IT'S A PLEASURE TO WELCOME ROS SEGAL FROM THE HARVARD MEDICAL INSTITUTE AS OUR NEUROSCIENCE SPEAKER TODAY, I'VE KNOWN ROS SINCE WE WERE A RESIDENT IN NEUROLOGY AS SHE WAS ATTENDING THE INSTITUTE IN ISRAEL, SHE GOT HER UNDERGRADUATE DEGREE FROM HARVARD AND THEN M.D. Ph.D. FROM ROCKEFELLER AND DID CLINICAL TRAIN ING IN NEUROLOGY AND HAS BEEN A FACULTY HARVARD AND NOW AT THE DANA FARBER CANCER INSTITUTE FOR THE LAST 23-25 YEARS OR SO, AND SHE'S BEEN A LEADER IN BOTH ACTOR SIGNALING AS THE INVOLVEMENT OF GROWTH FACTOR SIGNALING PATHWAYS IN NORMAL DEVELOPMENT AS WELL AS IN DISEASES OR THE PARTICULAR FOCUS ON PRIMARY TUMORS OF THE NERVOUS SYSTEM. SO CURRENTLY SHE'S NOT ONLY A PROFESS ONOR OF NEUROBIOLOGY AT HARVARD BUT TED WILLIAMS FOR THOSE WHO ARE BASEBALL FANS, AS WELL AS DIRECTOR OF THE NEUROSCIENCE PROGRAM HARVARD MEDICAL SCHOOL, SO ALL OF US THAT ARE BASEBALL FANS WOULD LOVE THAT HAVE THAT TITLE, ESPECIALLY THE ONES FROM BOSTON SO IT'S A PLEASURE TO HAVE YOU AGAIN. >> WELL THANK YOU FOR THE INTRODUCTION AND THANK YOU FOR BEING HERE ON CHRISTMAS WEEK AND I SHOULD SAY THAT THE TED WILLIAMS INVESTIGATOR USED TO COME WITH OPENING DAY TICKETS AND SOMEBODY TOOK THEM AND I HAVEN'T FIGURED OUT WHO BUT I'M STILL TRYING TO FIND OUT. SO WHAT I WOULD LIKE TO DO TODAY IS TELL YOU ABOUT THE WORK THAT WE'VE BEEN DOING ON NEUROTAUPEINS AND HOW THEY REGULATE AXONAL VIABILITY AND TALK ABOUT THE APPLICATION OF THAT TO DISEASE. NOW WE'VE DONE SINCE THE TIME OF LEVY MONITOR TELECHANEY AND VICTOR HAMBERG, THAT THE NEUROTROVEINS ARE IN THE CRITICAL FOR DEVELOPMENT AND SURVIVAL OF SENSORY NEURONS IN THE ONS IN THE DORSAL ROOT GANGLIA. --YOU HAVE TO MAINTAIN THE LONG AX WITH THE TARGET IN ORDER FOR THE--FOR THIS TO BE A FUNCTIONAL CIRCUIT AND FOR THERE TO BE THE ABILITY OF THIS SENSORY SYSTEM TO RESPOND TO STIMULATION. AND SO HOW DO THESE TARGET DRIVE NEUROTROPHIC FACTORS REGULATE THE CIRCLE NOT JUST OF THE NEURON BUT THIS COMPLEX MORPHOLOGY OF THE SENSORY NEURONS. AND SO WE KNOW A LOT OF THE INITIAL STEPS THAT ARE INVOLVED, NEUROTROUGHINS, EGS, BDNF AND NEUROTROUGHIN THREE AND FOUR, BOND TO THE TYROSINE KINASES IN THE PLASMA MEMBRANE. FOR EXAMPLE, NG F TO TRACK A BDNF TO TRACK THE TETRAAND THE BINDING INITIATES PHOSPHORYLATION AND ACTIVATION OF THE TYROSINE KINASE AND THIS IN TURN INITIATES SEVERAL INTRACELLULAR SIGNALING CASCADES INCLUDING TLC GAM ANDA THE RAS MAPP K KINASE PATHWAY AND THESE ARE CRITICAL FOR INITIATING RESPONSES TO THE TARGET DRIVE NEUROTAUPEIN. BUT THIS DOESN'T EXPLAIN HOW THE SIGNALING PATHWAY IS ADAPTED, THE ANATOMY OF STIMULATING THE END, THE DISTAL AXON AND EFFECTING A CELL BODY AND MAINTAINING SURVIVAL, BOTH OF THAT CELL BODY AND OF THAT LONG INTERVENING AXON. AND SO TODAY WHAT I'D LIKE TO DO IS TO BEGIN BY TELLING YOU ABOUT A WORK OF MANY YEARS LOOKING AT HOW NEUROTAUPEIN STIMULATION OF AXONS REGULATES GENE EXPRESSION AND IN PARTICULAR HOW THEY'RE A SET OF WHAT WE CALL RETROGRADE RESPONSE GENES WHOSE INDUCTIONS DEPENDS ON NEUROTROUGHIN STIMULATION OF THE AXON SPECIFICALLY. I'LL THEN TALK ABOUT THE FUNCTION OF ONE OF THESE RETROGRADE RESPONSE GENES, BCL W AND HOW IT PROMOTES THE SURVIVAL OF THE AXON ITSELF AND TALK ABOUT THE MECHANISMS REGULATING THE BCLW AND IN THE END, I WILL TURN FROM UNDERSTANDING OF THE BASIC QUESTION OF HOW NEUROTROUGHINS REGULATE AXONAL SURVIVAL, TALK ABOUT THE RELEVANCE FOR DISEASE, PARTICULARLY CHEMO INDUCED NEUROPATHY. WHAT WE USE TO STUDY THE MECHANISMS BY WHICH TARGET DRUG NEUROTROUGHINS EFFECT SENSORY NEURONS ARE COMPARTMENTED CULTURES ARE DEVELOPED BY BOB CAMPENOT, AND THIS LEADS TO THREE SEPARATE ISOLATED COMPARTMENTS, WE THEN PLATE SENSORY NEURONS IN THE CENTER COMPARTMENT AND THEY EXTEND THEIR AXONS, INSIDE THE COMPARTMENT AND THIS ALLOWS US TO SELECTIVELY STIMULATE THE DISTAL AXONS WITH NEUROTROPIC FACTOR BECAUSE THIS REDUCES REPPIZATION BETWEEN CELL BODIES AND AXON THAT THAT ARE OBSERVED IN VIVO, THESE ARE GOOD FOR DOING BIOCHEMISTRY, HOWEVER MORE RECENTLY WE TURNED TO USING MICROFLUIDIC CHAMBERS TO AGAIN REPLICATE THE SEPARATION BETWEEN CELL BODIES AND DISTAL AXONS OF THE SENSORY NEURONS IN A WAY THAT IS MUCH MORE AMENABLE TO IMAGING BECAUSE THIS IS--THIS CULTURE IS DONE ON A GLASS SLIDE. SO USING THESE METHODS WHAT WE--WE IDENTIFIED A SET OF WHAT WE CALL RETINAL LOCATION PROGRADE RESPONSE TUNES SO THESE ARE CHAINS THAT ARE INDUCED WHEN WE SELECTIVELY STIMULATE THE SENSORY AXONS WITH NEUROTROUGHINS, IN OUR CASE NG F AND BDNF AND NOT INDUCED WHEN WE STIMULATE THE CELL BODY DIRECTLY, YOU CAN SEIZE THESE ARE AGAIN INDUCED IN A VERY SPACIALLY LIMITED MANNER AND OUR CONTROL HERE AND THROUGHOUT THE TALK IS C-CROSS, THERE'S AN IMMEDIATE GENE THAT INDUCED BY NG F AND OTHER NEUROTROUGHINS REGARDLESS OF THE LOCATION OF STIMULATION. SO TO UNDERSTAND THE RETROGRADE RESPONSE GENES AND HOW THEY'RE REGULATED AND THEIR FUNCTION, WE FOCUSED ON ONE OF THESE WHICH IS BCL W, ALSO KNOWN AS BCL, TWO, LTWO. AND WORK FROM OUR LAB AND OTHERS OVER MANY YEARS HAS REALLY DEMONSTRATED THE MECHANISM BY WHICH TROUGHINS SIGNAL BACK TO THE NUCLEUS TO EXCHANGE GENE EXPRESSION. NG F OR OTHER NEUROTROUGHINS AT TYROSINE KINASE AT THE OXON, THIS IS ENDOSOME THAT'S TRANSPORTED BACK BY DINING BASE TRANSPORT TO THE CELL BODY. THERE THE ACTIVATED RECEPTOR WITHIN A VESICLE INITIATES CHANGES IN TRANSCRIPTION FACTORS AND CHANGES IN GENE EXPRESSION. VIA NUCLEAR CASCADE. OKAY. SO, SINCE THIS IS THE MECHANISM THAT REGULATES THE CHANGES IN BCL W EXPRESSION, SO TO UNDERSTAND WHAT--WHY YOU HAVE RETROGRADE RESPONSE GENES, GENES THAT ARE ONLY INDUCED WHEN YOU STIMULATE THE AXONS AND NOT WHEN YOU STIMULATE THE CELL BODY, WE WANT TO UNDERSTAND WHAT THE FUNCTION OF BCLW IS, AND SO STEPHANIE WHO'S A GRADUATE STUDENT IN MY LAB AND NOW WORKS IN FRANCIS COLLINS OFFICE WANTED TO LOOK AT THIS QUESTION, SO JUST TO REMIND YOU BCL W OR TWO LIFE TWO IS NOT SURPRISINGLY LIKE BCL, TWO. IT'S A PROSURVIVAL MEMBER OF THE BCL TWO FAMILY MEMBER AND IT BINDS, IT BINDS TO THE ONLY COMPONENTS AS WELL AS TWO BACKS AND BCL TO LTWO, BCL W INHIBITS THE ACTIVATION OF BACK AND THE SUBSEQUENT INITIATION OF THE APOPTOTIC MACHINERY AND CASPASE DEPENDENT APOPTOSIS. SO WHAT'S SO SPECIAL ABOUT BCL W AND WHY WOULD YOU HAVE IT INDUCED ONLY WHEN YOU STIMULATE THE AXON? AND WHEN STEPHANIE LOOKED AT THE BCL KNOCK OUT, AND AT TWO MONTHS OF AGE, SHE WAS--YOU CAN CAN SEE SOME OF THESE. SO WHAT YOU CAN SEE IS LOOKING AT THE INNOVATION OF THIS SKIN, CAN YOU LOOK AT THE LABELED SENSORY NEURON ENDING PGP9.5, CAN YOU SEE AT TWO MONTHS OF AGE THE MUTANT IS LIKE THE WILD-TYPE. BY SIX MONTHS OF AGE YOU SEE LOSS OF INNERVATION OF THE SKIN IN THE MUTANTS AND THIS IS QUANTIFIED HERE. THERE'S PROGRESSIVE LOSS OF INNERVATION OF THE SKIN AND.LY THIS OCCURS WITH NO LOSS OF DORSAL ROOT GANGLIA NEURONAL CELL BODIES. SO THIS IS A LOSS OF THE AXON ENDING THAT OCCURS WITH AGE, A PROGRESSIVE AXONAL NEUROPATHY. STEPHANIE WANTED TO KNOW IF THE LOSS OF INNERVATION WAS SIGNIFICANT TO BE MEANINGFUL. SO SHE LOOKED AT SENSATIONS WITH A HEAT PLATE AND MECHANO SENSATION WITH HAIRS AND WHAT SHE FOUND WAS THAT OVER TIME, AT TWO MONTHS OF AGE, THE ANIMALS WERE NORMAL, BY FOUR MONTHS OF AGE, THEY HAD LOSTOT RESPONSE TO NOXIOUS HEAT AND BY 12 MONTHS OF AGE THEY HAD ABNORMALITIES IN MECHANICAL SENSATION. SO, IN OTHER WORDS, WHAT BCL W IS DOING IS MAINTAINING THE AXON IN A PHYSIOLOGICALLY RELEVANT MANNER. SO IF BCL W IS PROMOTING AXONAL SURVIVAL, HOW ARE NEUROTROUGHINS REGULATING BCL W TO PROMOTE THIS SURVIVAL? AND AGAIN TO THE TO MAKE TOO MUCH OF TED WILLIAMSS, BUT YEAH, THIS IS THE ACTUAL TROPHY, I WANT YOU TO KNOW, AND THIS IS THE GROUP WHO DID THIS. SO THIS HAS BEEN SPEAR HEADED BY KAT KOSKER A POST DOC IN THE LAB AND THEN TWO GRADUATE STUDENTS, SARAH AND SARA AND MY LONG TIME LAB MANAGER MARIA. SO OUR FIRST QUESTION WAS WHERE IS THE--WE KNOW THAT NEUROTROUGHINS ARE UPREGULATES BCL RNA IN THE BODIES SO WE WANT TO SEE WHERE THE BCL W RNA IS LOCATED IN VIVO AND IS IT AGAIN EXPRESSED IN THE SENSORY NEURONS IN VIVO. SO WHEN WE LOOKED AT THIS, WHAT WE FOUND IS THAT BCL W BY INSITU HYBRIDIZATION IS CLEARLY EXPRESSED IN THE DRGs. BUT WHAT WAS MORE SURPRISING TO US IS THAT LIKE BETA ACTIN, WE ALSO SAW SIGNAL, ALONG THE NERVE SUGGESTING IT MIGHT BE RNA PRESENT IN THE AXONS AS WELL. AND WHAT WAS MORE STRIKING FOR US WAS AS WE LOOKED AT IT, IT WAS PRESENT IN THE PERIPHERAL BRANCH AND NOT IN THE CENTRAL BRANCH AND BETA [INDISCERNIBLE]. SO, OBVIOUSLY IN VIVO, THESE NERVES CONTAIN MUCH MORE BEYOND THE AXONS AND THEY'RE PRESENT SO TO FIND OUT IF BCL W RNA IS PRESENT IN THE AXON AS WELL AS IN THE CELL BODIES, WE TURN TO OUR INVITRO SYSTEM. AND IN THE CULTURES, BCL W LIKE THE ACT SIN RELATIVELY ENRICHED IN THE AXONS COMPARED TO THE CELL BODIES, AND BY INSITU HYBRIDIZATION AND AND IN THE AXON WHICH IS SIMILAR TO BETA ACTIN AND DISTINCT FROM GAMMA ACTIN, IT'S FOUND IN BOTH LOCATIONS ARE NEUROTROUGHINS REGULATING THE BCLRW IN BOTH OF THESE PLACES. SO WE STIMULATE THE DISTAL AXONS AND NOW WE LOOK AT THE RNA, BOTH IN THE CELL BODIES BOTH IN THE AXON AND WHAT YOU CAN SEE HERE, WE STIMULATE THE DISTAL AXONS, YOU CAN SEE FALSE NICELY AND IT'S IN THE DISTANT AXONS. BCL W INCREASES BOTH IN THE CELL BODIES, AND IN THE AXONS AND BETA ACTIN AS PREVIOUSLY SHOWN INCREASES IN THE AXON, IN CONTRAST IF WE STIMULATE THE CELL BODIES AGAIN, BCL IS A RETROGRADE RESPONSE GENE, SO YOU SEE NO INDUCTION WHEN YOU STIMULATE THE CELL BODIES, AND THERE'S NO CHANGE IN THE CELL LEVEL OR IN THE AXON AND THIS CONTRAST AND BETA ACTIN WHICH DO WHAT ONE WOULD EXPECT. >> PGH OKAY, SO BOTH OF THESE ARE RESPONDING NEUROTROUGHINS SO SINCE WE HAD SHOWN PREVIOUSLY THAT THE NEUROTARGETS, NEUROTROUGHINS WERE INITIATING TRANSCRIPTIONS OF RCLW WHICH TAKES PLACE IN THE NUCLEUS, WE WANTED TO KNOW WHETHER IT WAS IN FACT THESE NEWLY TRANSCRIBED RNAs THAT ARE BEING TRAFFICS OUT TO THE AXONS OR WHETHER YOU'RE AFFECTING BOTH IN A DIFFERENT MANNER. SO TO DO THIS, WE USED FOUR-THIOURIDINE, AND THEN CHASE FOR TWO HOURS IN THE ABSENCE OF URIDINE, WE NOW STIMULATE THE AXONS AND PURIFY THE RNA FROM EACH COMPARTMENT AND THEN WE LOOK AT THE STYLE LABELED NEWLY TRANSCRIBEDDED MRNA. OKAY SO IF WE STIMULATE THE DISTAL AXON AND LOOK IN THE CELL BODIES, WHAT YOU SEE IS AGAIN AS A NICE CONTROL ROBUST INITIATION, AND YOU INITIATE THE TRANSCRIPTION AND BETA ACTIN, AND NOW WHEN WE LOOK IN THE DISTAL AXON WE SEE, THERE'S NO C-PULSE THERE, THE NEWLY MADE BCLW IS ACTIVE WITHIN THE TWO HOURS OF STIMULATION AND THIS IS NOT THE CASE FOR BETA ACTIN AND COULD RESULTS THAT ARE CONSISTENT WITH JEFFREY TWIST'S PREVIOUS STUDIES. SO WHAT THIS SAYS TO US, THEN IS THE FOLLOWING: IS THAT THERE AND BACK AGAIN MODEL, NEUROTROUGHIN STIMULATION IS DISTAL AXON AND INITIATES THE SIGNALING ENDO STUDIES OF MULTIPLE ENDOCRINE AND TRAVEL BACK TO SELL BODY, GENERATES NEW TRANSCRIPTS THAT ARE IMMEDIATELY TRANSPORTED BACK OUT TO THE AXON IN A WAY THAT SEEMS SOMEWHAT SURPRISING AND VERY ENERGETICALLY DIFFICULT. NOW IT'S HARD TO IMAGINE YOU WOULD DO THAT UNLESS YOU WERE GOING TO TRANSLATE THE RNA THAT YOU GOT OUT THERE AT SUCH COST INTO PROTEINS. AND SO, SINCE THERE'S SOMETIMES A KOPBT ROUGH ATOM VERSEY ABOUT RIBOSOMES IN AXONS, I WANT TO SHOW YOU IN THE AXONS THAT WE'RE LOOKING AT INVITRO, THIS IS LOOKING AT A LABELED LARGE RIBOSOMAL SUBUNIT, WITH A CONTROL, THIS IS LOOKING AT LARGE SUBUNIT, A SMALL SUBUNIT OF THE SMALL OTHER OF THIS SMALL IBE SO --RIBOSOMAL SUBUNIT AND THEN THE RIBOSOMAL RNA AND THE TRNA SYNTHASE THAT LOAD UP THE AMINO ACIDS SO THEY CAN BE TRANSLATED AND SO ALL OF THESE ARE PRESENT AND WE THINK THAT WITHIN OUR SYSTEM, THERE CLEARLY IS TRANSLATION GOING ON IN THE AXONS SO WHEN WE LOOK AT BCL W PROTEIN WHAT WE SEE IS BCLW PROTEIN IS PRESENT IN THE CELL BODIES AND IN THE DISTAL AXON AND INTERESTINGLY THIS CONTRAST WITH BCL TWO WHICH IS PRESENT IN THE SELL BODY BUT REALLY HARD TO SEE AT ALL IN THE AXON AND LOOKING BIOCHEMICALLY WE SEE THAT AGAIN BCL W PROTEIN IS RELATIVELY ENRICHED IN THE AXONS, WITH BCL TWO IS CONCENTRATED IN THE SELL BODY. SO THESE SUGGEST TO US THAT BCL, LW HAS A ROLE IN AXONAL SURVIVAL IS REALLY THE FAMILY MEMBER, THAT IS SELECTIVELY LOCATED IN THE AXONS TO PROMOTE SURVIVAL. SO ALTHOUGH THE PROTEIN IS THERE, THIS DOESN'T TELL THAWS IT'S BEING TRANSLATED THERE, AND FRANKLY WE CAN'T AT THIS POINT UNEQUIVOCALLY STATE THAT BCL IS LOCALLY TRANSLATED AND WHAT WE CAN SAY, IS THAT WHEN YOU STIMULATE WITH NEUROTROUGHINS YOU GET AN INCREASE IN BCL PROTEIN IN THE CELL BODIES AND IN THE AXON. AND WE NOW ADD PSYCHE LOW HEXIMIDE TO THE AXONS TO BLOCK LOCAL SYNTHESIS WE BLOCK THE INCREASE IN THE AXONS SO WE DO NOT EFFECT THAT SCENE IN THE CELL BODY AND THIS IS SHOWN QUANTITATIVELY HERE. OKAY, SO, THESE DATA ARE VERY SUGGESTIVE THAT THERE IS INDEED LOCAL TRANSLATION OF BCLW AND WE'RE WORKING ON METHODS OF DEMONSTRATING THIS IN A MORE DEFINITIVE MANNER. NONE THE LITTLE, WE WANT TO KNOW WHETHER THESE PROCESSES OF LOCAL SYNTHESIS, INCLUDING AND ARE CLEARLY IMPORTANT FOR INCREASING BCLW LEVELS ARE IMPORTANT FOR SURVIVAL AND WHAT WE DID AS CYCLOHEXIMIDE TO THE AXONS AND WHAT WE FIND IS WE NOW BLOCK NEUROTROUGHIN DEPENDENT SURVIVAL. AND IN THIS FIGURE, WHAT WE ARE DOING IS QUANTIFYING NEURONAL SURVIVAL BY LOOKING AT FRAGMENTATION OF THE DISTAL AXONS AS SHOWN HERE IN THIS VIEW, WE THEN SEE HOW FRAGMENTED THE AXONS BECOME. SO TOGETHER THAT'S FOR OUR DATA REALLY PROVIDE A MODEL OF WHAT'S HAPPENING, NAMELY THAT NEUROTROUGHIN STIMULATION INISSUEIATES THE SIGNALING ENDO STUDIES OF MULTIPLE ENDOCRINES THAT GO BACK AND INDUCE TRANSCRIPTION OF BCL W, AND NEWLY MADE BCL W IS TRANSPORTED BACK AND MADE INTO PROTEINS AND WE BUILDING THAT THE LOCAL TRANSLATION MAY ALSO BE REGULATED BY NEUROTROUGHINS. ONE OF THE VERY INTERESTING FEATURES OF THIS IS THAT THIS PROVIDES A WAY IN WHICH YOU CAN HAVE BOTH SPACIAL AND TEMPORALLY DIFFERENT RESPONSES TO THE SAME GROWTH FACTORS. BECAUSE, OKAY. YOU HAVE THESE GROWTH, YOU HAVE A PROGRAM OF GENE INDUCTION THAT IS REALLY SELECTIVE FOR STIMULATION THAT DEPENDS ON THE SIGNALING ENDO STUDIES OF MULTIPLE ENDOCRINES, SO ONLY OCCURS IN RESPONSE TO NEUROTROUGHINS AND THAT YOU DON'T SEE WHEN YOU DIRECTLY STIMULATE THE CELL BODIES. BUT BECAUSE BCLW IS OTHER RESPONSE GENES ARE REGULATED AT MULTIPLE LEVELS, NAMELY IN RESPONSE TO THE NEUROTROUGHINS, YOU HAVE INDUCTION OF TRANSCRIPTION AND THEN THE TRANSPORT OF THE NEWLY MADE RNA WITH THE AXON AND WE THINK, LOCAL TRANSLATION OF THIS OF THE RNA INTERPROTEIN, THIS ALLOWS FOR A SPACIAL ASPECT TO REGULATION AS WELL. UNLESS THE NEUROTROUGHINS ARE PRESENT FOR A PROLONGED PERIOD OF TIME AT THE DISTAL AXONS, YOU WILL NOT SEE INDUCTION OF THESE RETROGRADE RESPONSE GENES. THIS MAKES SENSE PHYSIOLOGICALLY BECAUSE AS AN AXON IS GROWING OUT, AS IT GOES PAST THE INTERMEDIATE TARGETS, YOU DON'T WANT THAT TO BE SUFFICIENT TO STABILIZE THE AXON. YOU ONLY WANT TO GET STABILIZATION OF THE AXON WHICH REACH A TARGET WHERE IT WILL BE STABLE AND PRESENT FOR A LONG PERIOD OF TIME. OKAY, SO AS WE THOUGHT ABOUT THIS VERY INTERESTING FEATURE OF REGULATING EXPRESSION AT MULTIPLE LEVELS, TRANSCRIPTION RNA TRANSPORT AND POTENTIALLY TRANSLATION, WE STARTED THINKING ABOUT WHETHER THERE MIGHT BE MECHANISMS THAT COORDINATELY REGULATE DISTINCT PORTIONS OF THIS PROCESS. AND SO, WE FIRST LOOKED AT WHAT ABOUT BCL W TARGETS THE RNA FOR TRANSPORT INTO THE AXON. AND BCLW LIKE MANY AXONAL RNAs HAVE LONG AXONS AND WE FOUND THIS IS ESSENTIAL FOR NEUROTROUGHIN DEPENDENT TO THE AXONS AS SHOWN HERE WHEN WE ATTACH IT TO A--TO A GFP THAT RNA NOW IS TRAFFICKED TO THE AXONS IN A WAY THAT THE GFP WITHOUT THE THREE PRIME UTR IS NOT. SO WHAT ABOUT THE THREE PRIME UTR ALLOWED FOR THE TRAFFICKING AND AS YOU ALL KNOW THERE HAVE BEEN A NUMBER OF LARGE INVESTIGATIONS OF RNA BINDING PROTEINS AND THE SEQUENCE TO WHICH THEY BIND AND THESE INCLUDE AND HIPS AND CLIPS AND NEWER METHODS. SO WE LOOK THROUGH THE BIOINFORMATICS AND LOOKED THROUGH THE SEQUENCE AND FOUND THERE WERE MULTIPLE CONSENSUS SITES FOR BINDING OF AN RNA BINDING PROTEIN CALLED SFPQ, THERE WERE NO FRMP BINDING SITES THERE IS NONE THAT INTERACT WITH BETA ACTIN AND THEN THERE WERE A HUGE NUMBER OF PUTATIVE FMRP PREDICTED BINDING SITES. >> PGH SO ARE THESE ACTUALLY FUNCTIONAL? SO WE LOOKED AT THE RNA BINDING PROTEINS THAT INTERACT BCLW IN TWO WAYS. THE FIRST WAY WE DID IT WAS TO TAKE THE PORTION OF THE THREE PRIME UTR THAT EACH OF WHICH IS PREDICTED TO HAVE AN SFPQ BINDING SITE AND MULTIPLE FMRP BINDING SITES AND WE USE THAT TO PULL DOWN THE PROTEINS FROM THE CELL EXTRA. AND AS YOU CAN SEE HERE, THE THREE PRIME UTR ABUNDANTLY AND NICELY PULLS DOWN SFPQ FROM THE EXTRA AND WE SEE NO BINDING TO ZBP-ONE OR FMRP, DESPITE THE BINDING SITES. SO THIS SUGGESTS THAT IN FACT, SFPQ MIGHT BE BINDING TO BCL W. AND WE LOOKED AT THIS IN THE OTHER DIRECTION AND SO SO WE NOW PREPARED EXTRACTS AND WE PULL DOWN AND CROSS LINK AND THEN PULL DOWN SFPQ AND LOOK AT THE RNAs THAT ARE BOUND TO IT. SO WHAT YOU CAN SEE IS THAT BCL W IS BOUND BY SFPQ AND HONDURAS GABBA Ph AND GABBA ACTIN ARE NOT AND THESE ARE REGULATED NICELY BY FGF STIMULATION, SO SFPQ BINDS TO MRNA, WHERE IS THIS BINDING PROTEIN LOCATED? SO WE FIND THAT SFPQ IS PRESENT IN THE NUCLEI AND OF THE NEURONS AND IT'S ALSO PRESENT THROUGHOUT THE AXON AND IN THE GROWTH CONE OF THE AXON. NOW SFPQ IS REALLY AN INTERESTING RNA BINDING PROTEIN BECAUSE IT HAS BEEN IDENTIFIED AS EVERYTHING. IT'S CALLED SFPQ BECAUSE OF THE SLICING FACTOR, SF, IT'S ENRICHED, IT WAS ALSO IDENTIFIED BY HIRO KAWA AS A TRANSPORT MOLECULE IN DENDRITES SO IT PLAYS A ROLE IN TRANSPORT. IT'S ALSO BEEN IDENTIFIED IN THE RNA EXPORT MACHINERY OF THE NUCLEUS. SO WE THOUGHT THIS WAS A GREAT CANDIDATE TO TRY TO COORDINATE AND FINALLY IT HAS BEEN IMPLICATED IN TRANSCRIPTIONAL CONTROL BECAUSE IT BINDS TO THE NASCENT RNA CHAIN AND PROTECTS IT AS TRANSCRIPTION IS OCCURRING. SO THIS SEEMS LIKE A GREAT CANDIDATE FOR REGULATING AN RNA THAT NEEDS TO BE REGULATED AT MULTIPLE LEVELS. >> PGH SO WHAT HAPPENS TO BCLB RNA LEVEL IF WE ELIMINATE SFPQ. SO, THE POST DOC WHO I MENTIONED DID SFPQ KNOCK WN AND THEN SHE LOOKED AT THE NUCLEUS, THE CELL BODIES CYTOPLASM AND THEN THE DISTAL AXON. AND YOU CAN SEE HERE THAT AT BASELINE. WHEN YOU STIMULATE WITH NEUROTROUGHINS AGAIN, BCL W RNA GOES UP IN THE CELL BODY CYTOPLASM AND IN THE DISTAL AXON AS THOSE BETA ACTIN. WHEN SHE BLOCKS SFPQ, WHAT SHE FIND SYSTEM THAT THE RNA IS STILL BEING MADE BUT IT'S ACCUMULATING IN THE NUCLEUS AND DOES NOT GET OUT OF THE NUCLEUS, SO IT'S NOT IN THE CYTOPLASM OF THE CELL BODY AND NEVER MAKE ITS TO THE AXON. AND AGAIN BETA ACTIN IS NICE CONTROL BECAUSE THIS IS NOT BOUND OR EFFECTED BY SFPQ. SO, GIVEN THAT WE THINK THAT BCL W IS CRITICAL FOR THE SURVIVAL OF THE AXON, THIS WOULD PREDICT THAT SFPQ IS NECESSARY FOR AXONAL SURVIVAL. AND SO WE CARRIED OUT AGAIN SFPQ KNOCK DOWN AND THEN LOOKED AT AXONAL SURVIVAL USING THESE WINEARIZED IMAGES AND WHAT YOU CAN SEE HERE, AGAIN IS THAT, YOU NOW PREVENT NEUROTROUGHIN DEPENDENT SURVIVAL OF THE AXON WHEN WE KNOCK DOWN SFPQ. WHAT'S MORE IMPRESSIVE TO ME, ANYWAY, IS THAT WE CAN ACTUALLY RESCUE THIS IF WE REINTRODUCE BCL W PROTEIN INTO THE DISTAL AXON. SO SUGGESTING THAT AT LEAST ONE OF THE THINGS THAT KNOCKED DOWN OF WHAT THE SYSTEM IS DOING IS ELIMINATING LOCALLY--THE LOCAL BCL W AND INTERESTINGLY BCL TWO ADDED INTO THE AXON SYSTEM UNABLE TO RESCUE. SO AGAIN GOING BACK TO OUR MODEL WHAT WE THINK IS THAT THE INITIAL--THE INITIAL RESPONSE NEUROTROUGHINS AND TRANSCRIPTION, THE SFPQ BEGINS TO INTERACT WITH BCL W IN THE NUCLEUS AND OUR MODEL SO WE DO NOT YET HAVE GOOD DATA FOR THIS IS THAT SFPQ IS INVOLVED IN CONTINUES TO BE INVOLVED IN MOVING THE NEWLY MADE BCLW RNA INTO THE AXON AND POTENTIALLY IN REGULATING THE LOCAL TRANSLATION AS WELL. >> PGH SO IN THE LAST LITTLE BIT OF THIS TALK, WHAT I WOULD LIKE TO DO IS TURN FROM WHAT IS REALLY VERY BASIC BIOLOGY TO TRY TO UNDERSTAND THE IMPORT OF THESE STUDIES FOR DISEASE. AND WE HAD REALLY BEEN LOOKING AT HOW NEUROTROPHIC FACTORS REGULATE AXONAL SURVIVAL. SOMETHING THAT IS ESSENTIAL DURING THE--DURING THE LONG LIVES OF PEOPLE AND THERE ARE A NUMBER OF CASES IN WHICH YOU SEE LATER IN LIFE, AXONAL NEUROPATHY BUT I'M AT A CANCER INSTITUTE SO THE NEUROPATHY I CHOSE TO STUDY IS CHEMO INDUCED PERIPHERAL NEUROPATHY, IS ACTUALLY A MAJOR PROBLEM AND ONE THAT LOOKS A LOT LIKE OUR BCL W KNOCK OUT, THIS SHOWS CHEMO INDUCED PERIPHERAL NNG GLOBALLY RATY IS REPORTED TO BE IN APPROXIMATELY 30% OF PATIENTS TREATED WITH VARIOUS TRADITIONAL CHEMO CYTOTOXIC CHEMO THERAPIES. SO IF YOU EXAMINE PATIENT WHO IS HAVE BEEN ON CHEMO THERAPY, VIRTUALLY ALL OF THEM HAVE SOME NEUROPATHY. IF YOU LOOK AT THIS AND LOOK AT THE SKIN, WHAT YOU SEE IS JUST AS WAS THE CASE IN THE BCL W KNOCK OUT, YOU LOSE THE EPIDERMAL INNERVATION AS SHOWN HERE IN GREEN IN THIS FIGURE FROM A PATIENT WITH CHEMO INDUCED LOSS OF INNERVATION. ONE OF THE CYTOTOXIC CHEMO THERAPIES THAT IS WITH THIS IS CYTOTAX ILLEGALS AND IT'S IN PARTICULAR TO OVARIAN AND BREAST CANCER AND THE CHEMO INDUCED NEUROPATHY IS THE DOSE LIMITING TOXICITY OF THIS CYTOTOXIC DRUG. SO ABOUT 30 PERCENT OF PATIENTS EXPERIENCE CHEMO INDUCED NEUROPATHY AND PRIMARILY SENSORY NEUROPATHY SO HOW DOES TAXLE WORK, IT'S KNOWN TO BIND TO MICROTUBULES AND IT TABLIZES THE MICROTUBULE SO THAT THEY'RE NO LONGER ABLE TO TURNOVER. AND OBVIOUSLY THE MICROTUBULES ARE IMPORTANT IN AXONS, IT'S IMPORTANT FOR MANY THINGS, TRANSPORT OF BOTH PROSURVIVAL FACTORS AS WELL AS MITOCHONDRIA, AXONAL MICROTUBULES ARE A HUB FOR SEVERAL PRODEGENERATIVE CASCADE THAT REALLY DEPEND ON CYTOSKELETAL STABILITY AND SO, THIS--THIS IS THOUGHT TO BE, THERE ARE THOUGHT TO BE MANY WAYS IN WHICH TAXAL MAY BE IMPINGING ON AXONS AND CAUSING AXON DEATH. WE WERE INTERESTED IN WHETHER OUR MODEL WAS ABLE TO SURVIVE AND WAS EFFECTED BY AXAL AND WHETHER THE TREATMENT OF IT MIGHT LEAD TO THE CHEMO INDUCED NEUROPATHY AND WE USE THE TWO CULTURE SYSTEMS, THE CAMP NO CULTURES AND MICROFLUIDIC CHAMBERS TO LOOK AT P A XATOXAL INDUCED NEUROPATHY INVITRO AND AGAIN WE CAN SELECTIVERY TREAT THE AXONS AND/OR CELL BODIES WITH TAXOL AND THEN LOOK AT RESPONSES. SO IN BOTH SYSTEMS WHAT WE SEE IS WHEN WE TREAT THE AXONS AND NOT THE CELL BODIES WITH TAXOL, WE SEE AN INCREASED IN AXONAL DEGENERATION AS GENERATED AND PRACTICINGMENTATION AND BY RETRACTION OF AXONS TO THE DYING BACKSA AXON OPERATING GLOBALLYATHY AS SHOWN HERE. THIS EFFECT IS DOSE DEPENDENT AS YOU SEE HERE AND IF WE INSTEAD ADD P A XOL TO THE BODIES TO THE STAINED DOSES WE SEE NO EFFECTS ON AXON DEGENERATION AND EVEN WHEN WE'RE ADDING, AND ADDING THE TAXOL TO THE AXON THAT IS CAUSED DEGENERATION WITHOUT IMMEDIATELY EFFECTING THE CELL BODIES THEMSELVES. SO WE HAVE A SYSTEM WHERE WE LOOK AT TAXOL INDUCED DEGENERATION, WHAT HAPPENS TO OUR CASCADE THAT WE FIND TO BE IMPORTANT FOR AXONAL SURVIVAL AND SO WHEN WE ADD TAXOL JUST TO THE AXONS, WHAT WE SEE IS A DECREASE IN BCLW RNA IN THE AXONS AND DECREASE IN PROTEINS IN THE AXONS. >> PGH SO CLEARLY IT'S IMPINGING ON THE AXONALs AND THERE ARE A LOT OTHER RNATHAT IS WILL BE EFFECTED AND WE STARTED BY ASKING ARE THERE OTHER BCL-TWOS THAT ARE AFFECTED? AND JUST TO REMIND YOU BCL TWO AND BCLXL, AND BCLW ARE ALL PRESENT IN SENSORY NEURONS. WE LOOKED AT THE OTHER COMPONENTS AND WHAT WE SEE IS THAT TAXOL DECREASED BCL W LEVELS BUT THERE'S NO APPRECIABLE EFFECT ON THE--ON EITHER BCL TWO OR BCL XL IN THE TIME WE'RE LOOKING. >> PGH SO THIS SEEMS TO BE FAIRLY SPECIFIC. THIS SUGGESTED MAYBE BCL W OR IT'S DERIVATIVE MIGHT BE ABLE TO RESCUE SOME OF THE CHEMO INDUCED NEUROPATHY AND SO WE DID THE FOLLOWING EXPERIMENT, WE AGAIN ADD IT BEFORE WE TREAT IT WITH TAXOL, WE ADDED BCL W ADDING DIRECTLY THE PROTEIN TO THE AXONS BY A METHOD OF CHARIOT PROTEIN TRANSFECTION, SO BCL W GOES INTO THE AXONS, WE HAD TAXOL AND THEN WE LOOK AT DEGENERATION WE DID THE SAME THING WITH BCL-TWO AND XL AND YOU CAN SEE THEY ALL GET INTO THE TAGS AND YOU CAN SEE IT'S PRESENT AT LOWER LEVELS THAN TWO OR XL. AND AGAIN IN WE ADDED THAT INTO BCLW INTO THE AXONS WE CAN NOW PROTECT FROM CHEMO INDUCED NEUROPATHY FOR TEXT AXOL WHERE BCLTWO AND THE OTHER DO NOT. MORE OVER IF WE INSTEAD ADD BCL W INTO THE CELL BODIES OR TWO OR XL, IT AGAIN DOES NOT PROTECT FROM TAXOL INDUCED NEUROPATHY. SO THESE BETA INDICATE THAT BCL W IS ACTING LOCALLY IN THE AXON TO PROTECT FROM TAXOL NEURODEGENERATION AND THE FUNCTION IS QUITE UNIQUE AND WE DO NOT SEE IT. BCL-TWO OR XL, EVEN WHEN WE INTRODUCE THEM DIRECTLY INTO THE ACONS. >> PGH SO WHY BCL W? WE STARTED THINKING ABOUT THIS STRUCTURALLY AND AGAIN, BCLW TWO AND XL ARE QUITE SIMILAR BUT THEY'RE ONLY ABOUT 30 TO 37-40% IDENTICAL SO THERE ARE A LOT OF AREAS THAT ARE DIFFERENT. AND SO WE STARTED THINKING ABOUT THE DIFFERENCE, THE AGE DOMAINS WITHIN THE BCL TWO FAMILY. IN PARTICULAR, WE FOCUSED ON THE BH-FOUR DOMAIN WHICH IS KNOWN TO BIND AND INHIBIT SEVERAL APOPTOTIC PROTEINS SUCH AS THAT. AND FORTUNATELY FOR US, OUR NEIGHBOR LAUREN WOLINSKY HAS DEVELD THESE STABLE PEPTIDES FOR THE BCL TWO FAMILIES, THE DH-FOUR DOMAINS SO WE ALREADY HAD THE BCL W BH-FOUR DOMAIN AS WELL AS THE BCL TWO AND XL, THE BHL DOMAIN AND THE PEPTIDE THAT MAINTAINS THE ALPHA HELICAL STRUCTURE. THOUGH WHAT WE DID WAS TO STKPWRAOUS THE STABLE PEPTIDE FOR THE BCL W DOMAIN INTO THE AXON AND YOU CAN SEE HERE THE AXONS WITH THE FITC LABELED PEPTIDE AS SHOWN HERE. AND WHAT WE FIND IS THAT THE BH-FOUR DOMAIN ALONE IS ABLE TO RESCUE FROM TAXEL NEUROPATHY AND AT THE TIME THIS SLIDE WAS MADE WE HADN'T MADE THE BCL RESCUE BUT IT DIDN'T REST EITHER. SO THEN REALLY THIS IS VERY EXCITING FOR US IN THAT IT SUGGESTS THAT YOU MIGHT BE ABLE TO IMAGINE A SMALL MOLECULE BASED ON THIS STABLE PEPTIDE STRUCTURE AS A WAY OF PREVENTING CHEMO INDUCED NEUROPATHY. IT ALSO GIVES US METHODS FOR STARTING TO LOOK AT HOW THE TAXEL IS ENGAGING THE DEGRADATION GRATES--GRATUEIDATIVE MACHINERY IN THE AXONS AND I'LL JUST SUMMARIZE BY SAYING THAT WE THINK THAT TAXEL IS EFFECTING THE MICROTUBUALULES IN THE AXON AND WHILE WE THINK IT'S DELAYING THE RETROGRADE TRANCE PORT OF SIGNALING ENDOSOMES WE THINK THAT IT REALLY IS CAUSING PROBLEMS IN THE INTRA GRADE TRANSPORT AND THAT THE LACK OF BCL-W ALLOWS P A C AXON DEGENERATION AND THIS IS SOMETHING THAT WE'RE ACTIVELY WORKING ON. >> PGH SO JUST TO SUMMARIZE WITH WHAT I TOLD YOU, I STARTED TO WITH BASIC SCIENCE TRYING TO UNDERSTAND HOW NEUROTROUGHINS ARE FUNCTIONING AS TARGET DRIVE FACTORS REGULATE THE SURVIVAL OF THE NEURONS BUT EXTENDED MORPHOLOGY AND SENSORY NEURONS, WE IDENTIFIED BCLW AS A RETROGRADE RESPONSE GENE THAT'S IMPORTANT FOR AXONAL SURVIVAL AND WE'VE BEEN LOOKING AT THE RNA BINDING PROTEIN THAT'S RESPONSIBLE FOR ORCHESTERATING SOME OF THE FUNCTIONS OF BCLW IN MEDIATING AXONAL SURVIVAL AND WE TURN BASIC STUDIES TO TRY AND SEE HOW THIS GAVE US INSIGHT INTO A DISEASE PROCESS, CHEMO INDUCED PERIPHERAL NEUROPATHY AND WHETHER IT MIGHT BE USEFUL FOR DEVISING NEW TREATMENT AND THIS IS THE ENTIRE LAB AND I JUST WANT TO POINT OUT COLLEAGUES, SARAH, SARA, MARIA AND KAT, WHO WERE RESPONSIBLE FOR THE WORK THAT I TOLD YOU ABOUT TODAY AND MOST OF OUR FUNDING IS FROM NINDS. >> PGH AND WE REALLY APPRECIATE IT. [ APPLAUSE ] >> PGH [INDISCERNIBLE] >> WE ARE STARTING TO LOOK AT THAT. SO THE QUESTION WAS HOW DOES TAXEL EFFECT OTHER RRGs? WE'RE LOOKING AT THAT AND IT CLEARLY EFFECTS A NUMBER OF AXONALLY TRANSPORTED RNAs. WHAT WAS REALLY SURPRISING TO US WAS THAT BCLW AT LEAST IN THE SHORT-TERM IS SUFFICIENT FOR RESCUING. I DON'T KNOW--I THINK IN SOME WAYS, THE WAY WE LOOK AT CHEAPEE INDUCED NEUROPATHY IS THE BEST THING TO LOOK AT BECAUSE IF WE CAN PROTECT FOR A SHORT TIME AFTER CHEMO IS GIVEN THAT MAY BE ALL YOU NEED. BUT I'M SURE IT'S EFFECTING OTHERS TO SAY NOTHING OF MITOCHONDRIAL TRANSPORT. >> [INDISCERNIBLE]. >> BCLW, YEAH. >> [INDISCERNIBLE]. >> YEAH, GREAT QUESTION SO WE ACTUALLY LOOKED AND WHEN WE PUT BACK IN THE BCLW IS WE USE TODAY FOR A HIST, TAG SO WE COULD PULL IT DOWN THE ONLY THEY THINK WE FOUND THIS FAR ASSOCIATED WAS PUT ON THE AXON IN THERE, AND WE DON'T HAVE ACCENT BODY SO IT COULD BE A CIRCULAR ARGUMENT, WE DON'T HAVE ENOUGH MATERIAL TO DO IT BY MASS SPEC. YES IT DOES. ABSOLUTELY. YEAH. WE ACTUALLY THINK--WE THINK THERE MUST BE SOME MODIFICATION OF THAT THAT'S HAPPENING, SOME POST TRANSLATIONAL MODIFICATION THAT'S TAPPING SPECIFICALLY INTO THE MITOCHONDRIA. THAT' OUR MODEL TO DO THAT. THAT'S SURPRISING. >> [INDISCERNIBLE]. >> OKAY SO WHEN WE--OUR DIVIDERS ARE ON 500- MICRONS RIGHT? SO WITH THAT--THE TIME THAT WE SEE FROM STIMULATION TO SEEING RNA INCREASE IN THE SELL BODY IS HALF AN HOUR, IS AT FIRST AND IT PEAKS AROUND ONE HOUR, SO, ABOUT WHAT--AND THEN YOU SEE IT, THE RNA BACK OUT IN THE AXON LATER, ONE AND HALF TO TWO HOUR SYSTEM WHEN YOU SEE IT AND THEN THE PROTEIN IN THE AXON WE SEE AT FOUR HOURS. >> [INDISCERNIBLE]. >> I MEAN THEN CHRISTINE IS SORT OF THE OPPOSITE OF TEXT AXEL, INSTABLE OF STABILIZING THEM, IT DESTABILIZES THEM. I SUSPECT THAT IT WOULD BE DOING THE SAME THING, BECAUSE IT WOULD ALSO BE INTERFERING WITH TRANSPORT OF ANOTHER GROUP OF MOLECULES. PLATINS ALSO CAUSE NEUROPATHYS AND WE--WE--THERAPIES WHO ARE DOING THESE STUDIES INITIALLY, HER IDEA WAS TO LOOK AT MULTIPLE DIFFERENT TYPES OF CHEMO INDUCED NEUROPERATING GLOBALLY AT SCHESTUDY ALL OF THEM AND AS SHE'S GOTTEN ALONG IN GRAD SCHOOL, SHE REALIZED THAT SHE'S NOT GOING TO DO ALL OF THEM BUT DO ONE AND THAT'S SOMETHING WE WOULD BE REALLY INTERESTED IN. >> [INDISCERNIBLE]. >> SO THE ANSWER TO YOUR SECOND QUESTION IS YES, WE DO RESCUE THE DEGENERATION IN THE BCLW KNOCK OUT BY ADDING BACK JUST TO THE AXON. YES, SO AGAIN--SO HOW MUCH WE INCREASE WE'VE BEEN JUST DEBATING THIS AND TRYING TO FIGURE IT OUT BECAUSE IT'S NOT EXACTLY THE SAME TIME AND THE PROTEIN TRANSFECTION DOESN'T EQUALLY EFFECT EVERY AXON SO THERE'S A LOT OF--IS IT AS EFFECTIVE AS? YES, IT IS. >> [INDISCERNIBLE]. >> YES, WE WOULD LOVE TO KNOW THAT. THAT IS SORT OF THE MODEL THAT I'M SHOWING. WE DON'T KNOW THAT AT ALL. WE HAVE BEEN VERY ACTIVELY WORKING ON THIS COOL TECHNIQUE THAT'S ALMOST WORKING BUT NOT QUITE SO THAT WE CAN VISUALIZE THE LOCALLY--SPECIFICALLY VISUALIZING REALTIME LOCALLY TRANSLATED BCLW, AND WHEN THAT'S WORKING WE'LL LET YOU KNOW BUT RIGHT NOW WE DON'T KNOW. >> [INDISCERNIBLE]. YEAH THOSE ARE ON TWO DIFFERENT SYSTEMS AND IT ALL DEPENDS ON THE SUBSTRATE WHICH IS CRITICAL FOR AXONS OF COURSE, SO IN THE CULTURES WHERE WE GROW THEM ON ACTUALLY A COLLAGEN SUBSTRATE WHAT WE FIND IS IN RESPONSE TO TAXEL THEY UNDERGO THAT FRAGMENTATION THAT WE SEE AND THAT IS WHERE WE DID THE ADD BACK EXPERIMENT. THE OTHER IS IN THE MICROFLUIDIC CHAMBERS WHERE THEY'RE GROWING INSTEAD OF THE--ON A LAMMENIN SUBSTRATE. AND THERE IN RESPONSE TO TAXEL IS WHAT WE SEE THE ATTRACTION AND WE HAVE STARTED LOOKING AT THE AD-BACK OF BCLW IN THAT SYSTEM AS WELL THAT I DON'T HAVE AN ANSWER FOR WHAT IT DOES. BUT IT REALLY DEPENDS ON THE SUBSTRATE WHICH IS CRITICAL. >> [INDISCERNIBLE]. >> OH, WELL THIS IS A LITTLE BIT OF A PROBLEM BECAUSE IF WE BLOCK ALL TRANSPORT, YOU WILL BLOCK THE INITIATION OF TRANSCRIPTION BECAUSE WHICH WE DO AND ADD LIKE CULTURES IN AND JUST BLOCK MICROTUBULE DEPENDENT TRANSPORT WE GET NO TRANSCRIPTION BECAUSE THERE'S NO SIGNAL GETTING BACK, SO WE CAN'T DO THAT. WE--WE DON'T--THERE AREN'T GREAT SELECTIVE ANTAGONISTS OF ALL KINASEINS AND WE DON'T ACTUALLY KNOW WHICH KINASIN IS INVOLVED SO FOR THE MOMENT WE HAVE NOT BEEN ABLE ON DO THE EXPERIMENT THAT YOU'RE ASKING FOR. SO, ALL WE KNOW, WHAT WE DO KNOW IS THE TIMING THAT THERE IS THIS INCREASE BETWEEN TWO AND FOUR HOURS. SO IN ONE CASE WE'RE LOOKING AT RNA BY PC R, BY QUANTITATIVE RTPC R, AND IN THE OTHER CASE WE'RE LOOKING AT PROTEIN BY WESTERN WHICH OF COURSE DEPENDS ON THE ANTIBODY AND SO, I DON'T KNOW THAT CAN YOU SAY EXACTLY HOW TO THROW IT TOGETHER. >> [INDISCERNIBLE]. >> WELL BECAUSE OF THE POLARITY HAD IT HAS TO BE AKINASEIN, BUT THERE ARE A LOT OF KINASINs BUT WE DON'T KNOW WHICH ONE THERE IS. >> [INDISCERNIBLE]. >> YEAH SO I DIDN'T GO INTO THIS BUT BCLW UNLIKE BCL TWO IS NOT HIGHLY EXPRESS INDEED THE EMBRYO. YOU FIRST SEE IT IN THE SENSORY NEURONS DETECT EXPRESSION AT ABOUT E16, RIGHT AS THE FIRST SENSORY NEURONS ARE REACHING TARGETS AND IT GOES UP THROUGH TO P-SEVEN AND THEN IT STAYS HIGH THROUGHOUT LIFE. BCL-TWO HAS VERY DIFFERENT DEVELOPMENTAL PROFILE SO IT'S QUITE HIGH EARLY AND THEN IT GOES DOWN IN ADULTHOOD. SO THAT'S THE REASON THAT IT'S NOT--IT'S NOT REALLY AN ANSWER TO A QUESTION PER SE BUT THAT'S THE REASON WE THINK OF IT MORE OF BEING INVOLVED ONCE YOU REALLY REACH YOUR TARGET AND FOR STABILIZATION. >> [INDISCERNIBLE]. >> YEAH, I KNOW. YEAH, I HAVE A WHOLE OTHER PROJECT ON PROTEOGLYCANS AND I LOVE THEM BUT THEY'RE SO HARD. [LAUGHTER] I LOVE THEM BUT THEY'RE VERY DIFFICULT. IT IS THE MOST--I DON'T KNOW IF YOU NOTICE THIS OR NOT, NOT ONLY WAS IT THE MOST INDUCED BUT ALSO HAS BY FAR THE BIGGEST ERROR BAR WHICH IS CHARACTERISTIC OF PROTEOGLYCANS IN MY EXPERIENCE THAT THERE'S A LOT OF VARIABILITY THAT WE DON'T UNDERSTAND AND I'M SURE IT'S--IT'S VERY IMPORTANT IN SOME ASPECT OF AXONAL EITHER GROWTH OR STABILIZATION BUT WE HAVEN'T LOOKED INTO THAT AT ALL. I THINK IT'S ONE EVER THE NEW PROTEOGLYCANS THAT'S SUPPOSED TO BE INVOLVED NOT IN PREVENTING REGENERATION BUT IN PROMOTING, YEAH, YEAH, YEAH. YEAH. >> [INDISCERNIBLE]. >> YEAH, CERTAINLY AND WE HAVEN'T LOOKED AT IT AT ALL. IT'S A REALLY GOOD QUESTION. WE JUST HAVEN'T DONE IT. PEOPLE IN MY LAB, I LIKE PROTEOGLYCANS BUT PEOPLE IN MY LAB, PARTICULARLY GRADUATE STUDENTS WHO ARE TRYING TO FIGURE OUT HOW TO FINISH GET WORRY BODY PROTEOGLYCANS. >> WE'VE ONLY FOUND ONE, WE'VE SPENT A LOT OF TIME FOR THREE ALTERNATIVES IN PARTICULAR, WE DID NOT FIND THEM IN THE DATABASE, THERE'S ONLY ONE THAT'S BEEN IDENTIFIED. [ APPLAUSE ] THANK YOU.