LEARNING, TO TAKE THIS OPEN FIELD WITH ALL THESE HOLES AROUND THE MIDDLE, AND AROUND THE OUTSIDE, AND THERE'S A BLACK BOX THERE, THEY ARE COLORED SYMBOLS TO FIND THEIR WAY, AND THEY LEARN OVER TIME HOW TO FIND THE BOX AND HIDE. SO BOTH -- ALL THREE LINES LEARN, HETEROPLASMIC LEARNS MORE SLOWLY. MEGAN LET THE ANIMALS REST FOR A DAY AND ASKED THEM IF THEY COULD REMEMBER THE TASK, 129 AND NZB IMMEDIATELY RAN IN THE HOLE. THE HETEROPLASMIC ARE COMPLETELY FORGOTTEN. HAVING TWO PERFECTLY NORMAL MITOCHONDRIAL DNA'S WE CAN GET MANY FEATURES OF LEARNING DISABILITIES AND OBSESSIVE COMPULSIVE BEHAVIOR AND DEPRESSION. OKAY. NOW LET'S LOOK AT A NUCLEAR MUTATION, THIS TRANSLOCATOR, AND ISOFORMS MAKE THIS NONLETHAL, THIS OCCURRED IN SWITZERLAND 500 YEARS AGO, WE TRACED A HUGE PEDIGREE IN NORTH AMERICA AND ULTIMATELY WE GET THESE PEOPLE HAVING HEART DISEASE. THAT MAPS THE CHROMOSOME FOUR, A FRAME SHIFT MUTATION BOODLE. THEY HAVE HEART DISEASE AND MYOPATHY. EVERY HEART BEAT IN THE WILDTYPE HUMANS IS NORMAL, BUT OF THE ANT WE SEE HIGHLY DISRHYTHMIC EFFECT. GREEN IS NORMAL, RED IS ABNORMAL. BUT THERE'S A MAJOR DIFFERENCE. SOME MUCH THE INDIVIDUALS CAN LIVE TILL THEIR 40, WITH HYPERTROPHIC CARDIOMYOPATHY. SOME OF THE INDIVIDUALS AT VERY EARLY CHILDHOOD DEVELOP A MASSIVE DILATED CARDIOMYOPATHY REQUIRING A HEART TRANSPLANNED AND HAVTRANSPLANTSAND WOULD DIE. WHY THE DIFFERENCE? IT'S ALL ABOUT THE MITOCHONDRIAL GROUP, H HAS NORMAL, AND THOSE WITH U PROGRESS TO TRANSPLANTATION. TO PROVE TO YOU THAT THAT'S TRUE, WE CREATED A MOUSE WITH THE SAME -- WITH AN ANT DEFECT, NORMAL MOUSE WILL RUN. OXYGEN USE IN RED, CO2 GIVEN OFF, USING AEROBIC EXERCISE, CAN RUN FOR LONG PERIODS OF TIME. THE KNOCKOUT MOUSE RUNS AND FALLS DOWN. IT HAS THEN AN ACCUMULATION OF MITOCHONDRIA, SUCCINATE DEHYDROGENASE, CREATING MYOPATHY. NOW, OOPS, THESE ARE THEN THE HEART ECHOES OF WILDTYPE CO 1, ND-6, ANT, ANT-C OCO 1. THIS IS HEART CAN HARDLY BEAT. THIS IS THE REPERTOIRE WITH H VERSUS U MITOCHONDRIAL DNA. SO WE CAN THEN QUANTIFY THAT BY VELOCITY VECTOR IMAGING, WILDTYPE WE GET UNIFORM HEART BEATS, AND THIS SHOWS THE SAME PHENOTYPE. OKAY. SO WE CAN THEN NOW QUANTIFY ALL THIS SO NOW WE'RE LOOKING AT DIFFERENT CARDIAC EFFECTS. IN THIS CASE, DIASTOLIC DIE AMOUNTER OF THE LEAP VENTRICLE THEY ARE UNIFORM. THIS IS BAD. THIS IS EJECTION FRACTION, AND IF WE NOW LOOK AT THE VELOCITY VECTOR IMAGES YOU CAN SEE WE IN FACT HAVE A DIRECT RELATIONSHIP BETWEEN THE MITOCHONDRIAL DNA AND NUCLEUS. AND IF WE LOOK AT LIFESPAN, THESE GUY QUICKLY RELATIVE TO WILDTYPE, THE OTHERS INTERMEDIATATE, OTHERS ARE NORMAL. C, YOU CAN SEE A PROGRESS OF ABNORMAL MITOCHONDRIA. SO WHAT THAT SHOWS THEN IS THAT MITOCHONDRIAL VARIATION NATURALLY OCCURRING HAS ALSO HAD A BIG EFFECT ON THE PENETRANCE OF EVEN CLASSIC MENDELIAN NUCLEAR GENES. LAST POINT TO BE MADE, THIS IS THE FAMILY WITH THE 3243 MUTATION, I SAW THIS FAMILY IN THE EARLY '80s. THIS WOMAN HAD LACK PARTICULAR ACIDOSIS, THESE ARE THE CHILDREN, WE COULD EXAMINE THEM. THESE ARE NOT. THEY ALL DIED IN LATE TEENS OR EARLY 20s, MUSCLE FIBERS WERE DEGENERATING, WOLF PARKINSON WHITE CONDUCTION DEFECTS. WE TOOK THE HETEROPLASMIC CELL LINES AND THIS WITHOUT MITOCHONDRIAL DNA AND FUSED THAT TO THIS CELL CREATING DIFFERENT PERCENTAGES. THIS FAMILY WAS 70% MUTANT HAD CARDIOMYOPATHY, 10 TO 30% TYPE ONE, TYPE TWO DIABETES OR AUTISM, THIS IS BIZARRE, THIS IS SUPPOSED TO BE AN AUTO-IMMUNE DISEASE, THIS IS METABOLIC BUT THE SAME MUTATION CAUSES BOTH DISEASES, YET AT HIGH LEVELS WILL KILL YOU AS AN INFANT. WE COULD ASK WHAT'S DIFFERENT ABOUT THESE DIFFERENT GENOTYPES? WE CAN SEE AS THE PERCENTAGE OF MUTANT MITOCHONDRIAL DNA AS THE PERCENTAGE OF MUTANT INCREASES THE PROTEIN SYNTHESIS INCORPORATION IN THIS MITOCHONDRIAL DNA CODED SUBUNIT DECLINES, THERE'S A THRESHOLD EFFECT, OXYGEN CONSUMPTION STAYS CONSTANT UNTIL 60% AND FALLS OFF. WE SEE ALTERATIONS IN MITOCHONDRIAL MORPHOLOGY AND FOR THE -- FROM ZERO, 20, 30% A REMARKABLE DECLINE IN CELL SIZE. WE'RE SEEING SIMPLE CHANGES IN MITOCHONDRIAL GENOTYPE, BY ONLY 10%, HAS A BIG EFFECT ON CELL PHYSIOLOGY. TO CUT A LONG STORY SHORT WE'VE HAVE DONE RNA SEQ, IN FACT AUTISM, THAT'S ONE TRANSCRIPTIONAL PROFILE FROM THE NUCLEUS. 50 TO 90%, THAT'S ANOTHER TRANSCRIPTIONAL PROFILE, THE THIRD KILLS THE INFANTS. THIS IS THE NORMAL GENE EXPRESSION PROFILE. THIS IS THE 20 TO 30% HERE. COMPLETELY OPPOSITE IS THE 50 TO 90%, AND THEN 100%. SO WHAT THIS IS SAYING IS SUBTLE CHANGES IN MITOCHONDRIAL BIOENERGETICS IS CAUSING PHASE SHIFTS IN THE EPI GENOME EXPRESSION, AND THIS EXPRESSION WE BELIEVE CREATES THESE PHENOTYPIC CHANGES RELATED TO CANCER, METABOLIC SYNDROME, DIABETES AND SO ON, IT'S THE NUCLEAR CYTO PLASMIC CROSS TALK THAT'S IMPORTANT. THE NUCLEAR ORGANISM, THE ANATOMY OF ENERGYICS, THIS CAN TOLERATE A HIGH MUTATION RATE, THIS IS LOW BECAUSE ALL THE MUTATIONS HAVE TO GO THROUGH DEVELOPMENT BEFORE THEY CAN BE ACTED ON BY SELECTION, AND THEREFORE A HIGH MUTATION RATE WOULD BE DILATARIOUS. THE ENERGETICS. MITOCHONDRIA MAKE THE ATP AND ACETYL, WHY WOULD THAT BE? THE NUCLEUS CAN'T DO ANYTHING WITHOUT ENOUGH ENERGY AND HAS TO KNOW THE ENERGY FLUX THROUGH THE NTSB. MITOCHONDRIA. IT USED HIGH ENERGY INTERMEDIATATES TO CHANGE THE EXPRESSION TO BE REPLICATED, TRANSCRIBED AND TRANSLATED WHEN THERE WAS ENOUGH ENERGY. NOW THAT SAYS IF THIS CONCEPT IS CORRECT, THEN THESE ARE THE MAJOR INTEREST SPECIFIC VARIABLES RELATED TO PHENOTYPE, PRIMARILY INTRASPECIFIC. I'D LIKE TO END BY THANKING PLANE YEARS OF WONDERFUL COLLEAGUES AND I DON'T EVEN KNOW THAT'S THAT CLEAR, BUT PEOPLE HERE, ALICIA, DANIELLE, KIRSTEN, PIETRO, BRIAN MORROW, MARK AND KATRINA, THE STUDIES IN TIBET DONE WITH THE PEOPLE AT THIRD MILITARY MEDICAL UNIVERSITY, AND THE OPHTHALMOLOGY STUDIES, OUR EPI GENOMIC STUDIES BY PAULO CORSI, THE CARDIOLOGY STUDIES, THE FAMILY WE STUDIED IN PENNSYLVANIA, WITH KEVIN STRAUSS OF THE CLINIC FOR SPECIAL CHILDREN AND OUR LONG-TERM ANTHROPOLOGIC STUDIES. THANK YOU VERY MUCH. [APPLAUSE] >> WE HAVE TIME FOR A FEW QUESTIONS. THE MICROPHONES ARE IN THE AISLES. USE THEM SO PEOPLE WATCHING ON THE VIDEO CAN HEAR THE QUESTIONS. WE'RE HOPE FOR QUESTIONS. YOU SHOWED THE DATA WHERE THE HETEROPLASMY WAS THE PROBLEM. THE HOMOPLASMY WAS FINE. >> SO, THANK YOU, FRANCIS. AGAIN, LET ME REITERATE. THE MITOCHONDRIAL DNAS ARE PERFECTLY NORMAL MICE, JUST COLLECTED FROM DIFFERENT ENVIRONMENTS. BUT THEY STILL DIFFER BY ABOUT AS MANY NUCLEOTIDES AS YOU DO. BUT THOSE, SOME OF THOSE ARE AMINO ACID SUBSTITUTIONS. REMEMBER, ANYTHING IN A DIFFERENT ENVIRONMENT WILL HAVE A SLIGHTLY DIFFERENT AMINO ACID SUBSTITUTION. WE'VE TAKEN ONE MITOCHONDRIAL DNA WITH TWO OR THREE CHANGES AND ANOTHER MITOCHONDRIAL DNA WITH THREE OR FOUR DIFFERENT ONES, PUT THEM TOGETHER AND ASKED THEM TO MAKE THE SAME MULTI-SUBUNIT COMPLEXES. AND SO NOW YOU HAVE A PROBLEM OF A UNIFORM NUCLEUS BUT TRYING TO INSERT DIFFERENT POLY PEPTIDES INTO THE SITE. >> OVER HERE. >> SO WHEN YOU HAVE CELLS THAT HAVE SOME MITOCHONDRIA WITH MITOCHONDRIAL DNA WITH AND WITHOUT MUTATIONS HAVE YOU TRIED TO MANIPULATE IN HANDS, MITT OH MITOPHAGY, MANIPULATIONS DONE IN RECEIVVIVO, AUTOPHAGY WOULD HAVE A BENEFIT IN ANY PATIENT? >> VERY GOOD QUESTION, MARK. I DIDN'T HAVE TIME, OBVIOUSLY, FOR OBVIOUS REASONS, TO GO INTO OUR EFFORTS TO DEVELOP DIAGNOSTICS AND THERAPEUTICS. THIS IS IN THE THERAPEUTIC AREA. I WOULD LIKE TO MENTION CARLOS' WORK, CREATING ENZYMES THAT WILL SEE SPECIFIC MITOCHONDRIAL DNA MUTATIONS, LET'S SAY PATHOGENIC, PUT THAT ENZYME IN THE NUCLEUS AND THAT PROTEIN GOES INTO THE MITOCHONDRIAL DNA, SEES IT AS FOREIGN AND DIGESTS IT. HE CAN CHANGE THE HETEROPLASMY BY THAT PROBLEM. WE HAVE A PROBLEM, HOW TO DELIVER SUCH A VECTOR TO ALL THE CELLS IN THE BODY BUT WE'RE ACTIVELY WORKING ON WAYS OF DOING THOSE THINGS. ERIC SHONE HAS PUT THEM INTO A KETO GENIC DIET BURNING FAT, HE CAN SHIFT THE HETEROPLASMA THAT WAY, A MORE CREDIBLE WAY OF TREATING PATIENTS BUT WE DON'T HAVE THE SAMPLE SIZE AT THIS POINT. THE NATIONAL INSTITUTE OF CHILD HEALTH HAS BEEN SUPPORTING A MAJOR EFFORT TO BEGIN TO GET A PATIENT REGISTRY SO THAT WE CAN DO CLINICAL TRIALS AND WE'RE GRATEFUL FOR THEIR SUPPORT ON THAT. >> OVER HERE. >> THANK YOU VERY MUCH, PROFESSOR, FOR YOUR PRESENTATION AS WELL AS YOUR CONTRIBUTIONS IN THIS FIELD. I HAVE THE SAME QUESTION AS I ASKED LAST YEAR. HOW TO GET THE DEFINITION OF MITOCHONDRIAL DISEASE, FOR EXAMPLE SOME OF THE DISEASES ARE BASED ON THE MUTATION OF MITOCHONDRIA, ENCODING PROTEINS, WE CAN SEE IT'S A DEFINITE DISEASE, BUT FOR SOME OTHER DISEASES SUCH AS MUTATION OF THE OTHER PROTEINS IN THE MITOCHONDRIA, IT CAN AFFECT THE MITOCHONDRIAL FUNCTION AS WELL AS THE MITOCHONDRIA ACTIVITY. SO HOW DO YOU THINK THOSE DISORDERS BELONG TO MITOCHONDRIA DISEASE OR ARE THEY DISEASES OF THE MITOCHONDRIAL PHENOTYPES? THANK YOU VERY MUCH. >> SAY YOUR NAME? >> [ INAUDIBLE ] >> AND THIS GENTLEMAN IS WORKING WITH WILL BORE AT NIA, DOING A VERY IMPORTANT STUDY, TRYING TO USE INFORMATION SCIENCES TO GO THROUGH THE LITERATURE AND DEFINE THE PHENOTYPES OF KNOWN MITOCHONDRIAL DISEASE TO TRY AND DEVELOP ALGORITHM FOR BETTER DIAGNOSIS. THE -- SO THIS IS AN AREA OF DISCUSSION IN THE FIELD, THERE ARE SOME PEOPLE THAT WOULD LIKE TO SPLIT THE FIELD OF MITOCHONDRIAL MEDICINE INTO WHAT THEY CALL PRIMARY DISEASE AND SECONDARY DISEASE. I ACTUALLY FEEL THIS IS REALLY MORE OF A CONTINUUM, AND THAT THERE ARE DISEASES THAT ARE DUE TO MITOCHONDRIAL DNA MUTATIONS, DISEASES DUE TO NUCLEAR MUTATIONS AND MITOCHONDRIAL GENES, ALSO THE INTERACTIONS OF THE GENES AND OF ANATOMICAL NUCLEAR GENES AS WELL. SO AT THIS PARTICULAR POINT, WE HAVE A BIG PROBLEM, BECAUSE WHAT WE SEE IS DIFFERENT MITOCHONDRIAL DNA MUTATIONS, OR DIFFERENT MUTATIONS IN NUCLEAR GENES, THEY CAN GIVE QUITE RADICALLY DIFFERENT PHENOTYPES. THAT IS ONE OF THE MOST QUESTIONS, WHY WE SPEND SO MANY YEARS TRYING TO MAKE MOUSE MODELS TO TRY AND LOOK AT ONE MUTATION TO FIND THE PHENOTYPE. BUT WHY WOULD THERE BE SUCH PHENOTYPIC VARIATION, WHY IS THE BRAIN AFFECTED IN SOME INDIVIDUALS IN THE HEART, SOME EYES, SOME KIDNEY. I THINK THE MITOCHONDRIA IS PIVOTAL TO SO MANY PARTS OF THE BODY. YOU CAN HAVE ENERGETICS, YOU COULD HAVE CHANGES IN THE MA TAB LIGHTSMETABOLITES, WE WANT EVERYTHING TO BE LINEAR BUT I THINK THIS IS SUCH A PIVOTAL SYSTEM IT AFFECTS EVERY ORGAN SYSTEM, EVERY INSTITUTE HERE AT NIH, AND WILL AFFECT THEM IN DIFFERENT WAYS, WE CAN ONLY UNDERSTAND THAT BY TRULY UNDERSTANDING THE PHYSIOLOGY OF THIS. THAT'S WHERE WE'RE AT RIGHT NOW. >> GREAT. WE'RE OVER TIME BUT WE'LL TAKE TWO MORE QUESTIONS. HERE? >> ALL RIGHT, THANK YOU VERY MUCH. VERY INTERESTING TALK. I WAS WONDERING ABOUT THE CONNECTION BETWEEN MITOCHONDRIA AND AGING, AND SO IN MITOCHONDRIAL DISEASES YOU NEED SOMETIMES 70% EXPANSION OF THE MUTATION TO GET A PHENOTYPE, WHEREAS IN AGING, IT'S UPON POSTULATED OR PROPOSED WITH AGING YOU GET ACCUMULATION OF DNA DAMAGE THAT LEADS TO MITOCHONDRIAL DYSFUNCTION, BUT THE ACCUMULATIONS THAT YOU SEE IN AGING IS MUCH, MUCH -- ON A MUCH, MUCH LOWER FREQUENCY. IS THAT SOMETHING YOU COULD ELABORATE ON? >> RIGHT. SO THAT'S A REALLY GOOD QUESTION. BUT REMEMBER, THE CELL HAS FIVE THOUSAND FAR GETS. AND SO THE CONCEPT THAT WE HAVE FROM BEING A MENDELIAN GENETICIST, TWO ALLELES, YOU GET TWO, YOU'RE DONE. WE COULD HAVE ONE OUT OF FIVE THOUSAND THAT ARE MUTANT HERE. SO YOU COULD HAVE FIVE THOUSAND MUTANTS, A DIFFERENT ONE IN EVERY MITOCHONDRIAL DNA. AND THAT CONCEPT IS ALSO VERY RELEVANT IN CANCER. WE'RE WORKING WITH CANCER, HOW IT AFFECTS CANCER CELL GROWTH, IT'S A REALLY BIG AREA. HOW DO YOU QUANTIFY THAT WHEN YOU HAVE MANY DIFFERENT MUTATIONS? SO ONE OF THE THINGS WE SPENT A LOT OF TIME AND MONEY ON IS TRYING TO DEVELOP WAYS TO LITERALLY ANALYZE EVERY SINGLE MITOCHONDRIAL DNA, DEFINE ITS MUTATIONS AND ADD THAT UP AS A POPULATION BIOLOGY. AND THEN YOU'LL SEE THAT IN FACT EVEN THOUGH ANY ONE MITOCHONDRIAL MUTATION IS VERY LOW, THE SUM TOTAL IS ACTUALLY QUITE PRODIGIOUS. WE HEARD EARLIER TODAY THE STUDIES BY LARSON AND POCOVV WHERE THEY PUT IN A POLYMERASE CAUSING THE ANIMALS TO AGE PREMATURELILY. THEY DON'T GET ONE MUTATION, THEY GET MANY MUTATIONS, AGAIN, YOU HAVE TO QUANTIFY EVERY MITOCHONDRIAL SEPARATELY. >> THANK YOU. >> THE LAST QUESTION, YOU CAN ANSWER OTHER QUESTIONS IN THE LIBRARY IN A MINUTE. FINAL QUESTION OVER HERE, YES? >> YOU SHOWED IN YOUR MOUSE STUDIES THAT THE DILATARIOUS ALLELEE WERE LESS IN EVERY JAYS BUGENERATION BUT IN HUMANS IT DIDN'T HAPPEN. >> I DIDN'T MAKE THAT AS CLEAR AS I SHOULD HAVE. OUR WORK AT THE CURRENT STATE SHOWS -- IN HUMANS, THE FEMALE GENERATES ABOUT 3 TO 5 MILLION PROTO-OCYTES, OVULATING 400 VIABLE EGGS. WHERE ARE THE REST OF THE CELLS GOING? IT SEEMS EVERY TIME A FEMALE GOES TO THE CYCLE, MANY HUNDREDS, IF NOT A THOUSAND, OF THESE BEGIN TO MATURE. AND THEN IN FACT THEY ARE IN COMPETITION WITH EACH OTHER AND ALMOST ALL OF THEM WILL DIE BY APOPTOSIS, ONLY A FEW WILL FORM A MATURE FOLLICLE AND APOPTOSE. IT TURNS OUT THE ONES WITH THE HIGHEST UNDERGO APOPTOSIS. THE CELLS WITH THE MOST MITOCHONDRIA HAVE THE MOST OXIDATIVE STRESS, SO YOU HAVE AN INTRAOVARIAN COMPETITION, THAT COMPETITION IS NOT ON OR OFF. SO EVOLUTION WANTS VARIABILITY TO GO INTO THE SPECIES. I'M ARGUING IT'S THE EVOLUTIONARY RADIATION TOOL. SO THE THRESHOLD IS NOT AT ZERO. IT'S UP AT A LEVEL. WE GET A LOT OF 3243 MUTATION PATIENTS, THAT'S DILATARIOUS WHEN IT'S HOMOPLASMIC. WHEN IT'S LOW, IT GIVES YOU MIGRAINE HEADACHES. IT'S NOT ENOUGH TO CLEAR THE OVARY BUT NOT TO SEE THE PATIENTS IN THE CLINIC. THAT'S WHY WE HAVE SO MANY SPECIFIC MITOCHONDRIAL MUTATIONS. >> PLEASE JOIN THE CONVERSATION IN THE LIBRARY OVER COFFEE AND COOKIES AND PLEASE THANK OUR SPEAKER AGAIN. [APPLAUSE]