TODAY'S TALK WILL BE COMING FROM BALTIMORE DR. MORTEN SCHEIBYE-KNUDSEN AND DR. FANG SPEAKING NEURODEGENERATION AND DNA REPAIR (INDISCERNIBLE) BE SURE EVERYBODY MUTES. LET'S TAKE IT OFF IN BALTIMORE. >> CAN YOU HEAR ME FROM BALTIMORE? >> YES WE CAN HEAR YOU. WE HAVE A DIFFERENT VIDEO SYSTEM HERE. SO JUST BEFORE WE START, I WANTED TO SAY I'M HOPING THAT ALL OF YOU CAN PARTICIPATE IN THIS ADOBE CONNECT TEST THAT WE HAVE SET UP (INDISCERNIBLE) DEERBORN, CAN YOU MUTE UP THERE? OKAY. THANKS. THIS IS AN IMPORTANT OPPORTUNITY TO TEST THE SYSTEM AND KEN AND I WORKED ON THIS FOR A WHILE AND WE HAVE SOME GREAT HELP FROM OUR PEOPLE INVOLVED. SO THIS IS A VERY IMPORTANT OPPORTUNITY TO THE TEST THIS EQUIPMENT AND TO TEST WHETHER WE CAN MOVE TO ADOBE CONNECT AND I'M HOPING THAT EVERYONE PARTICIPATES IN THAT SO WE CAN MAKE A DECISION WHETHER TO MOVE THAT SYSTEM OR NOT IN THE NEXT SEASON. WITH THAT WE SWITCH HERE TO A TALK GIVEN BY MORTEN SCHEIBYE-KNUDSEN, AND EVANDRO FANG FROM THE LABORATORY OF MOLECULAR GERONTOLOGY HERE. MORTEN IS A SENIOR POST DOC RESEARCH ASSOCIATE HERE WHO HAS DEVELOPED SOME NEW IDEAS AND THINKING ABOUT METABOLISM AND IN PARTICULAR NUCLEAR MITOCHONDRIAL SIGNALING THAT WILL BE DISCUSSED. HE AND EVANDRO HAVE BOTH PARTICIPATED AND MADE SOME VERY INTERESTING OBSERVATIONS IN REGARD TO THIS SIGNALING PROCESS. WE ARE PLEASED THEY WOULD COME A LONG WAY HERE AND GIVE THE TALK. THANK YOU VERY MUCH. >> CAN YOU HEAR US? SO I HOPE YOU CAN HEAR ME, IF NOT, PLEASE -- >> KEN, CAN YOU HEAR THIS? >> WE CAN HEAR FINE. >> ALL RIGHT. GREAT. CAN YOU SEE THE SLIDES ALSO? >> EVEN THAT. >> FANTASTIC. THANK YOU VERY MUCH. OKAY SO FIRST THANK YOU FOR THE¨¨ INTRODUCTION. THANK YOU, KEN AND WILL, FORGIVING ME THE OPPORTUNITY, I'M SURE EVANDRO IS ALSO GRATEFUL FOR GETTING THIS OPPORTUNITY TO TALK TO THIS CROWD HERE. SO IT'S BEEN A VERY EXCITING -- SOME VERY EXCITING YEARS IN THE BORE LAB WORKING WITH THESE DNA REPAIR DISORDERS BECAUSE I THINK WE ARE GETTING¨c SOME INTERESTING DATA ON SOME -- MAYBE SOME BASIC MOLECULAR PATHOGENESIS OF THESE DISORDERS THAT HAVE IMPLICATION AND THE IMPOSSIBLE THERAPEUTICS OF THESE DISORDERS AS WELL SO IT'S BEEN VERY REWARDING I THINK TO BE WORKING HERE. SO IT'S CLEAR THAT THE DNA IS UNDER A LOT OF STRESS BOTH FROM EXOGENOUS SOURCES. WEBuÖ HAVE IRRADIATION SUN EXPOSURE BUT ALSO FROM ENDOGENOUS FROM MITOCHONDRIA THAT PRODUCES FREE RADICAL. THEY CAN ALL CAUSE VARIOUS DIFFERENT TYPES DNA DAMAGE SO THE CONSEQUENCE OF DNA DAMAGE IS EITHER THAT IT GETS PREPARED OR THAT IT LEADS TO AN ACCUMULATION OF DAMAGE TO SENESCENCE OR POSSIBLY TRANSFORMATION. THESE ARE OF COURSE PATHWAYS, PROCESSES THAT CONTRIBUTE TO A NUMBER OF DIFFERENCE DISEASES. SO THE CELLS HAVE DEVELOPED NUMBER OF INTRICATE PATHWAYS TO DEAL WITH THESE. THESE INCLUDE DIRECT REVERSAL, BASIC REPAIR MISMATCH REPAIR AND THESE DEAL WITH SINGLE PHASE MODIFICATIONS. WE HAVE NUCLEOTIDE EXCISION REPAIR WHICH IS A LARGE FOCUS OF THIS TALK TODAY BUT THERE'S ALSO ENDOCRINE CROSS LINK REPAIR. NEW CLEO TIDE EXCISION REPAIR DEALS WITH HELIX LESIONS AND BULKY ADDUCTS. THERE ARE TWO SUBPATHWAYS TO THIS REPAIR PATHWAY, SO THERE'S A GLOBAL GENOMIC PATHWAY AND A TRANSCRIPTION COUPLED REPAIR PATHWAY. I'M GOING TO TALK A LITTLE BIT ABOUT CSB GENE MUTATED IN SYNDROME, AND I THINK EVANDRO WILL TALK ABOUT PIGMENTOSUM, THE XPA GENE. SO IF YOU HAVE A DEFECT IN SOME OF THESE DNA REPAIR PATHWAYS YOU CAN GET ACCELERATED ABLING. THE MOST NOTABLE SYNDROMES PROBABLY WARNER SYNDROME AND PROGERIA. AND WARNERS IS THE ONE THAT LOOKS THE MOST LIKE THE NORMAL HUMAN AGING. I HAVE BEEN PARTICULARLY INTERESTED IN COCHRAN SYNDROME AND MORE RECENTLY PIGMENTOSUM, BECAUSE THEY HAVE -- >> WE LOST BALTIMORE. CAN YOU SEE THERE DAVID? >> WE LOST THEM AS WELL KEN. WE ALSO LOST AT OHHU. WE SEEM TO HAVE LOST THE SPEAKER IN BALTIMORE. >> WE'RE BACK ON NOW. >> CAN YOU HEAR US? >> THAT'S BETTER. >> OKAY. SORRY ABOUT THAT. WE GOT DISCONNECTED. SO I WAS TALKING ABOUT -- >> A LITTLE PREMATURE AGING I THINK. >> PREMATURE AGING. SO I WAS TOLD ABOUT THESE ACCELERATEDDED AGING DISORDERS. I HAVE BEEN PARTICULARLY INTERESTED IN COCHRAN SYNDROME AND MORE REPRESENTLY (INDISCERNIBLE) PIGMENTOSUM AND MORE RECENTLY IN ATAXIA, THEY HAVE A VERY INTERESTING NEURODEGENERATIVE PHENOTYPE, PARTICULARLY CHARACTERIZED BY CEREBELLAR DEGENERATION, THIS IS MUTATION PATIENT YOU CAN SEE SIGNIFICANT ATROPHY OF THE CEREBELLUM. THE PATIENTS ALSO DEVELOP SENSORY NEURAL HEARING LOSS AND COCHRAN'S SYNDROME PATIENTS YOU SEE LOSS OF CELL TRANSLATING COCHRAN NERVE AND THIS IS PARTICULARLY TRUE FOR COCHRAN SYNDROME PATIENTS AND PIGMENTOSUM PATIENTS AND PREDOMINANT IN ATAXIA PATIENTS. THEY ALSO DEVELOP NEUROPATHY SEIZURE, PEG MEANT RETINOPATHY, MUSCULAR DISTROPHY AND PARTICULARLY THESE IN COCHRAN'S SYNDROME. WHAT'S INTERESTING IN THESE FEATURES IS YOU ALSO SEE THEM IN KNOWN BE BONA FIDE MITOCHONDRIAL DISORDERS. THIS IS A PATIENT WHERE YOU SEE PREDOMINANT VERY SIGNIFICANT CEREBELLAR ATROPHY. YOU SEE LOSS OF CELLS IN THE INNER EAR. THIS IS ALSO AN ATROPHY PATIENT WHERE YOU SEE SIGNIFICANT OPTIC ATROPHY. SO A QUITE STRAIK STRIKING OVERLAP IN THE DNA PHENOTYPE IN THESE DISORDERS WITH MITOCHONDRIAL DISORDERS. TOr QUANTIFY THIS, OR TO BETTER EXPLAIN THIS WE RECENTLY CREATED A DATABASE OF MITOCHONDRIAL DISEASES, WHERE WE HAVE GATHERED THE PHENOTYPE OF ALL, I THINK WE HAVE 45 KNOWN MITOCHONDRIAL DISEASES IN THE DATABASE NOW. YOU CAN LOG ON AND IF YOU HAVE A AREA YOU'RE INTERESTED IN TO QUANTIFY WHETHER OR NOT THAT DISEASE COULD HAVE MITOCHONDRIAL COMPONENTS. YOU DO THAT WHEN YOU DO THAT YOU GET A NUMBER OF READ OUTS, WHICH ARE THESE TOOLS THAT WE MADE TO DETECT WHETHER A DISEASE IS MODIFIED IN MITOCHONDRIAL DYSFUNCTION IN SILICONE. SO TWO IS CLUSTERING AND SUPPORT MACHINE AND THE SUPPORT MACHINE IS -- SUPPORT VECTOR MACHINE, ARTIFICIAL INTELLIGENCE WE TAUGHT THE COMPUTER WITH MITOCHONDRIAL DISEASES AND HAD NON-MITOCHONDRIAL DISEASES AND A SCORE. THESE TWO I'LL TALK ABOUT A LITTLE BIT ON THIS SLIDE HERE. SO IS HIRE ARCCAL CLUSTERING WHERE -- HIERARCHICAL CLUSTERING WHERE EACH DOT IS A DISEASE, THE RED ONES ARE BONA FIDE MITOCHONDRIAL DISEASES T GREEN ONES ARE DISEASES THAT PREDOMINANT IN NON-MITOCHONDRIAL AND THE THE BLUE ONES ARE DISEASES THAT WE ARE INTERESTED IN SO ACCELERATED AGING DISORDERS AND AGING ALSO HAS BEEN PUT INTO THIS DATABASE. TWO DISEASE APPEAR CLOSELY TOGETHER IN THIS. (INDISCERNIBLE) ARE CLOSE ASSOCIATES PEO IS NOT ASSOCIATED VERY WELL WITH COCHRAN 3 DEFICIENCY. WHAT YOU CAN SEE IS COCHRAN SYNDROME MUTATION, TO SOME EXTENT TO A SURPRISE WE FOUND ALSO PIGMENTOSUM GROUP A ASSOCIATED WITH THESE MITOCHONDRIAL DISEASES. THIS IS THE SUPPORT VECTOR SCORE, A POSITIVE SCORE OF ABOUT ONE INDICATES THAT THERE'S A LACK IN MITOCHONDRIAL INVOLVEMENT AND IT'S ACTUALLY LINKED TO ATAXIA COCKAYNE SYNDROME POSITIVE SCORES WHICH INDICATE MITOCHONDRIAL, CONTROL DISEASES HERE GET A NEGATIVE SCORE. SO ONE APPEARS TO MITOCHONDRIAL PHENOTYPES. I THINK EVANDRO WILL TALK ABOUT THAT NOW. (INDISCERNIBLE) VERY INTERESTED IN THE XE AREO DERMA PIGMENTOSUM WHICH IS A DISEASE THAT KRAMER HAS BEEN WORKING FOR MANY YEARS. I TRIED TO SHOW YOU GUYS A PICTURE WHICH IS CAME FROM HIS REVIEW SO IT GIVES A VERY SIGNIFICANT -- SOME OF THE TYPICAL CLINICAL MANIFESTATIONS OF XP INCLUDING SENSITIVITY TO UV AS WELL AS EARLY AGE CANCER, ESPECIALLY PEER GROUP SHOWS THE NEURODEGENERATION. HERE THE PEOPLE SHOW THE HEARING LOSS. SO BASED ON DATA FROM CLINICAL TRIAL WE SHOOT HIGH SCORE MITOCHONDRIAL DYSFUNCTION, WE'RE INTERESTED TO KNOW WHICH KIND OF MECHANISM INVOLVEDDED IS NEURODEGENERATION OF XPA. HERE WE RAISE THE HYPOTHESIS, WHERE THERE'S EFFECTIVE DEFECTIVE MITOCHONDRIAL FUNCTION WHICH CAUSE THIS NEURODEGENERATION XPA. TO ASK THIS QUESTION WE HAVE IN VITRO AS WELL AS IN VIVO MODELS INCLUDING WE USE FIVE GROUPS OF CELL LINES INCLUDING XPA DEFICIENT AND CORRECTED. WE ALSO KNOCK DOWN THE XPA IN TWO HUMAN PRIMARY FIBROBLASTS, ALSO CONSIDERING THE NEURODEGENERATION WE INCLUDED TWO GROUPS OF NEURONS INCLUDING RAT NEURONS AS WELL AS YFY SO WE COULDN'T KNOCK DOWN THEM, WE ALSO INCLUDE XPA WORM WHICH SHOWS LOWER LIFE SPAN THAN WILD TYPE. IN ADDITION WE ALSO INCLUDED A LIST OF MOUSE TYPE IN OUR EXPERIMENTS INCLUDING WILD TYPE (INDISCERNIBLE) SHOWED SIGNIFICANT NEURODEGENERATION SO WE HAVE COLLABORATION WITH PROFESSOR AT HARVARD MEDICAL SCHOOL INVOLVING KNOCK DOWN MICE WHICH IS SHOWN SIGNIFICANT NEURODEGENERATION AND GOOD AS A MODEL FOR OUR PERMIT. BY APPLICATION OF TWO GROUPS OF CELL LINES INCLUDING PRIMARY CELL LINE AS WELL AS IMMORTALIZED WE DID A MICROARRAY AND THE DATA SHOW HERE THAT IN BOTH TWO GROUPS THERE'S INCREASE MANY PATHWAYS ARE INVOLVED IN MITOCHONDRIAL METABOLISM. TO FURTHER INVESTIGATE WE USE ELECTRON MICROSCOPY TO FEED THE ALPHA STRUCTURE OF MITOCHONDRIALIA. THE XPA DEFICIENCY, THEMIDE MITOCHONDRIA ARE LARGER AND THERE'S MORE DENSE MITOCHONDRIA HERE AND AFTER MITOCHONDRIA OXIDATIVE TOXIC TREATMENT XPA CELLS ARE MUCH MORE SUSCEPTIBLE TO THIS TOXICANT, THAN WILD TYPE HERE GIVE A QUANTIFICATION OF OUR RESULTS WHICH IS SHOW XPA HAS MITOCHONDRIA THAN WILD TYPE AND MORE SENSITIVE TO RHODEN TREATMENT. BESIDES XPA DEFICIENCY IS NOT MUCH LARGE IRTHAN WILD TYPE SHOWN HERE. WE FURTHER TRIED TO INVESTIGATE OTHER MITOCHONDRIAL PERIMETERS INCLUDING OXIDANT -- HERE TO SHOW THAT XPA DEFICIENT HAS HIGH LEVEL OF OXIDANT CONSUMPTION. IT ALSO HAS A HIGH LEVEL OF MEMBRANE POTENTIAL AS WELL AS MITOCHONDRIAL LOSS. THOSE MITOCHONDRIAL PHENOTYPE XPA IS BASED ON THE DIRECT WAY OF XPA IN MITOCHONDRIA. TO ANSWER THESE QUESTIONS, WE PURIFIED THE THE DIFFERENT FRACTIONS OF XPA WILD LINES, WHICH IS A TWO TYPES OF TREATMENT INCLUDING THE NORMAL TYPE AS WELL AS MITOCHONDRIAL TOXICANT. SO FOR HERE YOU CAN SEE EITHER AFTER THE META DINE TREATMENT YOU CAN SEE THE BANDS IN THE MITOCHONDRIAL FRACTION TO FURTHER WE TRY TO INCLUDE ANY NUCLEAR OR CYTOPLASMIC CONCENTRATION OF THE FRACTION WE GOT, WE USE PROTEASE K TREATMENT TO DIGEST THE OUT MEMBRANE OF MITOCHONDRIA. SO THESE BANDS DISAPPEARED SO BASED ON THIS RESULT WE CONCLUDE THAT XPA MAY NOT APPEAR IN MITE CHON DREARIA BASED ON CELL LINES WE USE AS WELL AS ENVIRONMENT WE USE. SO IF XPA DOES NOT LOOK LIKE THE MITOCHONDRIA IS THIS MITOCHONDRIAL DYSFUNCTION DUE TO SECONDARY EFFECT OF NUCLEAR DNA REPAIR DEFECT. AS I SHOW YOU XPA DEFICIENCY THERE'S HIGH LEVEL OF POTENTIAL AND THE MEMBRANE POTENTIAL IS REGULATED BY DIFFERENT COMPONENTS. ONE GROUP CAUSE MITOCHONDRIA UNCOUPLING PROTEINS GOT OUR INTEREST,v ESPECIALLY UCP-2, THIS IS A PROTEIN IN NEURONS AND ALSO HAS SHOWN PROTECTION EFFECT FOR THE NEURONS ON OXIDATIVE STRESS AND OTHER CONDITIONS. THE PROTEIN WHICH REGULATES UCP-2 BASED ON PCD PATHWAY. SO WE WANT TO KNOW WHETHER THE INCREASE, MEMBRANE POTENTIAL XPA IS DUE DEFECT DNA ACTIVITY. WE DID IMMUNOBLOTTING -- YOU CAN SEE IN XP DEFICIENCY THERE IS A LOW LEVEL OF UCM 2 WHICH IS -- THERE IS INCREASED MEMBRANE POTENTIAL. WE ALSO FOUND THAT THERE IS A DECREASE IN THE ACETYLATION ACTIVITY INCREASE P-55 AS TELLATE LEVEL IN ESP DEFICIENCY THAN WILE TYPE. SO -- WILD TYPE. WE TRIED TO ASK WHETHER THE DECREASED DEACETYLATION ACTIVITY OF XPA IS DUE TO HIGH PIELATION IN THIS SEROTYPE THE HAWAIILATION FOR POP 1 CONSUMES NAD WHICH IS AN IMPORTANT COMPONENT FOR TO EXECUTE DEACETYLATION ACTIVITY. TO FIRST ANSWER THESE QUESTIONS WE DETECTED THE PIELATION IN DIFFERENT CELL TYPES INCLUDING MOBILIZED CELL LINES. WE FOUND THAT INCREASED LEVEL PIELATION INXP DEFICIENCY INCREASE LEVEL IN PRIMARY NEURONS KNOCK DOWN XPA AS WELL AS IN THE -- FOR HERE WE CAN SEE WE USE THE DAY ONE. DAY 17 WARM WHICH IS OLD AGE. WE FOUND THAT WHEN WORM GROWS OUT BOTH XPA AND WILD TYPE, THERE'S A HIGH LEVEL PIELATION THAN THAT OF WILD TYPE. IF WE COMPARE THE WORM FROM THE 7TH DAY XPA HAVE MUCH HIGHER PIELATION THAN WILD TYPE WHICH CAN VALIDATE OUR HYPOTHESIS. WE FURTHER BENEFIT FUNDING BY MOUSE MODELS, THESE XP MOUSE SHOWS VERY SIGNIFICANT NEURODEGENERATION, ACCORDINGLY WE FOUND A HIGH LEVEL OF PIELATION HERE, LOW COMPRESSION DEAS TELLATION ACTIVITY -- ACETYLATION ACTIVITY. WE FURTHER CONSOLIDATED THAT IN THIS KNOCK DOWN MOUSE, NAD LEVELS IN BELL ALMOST IS LOWER THAN THAT OF WILD TYPE. THEN WE TRY TO CHALLENGE OURSELVES WHETHER BY INTERVENTION STUDIES OR BY RESCUE THE PHENOTYPE USE THE MODELS IN OUR LAB. THE ANSWER IS YES, WE HAVE VERY GOOD RESULTS FOR THE IN VITRO STUDIES. THEN WE MOVED TO THE IN VIVO STUDIES. FIRST WE USE THE XPA-1 WORM WHICH HAS A SHORT LIFE SPAN THAN WILD TYPE SO WE TREAT THE WORM, WITH DIFFERENT SUPPLEMENTATION INCLUDING TWO NAD PRECURSORS INCLUDING THE RIBOSIDE, MONONUCLEOTIDE. WE ALSO USE POP 1 INHIBITOR ADD TO SPECIFY INHIBITOR PIELATION. THEN WE DATED THE LIFE SPAN. HERE A VERY INTERESTING YOU CAN SEE THAT THE RED LUNG IS DISEASE XPA WITH TREATMENT AND AFTER THE SUPPLEMENTATION OF (INDISCERNIBLE) SIGNIFICANT INCREASE OF LIFE SPAN TO THAT OF THE WILD TYPE LEVEL. THOSE COMMON TREATMENT ALSO INCLUDE WILD TYPE BUT ONS GO SIGNIFICANCE. OUR RESULT WERE FIRST CONSOLIDATED BY WARMth FEEDING THE POP 1 INHIBITOR. INCREASE LIFE SPAN IN XPA FIRST CONSOLIDATED THAT THE HYPOPIELATION MAYBE A REASON TO INCREASE LIFE SPAN XPA WORMS. WE FURTHER CHALLENGE OURSELF TO INVESTIGATE OUR FUNDINGS IN THE XPA MICE BY IP INJECTION WITH MICE WITH 500-MILLIGRAMS FOR 14 DAYS AN ACTUALLY YOU CAN RESCUE THE MITOCHONDRIAL PHENOTYPE, IF WE COMPARE THE TREATMENT, IT LOWERS „ DOWN THE PIE LIG HT LEVEL FILLING TREATMENT INCREASE THE DEAS TELLATION ACTIVITY OF -- AS WELL AS INCREASE EXPRESSION LEVEL OF UCP-2. VERY IMPORTANTLY RECENTLY FOR THE PROJECT WE USE SUPPLEMENTATION IN THE MOUSE IN BOTH YOUNG AN+‡h OLDER AGE. THIS SUPPLEMENTATION CAN RESCUE THE MITOCHONDRIAL PHENOTYPE WHICH IS THE SAME AS XPA. INCLUDING INCREASE ATP LEVEL AS WELL AS DECREASE OF MITOCHONDRIAL LEVELS. BASED ON THE DATA SHOWED HERE AS WELL AS LIMIT FROM OTHER DATA SO WE PROPOSE THIS MODEL IN THE WILD TYPE CELLS AFTER MITOCHONDRIAL STRESS THESE MITOCHONDRIAL STRESS CELLS INITIATE MITOCHONDRIAL WE CALL MYTOFAGE WHICH CLEAVE TO INHIBIT THE CELL TO GO TO APOPTOSIS. IN THOSE DNA REPAIR DEFICIENT CELLS, VINCE IS A HYPERPIELATION WHICH CONSUMES PATH LEVELS. THE SECOND -- IF THE CELLS UNDER MITOCHONDRIA STRESS INHIBIT THE CELL TO GO TO MYTO HAVE GIVE, CELLS SHOULD BE -- MYTO HAVE GIVE CELLS -- MYTOPHAGY, WE TRY TO EXPLAIN WHY DNA E REGENERATION IN DNA REPAIR DISORDERS. SO OUR FUTURE WORK ALSO INCLUDING SOME OTHER THINGS. THANK YOU. WHICH MORTEN WILL TELL YOU ABOUT. THANK YOU. >> THANKS, EVANDRO. I'M GOING TO GO BACK AND TALK A LITTLE BIT ABOUT THE COCKRAYEN SYNDROME AND A LOT OF STUFF EVANDRO EXPLAINED WE ALSO FIND IN COCKAYNE SYNDROME CELL LINES MOUSE MODEL. SO IT'S AN ACCELERATED AGING DISORDER. THERE'S TWO CARDINAL FEATURES, THE OTHER ONE IS NEUROGENRETIVE PHENOTYPE, ATTACKSIS SENSORY NEURAL HEARING LOSS, MUSCLE WEAKNESS AND YOU ALSO SEE LACTIC ACIDOSIS IN THE BRAIN AND THESE ARE WHAT YOU SEE IN MITOCHONDRIAL DISEASE. THE DISEASE IS CAUSED BY MUTATIONS IN THE CSA OR CSB. ISLE FOCUS ON CSB, RESPONSIBLE FOR MAJORITY OF THE COKEAYNE SYNDROME CASES. THERE'S A LOT OF LITERATURE AND IT'S SUPPOSED TO BE LOCALIZED TO THE NUCLEUS AND THE MITOCHONDRIA, IN THE KNEW COLLIE YOU IT'S INVOLVED IN TRANSCRIPTION NUCLEOTIDE EXCISION REPAIR, INVOLVED IN EXCISION REPAIR AND ALSO INVOLVED IN THE REGULATION OF TRANSCRIPTION. IN THE MITE CHON CREATE'S PROPOSED TO REGULATE MITOCHONDRIAL TRANSCRIPTION. AS WELL AS REGULATE MITOCHONDRIAL BASIC EXCISION REPAIR. SO WE FOUND A SIGNIFICANT MITOCHONDRIAL PHENOTYPE IN THE COKEAYNE SYNDROME MICE. AS WELL AS IN CSB DEFICIENT HUMAN CELL LINES. THE MICE HAVE THIS INTERESTING PHENOTYPE WITH INCREASE METABOLISM WHERE YOU SEE MRI SCAN OF THE MICE. IN OLD WILD TYPE MICE YOU GET ACCUMULATION OF FAT SO WITH AGE YOU GET A LITTLE BIT LARGER. ADS CSB MICE DON'T ACCUMULATE FAT VERY MUCH SO THEY ARE STILL SKINNY AT OLD AGE. LOOKING AT NEUROLOGY WE DIDN'T FIND MANY CHANGE -- >> JIM, WE LOST THE SIGNAL AGAIN. HAS THAT HAPPENED TO YOU OR ANYTHING ELSE? THIS IS DAVE IN KENTUCKY. >> LOST THE SIGNAL AT OHSU. >> WE'RE ON TOP OF THAT. I CALLED THE BRIDGE AGAIN. >> THINK IT'S THAT PREMATURE AGING SETTING IN AGAIN. >> ALL RIGHT. SORRY ABOUT THAT. WILL SAID IT'S MY VOICEB MAKES IT GO. >> WE HEAR YOU AGAIN. WE DIDN'T FIND MUCH NEUROLOGICAL PHENOTYPE IN THESE MICE BUT SIGNIFICANT LOSS OF CELLS IN THE SPIRAL GANGLION IN THE INNER EAR WHICH CORRESPONDS WITH HEARING LOSS AND WHAT3 COKEAYNE SYNDROME PATIENTS. BECAUSE OF LITERATURE SHOWS VARIOUS DIETS CAN EXACERBATE OR ATTENUATE NEUROLOGICAL PHENOTYPES AND OTHER MODELS NEURODEGENERATIVE DISEASE, WE THOUGHT THAT PERHAPS THAT WAS THE CASE ALSO WITH THESE COKEAYNE SYNDROME MICE. WE PUT THE MICE ON DIET CALORIC WELL AS STANDARD DIET SUPPLEMENT. AND WE THEN HAD THE MICE IN THESE DIETS FOR EIGHT MONTHS. THESE ARE THE BODY WEIGHT CURVES. YOU CAN SEE IS THE WILD TYPE MICE GAIN WEIGHT. B MICE ON HIGH FAT DIET INITIALLY GAIN WEIGHT BUT STAGNATE AND THEN ACTUALLY WEIGHING ABOUT THE SAME AS WILD TYPE MICE ON STANDARD DISEASE. THE C MICE ON STANDARD DIET WEIGHS LESS THAN THE WILD TYPE MICE ON STANDARD DIET. LOOKING AT THE LITERATURE WE DON'T SEE MUCH DIFFERENCE BETWEEN THE TWO GENOTYPES AND CALORIE RESTRICTION T B MICE ARE DEFINITELY SMALLER AND WEIGH LESS THAN THE WILE TYPE MICE. SO ONE INTERESTING INITIAL OBSERVATION WAS THAT THE HIGH FAT DIET INCREASE IN METABOLISM WE SAW IN THE B MICE. WE SHOW OXYGEN CONSUMPTION RATES SO WE HAVE THE MICE IN CAGES TO MEASURE THE OXYGEN CONSUMPTION RATE OVER 72 HOURS. THE CSB MICE HERE ON STANDARD DIET HAVE A SIGNIFICANT HIGHER OXYGEN CONSUMPTION RATE COMPARED TO WILD TYPE ON STANDARD DIET BUT IF WE PUT THEM ON HIGH FAT DIET WE GET RESCUE OF INCREASE IN METABOLISM. B MICE CALORIC RESTRICTION HAVE EXTREMELY HIGH OXYGEN CONSUMPTION RATE SO THAT EXACERBATES THE PHENOTYPE OF THE MICE. LOOKING AT HEARING LOSS WE SEE THE SAME PHENOMENON THE HIGH FAT DIET APPEARS TO RESCUE THE PHENOTYPE IN CSBU MICE, THIS IS THE CASE WHEN WE TESTED HEARING WE CAN SEE CALORIC RESTRICTION ARE FUNDALLY DEAF, DON'T REACT AT ALL TO SOUND WHEREAS HIGH FAT DIET CSB MY TAB MICE HAVE PRESERVED HEARING. LOOKING AT HISTOLOGY ON THE INNER EAR YOU CAN SEE THE HIS RICK RESTRICTED LOSS OF CELLS IN SPIRAL GANGLION AND HIGH FAT DIET APPEAR TO HAVE RESCUE OF THE CELLS AND SPIRAL GANGLION, HIGH FAT DIET RESCUE PHENOTYPE. THIS WAS THE CASE FOR MULTIPLE ANYWHERE WE LOOK WE SAW THE HIGH FAT DIET APPEAR TO RESCUE PHENOTYPE. WE DIDN'T SEE SIDE EFFECT AS DIABETES, THEY HAD A VERY GOOD GLUCOSE TOLERANCE, LOW INSULIN LEVELS AND THE LIVER LOOKED RELATIVELY NORMAL. SO THE DIET WAS ACTUALLY PRETTY L WELL. TO GET MORE TO THE MOLECULAR BASIS WHY WE SAW THESE CHANGES WE LOOK AT THE NUMBER OF DIFFERENT THINGS, ONE WAS LACTATE, FOUND TO BE INCREASE THE BRAIN OF CSB PATIENT AS WHILE BACK. CIRCULATING LACTATE LEVELS IN THE MICE WE SAW IT WAS INCREASED IN THE BLOOD OF CSB MUTANT MICE. WE DIDN'T SEE ANY CORRECTION WITH THE HIGH FAT DIET. LOOKING AT CELL LINES, WE SAW CSB MUTANT MICE, THIS IS CS 1AN CELLS WILD TYPE CSB ON SPECTOR SO THE DEFISH HUMAN CELL LINES HAVE INCREASE IN EXTRA CELLULAR RATE AS MEASUREMENT OF LACTATE PRODUCTION. LACTATE IS PRODUCED AS A BY-PRODUCT FROM GLYCOLYSIS, GLUCOSE IS CONVERTED BY NUMBER OF STEPS AND PIRUVATE TRANSPORTED TO THE MITOCHONDRIA WHERE IT'S CONVERTED TO ACETYL COA OR LACTATE SO THE PYRUVATE TO LACTATE IS VERY TIGHTLY REGULATED BY A NUMBER OF THINGS. ONE IS THIS NAD NADH RATIO. PERHAPS THERE WERE DIFFERENCES THAT COULD EXPLAIN IS T FINDINGS WITH INCREASE LACTATE. THAT APPEARED TO BE THE CASE. SO DNAD NADH RATIO WAS LOWER THAN DEFICIENT CELLS COMPARED TO THE WILD TYPE SO THAT LED US TO SUPPORT THIS IDEA PARP ACTIVATION MAYBE DRIVING THE CHANGES. PARP WAS ACTIVATED LOOKING AT THE MOUSE CEREBELLUM CSB MUTANT MICE, THIS CORRESPONDS WITH LOSS OF CERTAIN ONE. AND IN WORMS WE SEE THE¨’– SAME IN OLD CSB MUTANT WORM, WE SEE REALLY SIGNIFICANT INCREASE IN PIE RUELATION. CORRESPONDING WITH THE LOSS OF WORM CALLED S ER-2. WE INHIBIT PARP BY VARIOUS INHIBITORS. WE LOOK AT MITOCHONDRIAL SUPEROXIDE P ON THE LEFT WE CAN SEE CSB DEFICIENT CONTROL CELLS HAVE A MUCH HIGHER SUPEROXIDE PRODUCTION THAN WILD TYPE CELLS. IF WE USE PARP INHIBITORS WE CAN RESCUE THE OR ATTENUATE THIS PHENOTYPE TO SOME EXTENT IN VIVO WE CAN INJECT PARP INHIBITORS IN VIVO, WE HAVE OLD AND YOUNG WILD TYPE WITH PJ 34, A VERY POTENT PARP INHIBITOR. AND YOU CAN SEE THAT THIS HYPER IT'S ATTENUATED TO SOME EXTENT, TO THE FAR RIGHT, BASICALLY NUMBERS HERE. CSB MUTANT MICE, THE OLD MUTANT MICE HAVE A LARGE DECREASE IN OXYGEN CONSUMPTION RATE OVER TIME WHEN THEY'RE ON THIS PARP INHIBITOR INDICATING PARP 1 ACTIVATION MAYBE DRIVING, SOME OF THIS METABOLIC PHENOTYPE. SO WHAT THE CONSEQUENCE OF PARP INHIBITION LOOKING AT CSB RECRUITMENT IN CELLS, CB WHERE CELLS ARE RADIATED. WE DON'T SEE RECRUITMENT TO DNA DAMAGE SITE BUT THERE IS A DEFECT IN THE RETENTION OF CSB AT THE DNA DAMAGE SITE WHEN I KNOW INHIBITING PARP ACTIVATION. WHEN THERE'S NO PARP POLYMER CSB IS NOT PRESENT AT THE SITE. THIS INDICATES CSB IS INTERACTING WITH -- >> NOW WE LOST THE AUDIO. THIS IS DAVE AT LEXINGTON. >> CAN YOU HEAR ME IN >> WE CAN HEAR YOU NOW. >> WITH INCREASING DOSE LOADED YOU CAN SEE THAT THERE IS INCREASE SIGNAL IN THE PAR SIGNAL. THERE IS NO SIGNAL FROM NEGATIVE CONTROL, POSITIVE CONTROL SHOWS GOOD SIGNAL. CSP APPEARS TO INTERACT WITH THE PARP POLYMER, THAT MAYBE RETAINING CSB AT THE LAY SENT INDUCED DAMAGE SITE. WHAT THIS IS DOING AT THE SITE, ONE POSSIBILITY IS THAT ABLE TO DISPLACE ADB RIBOSE POLYMERIZED MODIFIED PARP ONE, USE ASSAY TO INVESTIGATE THIS. SO THIS IS A DNA OLIGOTHAT CONTAINS A DAMAGE ONE RESTRIPINGS SEE. WE PREINCUBATE WITH PARP OR PARP PLUS NAD. AND IF PARP IS DISPLACEED FROM THE DNA DAMAGE ONE CAN INSIZE -- YOU CAN SEE ON THE LEFT SIDE THERE'S NO NAD PRESENT. WITH INCREASING DOSE WE SEE LITTLE INCISION OF THIS OLIGO. HOWEVER, IF WE ADD NAD PARP 1 BINDS LESS TO DNA BUT IT DISPLAYSES FROM THE PARP 1 DNA INDICATING CSB MAYBE RECRUITED TO THE DAMAGE SITE RETAINED BY THE PAR POLYMER. WHEN PARP DOES ITS JOB POLYMERIZING ADP IN THE SRI IT REQUEST FACILITATE DOWNSTREAM REPAIR. SO ON THE RIGHT SIDE WE HAVE THIS IN VIVO EXPERIMENT TREATED CELLS WITH UV RADIATION, AND LET THEM RECOVER OVER TIME. WE SEE THE SAME PATENT SO PARP ACTIVATION WILD TYPE BRIEFLY THAT RETURNS TO NORMAL. THERE IS A PNAS PAPER THAT SHOWS DNA CAN ACTIVATE PARP. WE GET ACTIVATION OF PARP AND IT APPEARS TO BE STABLE FOR MUCH LONGER THAN THE WILD TYPE INDICATING CSB MAY NOT BE PRESENT TO DISPLACE PARP 1. HOW DOES THIS EXPLAIN THE HIGH FAT DIET STUFF? FLOSS ONE POSSIBLE EXPLANATION WHY HIGH FAT DIET IS BENEFICIAL COULD BE THROUGH A SECONDARY METABOLITE. YOU WILL INCREASE KETOGENESIS. THAT'S USED IN THE TREATMENT OF REFRACTORY EPILEPSY, YOU GIVE CHILDREN A KETOGENIC DIET WITH LARGELY FAT AND PROTEINS. AND YOU GET A VERY INCREASE IN KETONE BODIES. SO ONE POSSIBILITY WAS PERHAPS THE BRAIN CAN METABOLIZE KETONE BODIES SO IT CAN USE THE KETONE BODIES FOR THEIR FUEL AND THESE DISEASE B DEFICIENT MICE. KETONE GUYS WERE INCREASED. KETONE BODIES THIS IS THE MOST PREVALENT IN THE BLOODSTREAM. PREVIOUSLY SHOWN TO INCREASE EXPRESSION OF SERP 1. SINCE WE KNEW IT WAS ATTENUATED CELL LINES IN THE MICE WE THOUGHT MAYBE THIS COULD RESCUE DEFECT AND WHY HIGH FAT DIET RESCUE THE PHENOTYPE OF THE MICE AND INDEED THIS IS EXPERIMENT LOOKING AT VO-2 MAX WHICH IS SHOWN TO BE INCREASE IN CSB DEFICIENT CELLS. WE CAN SEE TREATED WITH KETONE GUYS WE THE GET SIGNIFICANT REDUCTION IN THIS. WE DON'T GET A COMPLETE RESCUE. LOOKING AT PROTEIN LEVELS WE CAN TREAT DEFICIENT CELLS WITH INCREASING AMOUNTS OF BETA HYDROGENASE BEAUTY RATE. WE GET INCREASE IN SETS 1 EXPRESSION LEVEL THAT LEADS TO DECREASE IN AMOUNT OF P-53 AND DECREASE IN ACETYL P-53. AT THE SAME TIME WE RESCUED LOSS OF PCP-2 THAT EVANDRO WAS TALKING ABOUT. SWITCHING TO WORMS CSB WORM WERE SHORT. WE CAN ALSO TREAT CSB WORMS WITH PARP INHIBITOR. WE USE PJ 34. THAT RESCUE IS LIFE SPAN DEFECT. WE CAN USE THE KETONE BODY THAT RESCUES THE LIFE SPAN DEFECT. WE CAN COMBINE THE PARP INHIBITOR AND THAT RESCUE IT IS LIFE SPAN DEFECT. THAT'S NO SIGNIFICANT DIFFERENCE BETWEEN THIS RESCUE AND THIS RESCUE HERE. INDICATING THEY COULD BE WORKING THROUGH THE SAME PATHWAY. SO THED IDEA IS THAT YOU HAVE DNA DAMAGE AND LESION RESPONSIBLE IS STILL AN OPEN QUESTION BUT IT LEADS TO PARP ACTIVATION, DECREASE IN SETS 1 ACTIVITY. THAT WILL SECONDARILY LEAD TO DECREASE IN UNCOUPLING PROTEINS AN INCREASE IN MEMBRANE POTENTIAL. THAT LEADS TO MOREOK¨ RAS PRODUCTION. WE THINK IT'S ABLE TO DISPLACE PARP 1. ONCE IT'S DONE PARLAYING WHATEVER IT NEEDS TO PAR LATE, CSB DISPLACES IT AND FACILITATES DOWNSTREAM REPAIR. KETONES ARE ABLE TO INCREASE SETS 1 EXPRESSION LEVEL AND THIS MAYBE THROUGH AN INCREASE IN AMOUNT OF ACETYL COA, I DIDN'T SHOW THAT SO WE THINK MANY N MAYBE WHAT IS GOING ON. WHY DO THESE CHANGES LEAD TO NEUROLOGICAL PHENOTYPE? ONE POSSIBILITY IS THE BRAIN IS PARTICULARLY SENSITIVE TO CHANGES IN PA GENETIC DEMAND TREATMENT NUMBER OF PROCESSES SPECIFIC FOR THE BRAIN, FOR EXAMPLE NEUROPEPTIDE SYNTHESIS BUT ALSO CHANGES IN ACTION POTENTIAL AND AXIAL TRANSPORT. SO IF YOU ALTER THE ENERGY METABOLISM IN THE BRAIN THEN YOU MAY ALTER THE PHENOTYPE OF THE BRAIN. PATHOLOGICAL PHENOTYPE OF THE BRAIN. PARP INHIBITION IS A VERY DEMAND HANDING PROCESS. FOR EACH NAD MOLECULE YOU CONSUME THROUGH PARP YOU HAVE TO RECYCLE AND USE FOUR AMPs -- FOUR ATP MOLECULES SO YOU CREATE TWO AMPs, SO THIS IS A VERY ENERGETICALLY DEMANDING PROCESS. A IT CANP CONSUMPTION IS INCREASE IN DISEASE DEFICIENT CELLS AND XBA DEFICIENT CELLS. THIS IS MEASURING THE HALF LIFE OF ATP IN THESE CELL LINES. THINKING ABOUT DEMAND ON THE BRAIN, NORMAL CONDITION YOU HAVE BALANCE BETWEEN SUPPLY AND DEMAND. IN MITOCHONDRIAL NEURODEGENERATION THERE'S DEFECT AND THE SUPPLY OF ATP TO THE BRAIN. DEMAND BECOMES IN DNA DEFICIENT NEURODEGENERATION YOU MAY HAVE INCREASE IN DEMAND THAT DRIVES THE SAME PHENOTYPE OF DISEASE MITOCHONDRIAL DURATION. SIMPLY BECAUSE YOU HAVE THE INCREASED A THETP CONSUMPTION. SO THIS MAYBE THROUGH DNA DAMAGE THROUGH PARP ACTIVATION THAT LEADS TO LOSS OF A THETP AND NAD PLUS. -- ATP AND NAD PLUS. SO WITH THAT I WOULD LIKE TO THANK THE PEOPLE THAT PARTICIPATED. WILL BOHR OF COURSE AND EVANDRO FANG WHO HAS DONE AN AMAZING AMOUNT OF WORK. DAVE WILSON. AND ALSO PEOPLE FROM THE NIA, METABOLISM STUDY. WITH THAT I WOULD TAKE ANY QUESTIONS. >> THANK YOU, NICE TALK. WEAL GO AROUND NOW AND ASK ONE QUESTION AT A TIME, ONE QUESTION PER SITE. THEN OTHER SITE SECOND ROUND. LET'S GO TO NIEHS. UNMUTE. >> HI, KEN, NICE TALK. I NOTED THAT YOU HAD DIFFERENT LEVELS OF OXYGEN CONSUMPTION BOTH IN TISSUE CULTURE AND WITH ANIMALS IN BOTTLINGS. DO YOU THINK HAVING DIFFERENT BASELINES OF OXYGEN CONSUMPTION IN A CSB OR XBA CULTURE OR ANIMAL CHANGES BURDEN FOR MITOCHONDRIA, THEREFORE MAYBE SHIFT WHEN YOU GET A MITOCHONDRIAL DYSFUNCTIONAL PHENOTYPE? >> I MEAN, I THINK IT'S VERY COMPLEX SYSTEM OF COURSE AND THERE ARE FEET FORWARD AND FEET BACKWARD IN ALL THIS. >> CAN YOU SWITCH THE CAMERA? >> CAN YOU HEAR ME? >> WE HEAR YOU BUT WE DONE SEE YOU, WE SEE THE SLIDE. YOU SHOULD BE ABLE TO SWITCH THE CAMERA. THERE WE GO. THAT'S BETTER. I THINK THE INCREASE IN OXYGEN CONSUMPTION, I DIDN'T SHOW THAT BUT IN THE CELLS IT GIVE PARP INHIBITOR INJECT PARP INHIBITOR, TREAT CELLS WITH PARP INHIBITOR. THEN WE GET A DECREASE IN OXYGEN CONSUMPTION, SO THE ATP DEMAND OF THE CELL HE CAN I THINK IS WHAT'S DRIVING INCREASE OXYGEN CONSUMPTION RATE. SAME IS TRUE WITH MICE PARTICULARLY THE OLD MICE WHEN WE PARP INHIBITOR AN OLD MICE AND WE SEE DECREASE IN OXYGEN CONSUMPTION, SO ATP DEMAND WHICH MAYBE RELATED TO PARP ACTIVATION IS DRIVING INCREASE IN GENETIC -- IN OXYGEN CONSUMPTION RATE. >> CLEARLY IT IS COMPLEX AND SAME IS TRUE FOR HIGH FAT DIET DECREASING NEED FOR OXYGEN.  >> THANK YOU. >> VERY GOOD. MUTE AT NIEHS. LIT'S GO UNIVERSITY OF PITTSBURG. >> HI. THAT WAS REALLY GREAT. COUPLE OF QUESTIONS BUT LET ME START WITH ONE. THE FIRST HALF, -- >> I'M SORRY, HI, THIS IS ROB SOLE, UNIVERSITY OF PITTSBURGH. >> THANK YOU. >> IN THE FIRST HALF OF THE TALK YOU DESCRIBE SUPPLEMENTATION EXPERIMENT WITH NICOTINE HIGH BOMB SIDE AND SUGGESTED -- OR SHOWED THAT INCREASED YOUR BASAL PARP ACTIVATION. THAT SORT OF BOTH COUNTER INTUITIVE AND IN PRACTICE NOT SEEM GENERALLY WHEN ONE DOES THAT. CAN YOU SPEAK TO THAT RESULT? FLOSS >> I GUESS NOT. >> ARE YOU THERE IN BALTIMORE? >> OKAY, KEN, SWITCH TOWS THE NEXT PLACE. >> DID YOU GET THE QUESTION THEN? >> NO. NO WE DIDN'T SORRY ABOUT THAT. >> LET ME REFEOFFMENT THIS IS ROB SOB L, UNIVERSITY OF PITTSBURG. VERY NICE PRESENTATION. TWO QUESTIONS STARTING WITH THE XPA SECTION. YOU SHOW AD FIGURE NR SUPPLEMENTATION REDUCED THE PARP ACTIVATION PROFILE, IF YOU WILL. THAT THE MECHANISTICALLY COUNTER INTUITIVE AND IN PRACTICE NO ONE EVER SEES THAT, MYSELF INCLUDED. CAN YOU SPEAK TO THAT RESULT? >> THE REASON I SAY THAT IS YOU WOULD EXPECT THAT MORE SUBSTRATE YOU SHOULD SEE THE SAME OR MORE PARP ACTIVATION. WE HAVE SEEN THAT, OTHERS HAVE REPORTED THAT. >> WE SAW DECREASE IN PRODUCTION WHEN WE TREATED THESE XPA DEFICIENT CELL LINES WITH THESE COMPOUNDS THAT'S A QUESTION WE ALSO GOT FROM THE REVIEWER, WE THINK THE REASON WE SAW THAT, THAT IT WAS A DECREASE IN ROSS PRODUCTION. >> THIS IS RELATED ON THE OTHER SIDE OF THE COIN, UNLESS I MISUNDERSTOOD IT, ELEVATED GLYCOLYSIS IN CSB DEFICIENT MICE FOR CELLS. NORMALLY ONE SEES DECREASE GLYCOLYSIS FROM PARP ACTIVATION. SO I THINK -- WHICH IS -- IT COULD BE THAT YOU HAVE A MUCH MORE COMPLICATED VERY LIKELY MUCH MORE COMPLICATED OUTCOME THAN SIMPLE PARP ACTIVATION. >> WHICH ARE YOU LOOKING AT THESE THING? >> HISTORICALLY GLYCOLYTIC BLOCK FROM PARP ACTIVATION IS SEEN EVERYWHERE. NEURONS, HUMAN CELLS, MOUSE CELLS, ET CETERA. WE HAVE BEEN STUDYING HUMAN -- TISSUE CULTURE MODELS AND WITH SEE A TREMENDOUS GLYCOLYTIC BLOCK, IT'S NOT REPORTED YET BUT WILL BE SOON. IT'S DEFINITELY ALWAYS A GLYCOLYTIC BLOCK FROM PARP ACTIVATION SO GIVEN THAT YOU'RE SEEING THIS SORT OF COUNTER OBSERVATION THAT I THINK JUST SPEAKS TO THE COMPLEXITY OF THE SYSTEM. >> SURE. I MEAN, I THINK THAT THE REASON WE SEE THIS IS -- SO WE HAVE ONLY MEASURED INDIRECT BY MEASURING AMOUNT OF -- OR THE EVANDRO COG PRODUCED SO WE DON'T KNOW INCREASE OR DECREASE BUT THERE'S MORE LACTATE PRODUCED, SO MY PERSONAL FEELING IS THAT THIS IS THROUGH PARP ACTIVATION, WE INHID PARP USING PJ 34 OR 3,AD. WE GET INCREASE IN THE OCAR TO UCRAR RATIO. >> I'M NOT QUESTIONING THE RESULT. I THINK IT'S TELLS US HOW COMPLICATED THESE THINGS ARE, ABSOLUTELY. >> YOU USE PARP INHIBITORSNA TOO? >> THAT ELIMINATE IT IS BLOCK ONE SEES. WE'RE LOOKING AT DNA DAMAGE DEPENDENT SO MAYBE A DIFFERENT PHENOTYPE, MAYBE SOME -- AGAIN, YOU'RE TALKING ABOUT MASSIVE AFFECTS THAT ARE GOING ON THERE. SO IT'S QUITE COMPLICATED. I AGREE. >> THANK YOU AT PITTSBURG, PLEASE MUTE THERE IN PITTS BURGLARY. BACK TO PORTLAND OREGON. OHSU. >> THANKS FOR THE TALKS. STEVEN LLOYD. I HAD A QUICK QUESTION IN TERMS OF XPA AND XPA CS MICE THAT -- DID YOU ALSO CONCOMITANTLY LOOK AT LEVELS OF OGG-1 AND SOME OF THE OTHER GLUCOSE LACES THAT WOULD BE BEING IMPORTED INTO THE MITOCHONDRIA AND WHETHER THOSE WERE BEING ACTED IN >> WE CAN DO THAT. >> THAT'S A GOOD SUGGESTION. WE (INAUDIBLE) IN OUR (INAUDIBLE) TRIAL. >> I'M SORRY, I WAS HAVING TROUBLE PICKING YOU UP ON THE MIC. CAN YOU REPEAT THE ANSWER? >> WE DIDN'T DO THAT. WE DIDN'T DO THAT IN THIS PROJECT. THANK YOU FOR FOR YOUR SUGGESTION, WE'LL CONSIDER THAT IN OUR FOREIGN PROJECT. THANKS. >> OKAY. THANK YOU. >> OKAY. THANK YOU AT OHSU. PLEASE MUTE THERE. LET'S GO TO UNIVERSITY OF KENTUCKY, LEXINGTON. >> HI (INDISCERNIBLE) FROM KENTUCKY. I HAVE A QUESTION ABOUT PATCH DISPLACEMENT BY CSB. SO THE CLASSIC PAPER BY LINDA WAS ASKED ADD LEGEND CELLS ACTUALLY ENHANCES DISPLACEMENT OF PARP FROM DAMAGE DNA. ANY COMMENT ON THAT? ALSO IS THIS DISPLACEMENT DAMAGE DEPENDENT DEPENDENT? THANK YOU. >> THANK YOU VERY MUCH. THAT'S A GREAT QUESTION. SO WE DEFINITELY SEE THE AFFINITY FOR DNA DECREASE IS WHEN PARP IS PIE LURRYLATED. SO -- PYRULATED. SO THAT CERTAINLY CONFIRMED.¨ WITH REGARDS TO -- WHETHER OR NOT THAT HAPPENS BY PARP ITSELF IN THE IN VIVO SITUATION, JUMPS OFF BY ITSELF OR WHETHER OR NOT IT NEEDS HELP, IT NEEDS HELP FROM FOR EXAMPLE CSB. WITH THE SPECIFIC -- SPECIFICITY TOWARD THE DAMAGE SITE WE HAVE DONE LOOKING AT UV DISCHARGE AL GO, IT DOES APPEAR CSB IS p ABLE TO DECREASE THE ACTIVATION OF PARP ONE ON A UV DAMAGING GO. -- DAMAGED OLIGO. BUT WE HAVEN'T DONE ANYTHING EXTENSIVE WHETHER I THAT. THANK YOU. >> THANK YOU. >> OKAY. THANK YOU. PLEASE MUTE THERE IN KENTUCKY. ANYONE AT BROOKHAVEN? ANYONE HERE HAVE QUESTIONS? I HAVE ONE. THE WE PUBLISHED AN AUTOPSY ON 4 XP PATIENTS WHO DIED, XP, TWO OF THE XPC PATIENTS HAD BRAIN TUMOR S AND OVARIAN TUMORS UTERINE TUMORS BUT WE HAD TWO XP PATIENT WHOSE DIED OF NEUROLOGIC DEGENERATION. BOTH IN THEIR 40s AND THEIR BRAINS WERE INFANTILE SIZE WITH DEGENERATION OF CEREBELLUM BUT ALSO MORE CEREBRUM. INTERESTING THING ABOUT THAT, ONE WAS XPA AND WAS VERY KICKECTIC BUT THE OTHER WAS XPD, AND WAS IF ANYTHING OBESE. BOTH HAD SIGNIFICANT NEURODEGENERATION. DOES THAT SAY ANYTHING TO YOUR THEORY OF FEEDING HIGH FAT DIET? >> SURE. IT'S A VERY -- THERE'S DEFINITELY MANY, MANY FACTORS THAT REGULATE OBESITY AND ENERGY INTAKE. I THINK IT'S VERY DIFFICULT TO CONCLUDE ANYTHING FROM A SINGLE CASE BUT THAT CERTAINLY IS -- IT WAS CERTAINLY FOLLOW FROM OUR THEORY THAT THE NEURODEGENERATION WOULD GO ALONG WITH THE WEIGHT LOSS. IN MANY OTHER NEURODEGENERATIVE DISORDERS SUCH AS PARKINSON DISEASE AND HUNTINGTON DISEASE YOU SEE A STRONG CORRELATION BETWEEN WEIGHT LOSS AND DISEASE PROGRESSION. SO WHILE THIS IS SPECIFIC, NEUROGENRETIVE OR SPECIFIC ASPECT OF CS NEUROGENERATION OR SOMETHING GENERAL I THINK IS STILL AN OPEN QUESTION. >> WELL THE OBESE PATIENTS ALSO HAD A SIMILAR NEURODEGENERATION, I DON'T THINK IT'S THAT SIMPLE. THE OTHER QUESTION LOOKING FOR DELETION JAY ROB AND PJ BROOK IS LOOKING AT CYCLOPURINE AND THE MEETING IN JAPAN DR. MORAY ANNOUNCED AN ANTIBODY TO -- HAVE YOU DONE THAT IN THE INVESTIGATION LOOKING FOR THAT? THESE WOULD BE A STABLE OXIDATIVE LESION THAT MIGHT BE A CANDIDATE. >> I MEAN, THAT WOULD BE EXTREMELY INTERESTING TO LOOK AT. IT'S NOT SOMETHING THAT WE HAVE DONE BUT I THINK THAT WOULD BE VERY INTERESTING. >> THANK YOU. ARE THERE ANY QUESTIONS ANY OF THE OTHER SITES? ANY QUESTIONS IN BALTIMORE? >> THIS IS DAVE IN KENTUCKY. DAVE WARREN. GOING BACK TO DR. (INDISCERNIBLE)'S QUESTION, RAISES AN INTERESTING POINT, CSB IS NEEDED TO DISPLACE PARP, HAVE YOU LOOKED AT WHAT HAPPENS IN CSA DEFICIENT? I KNOW YOU FOCUS ON CSB BUT CSA MAY HAVE A SORT OF DIFFERENT ROLE IN REPAIR THAN CSV AND NOT THE PHENO-- THOUGH THE PHENOTYPES ARE THE SAME. >> THAT IS SOMETHING WE ARE INVESTIGATING BUT THAT'S VERY INTERESTING. MITOCHONDRIAL PHENOTYPE IS RELATED TO THE ATP ASPECT OF THE DISEASE. WHETHER THROUGH PARP ACTIVATION I THINK WE HAVE SOME EVIDENCE FOR THAT BUT THERE MAY ALSO BE PROCESSES THAT ARE AT PLAY. WITH RESPECT TO CSA WE ARE INVESTIGATING THAT CURRENTLY. >> THANKS. >> SCOTT WILLIAMS NIHS. JUST CURIOUS, YOU WERE SEEING POTENTIALLY DIRECT INTERACTIONS OF CSB WITH PAR, IS THAT CORRECT IF I REMEMBER FROM THE SLOT BOTH EXPERIMENT? I WAS WONDERING, DOES CSB HAVE ANY DOMAINS FOR SIGNATURES FOR PARP BINDING MACRO DOMAINS PRCT DOMAINS FTA DOMAINS OR DO YOU THINK THAT'S MECHANISTICALLY IMPORTANT DIRECT INTERACTIONS THERE THROUGH RECRUITMENT FROM OTHER UNKNOWN PAR INTERACTING MODULES? CSB HAS FOUR PARP BINDING DOMAINS. THIS IS DONE IN COLLABORATION WITH (INDISCERNIBLE) LAB. WE ARE IN THE PROCESS OF MUTATING THESE DOMAINS TO SEE WHETHER OR NOT WE CAN DISRUPT THIS ASSOCIATION. >> ARE THESE NEW FOLDS OR OTHER PAR INTERACTING ONES? >> THESE ARE THE -- I CAN'T REMEMBER I CAN CURRENT -- CAN'T CURRENTLY REMEMBER THE TYPE OF DOMAINS BUT -- WE CAN EMAIL BACK AND FORTH AND I CAN SEND YOU THE INFORMATION IF YOU'RE INTERESTED. >> I WAS JUST CURIOUS. THANKS. >> THIS IS BEN (INDISCERNIBLE) UNIVERSITY OF PITTSBURG. CAN YOU HEAR ME? >> YES. DID YOU USE ATP IN THE EXPERIMENT THAT PUSHED OFF PARP? IT COULD BE A BAT OF THE KDs, IF YOU ADDED ANY DNA BINDING PROTEIN YOU PROBABLY DISPLACED PARP. SO WAS IT ATP DEPENDENT. SECOND POINT IS I LOVE YOUR NEW WEBSITE, EXCITING AND INTERESTING THAT ATAXIA CLUSTERS WITH XPA AND AT IN YOUR CLUSTER DIAGRAM. AND IT WOULD BE VERY INTERESTING TO PUT XPF AND ERCC 1 PHENOTYPE TO SEE P IF IT WAS THERE. THANK YOU. >> YEAH. WITH REGARDS TO THE ATP, THIS WAS DONE WITH ATP, WE HAVEN'T FOUND ANY -- THERE DIDN'T SEEM TO BE DEPENDENCY ON ATP ON THIS PROCESS. I THINK IN AGREEMENT WITH¨ OTJe$ LITERATURE SHOWING WHATEVER CSP IS DOING DOESN'T DEPEND ON ATP SO IT MAYBE A BATTLE OF KD AS YOU'RE SAYING. REGARD TO THE WEBSITE GLAD YOU LIKE IT. WE HAVEN'T ADD XPF YET BUT THAT'S INTERESTING.(d ARE THERE ANY MORE QUESTIONS? IF NOT, WE WANT TO THANK YOU FOR THAT WONDERFUL PRESENTATION. [APPLAUSE]