GOOD AFTERNOON, EVERYBODY. THE BOB WENTHOLD MEMORIAL SEMINAR BEGUN IN 2010 TO HONOR OUR COLLEAGUE WHO SERVED AS NIDCD SCIENTIFIC DIRECTOR WE HAVE THE PRIVILEGE OF CELEBRATING HIS DISTINGUISHED CAREER WITH A TALK ENTITLED: REGULATION OF GLUTAMATE RECEPTORS AND SYNAPTIC PLASTICITY IN THE BRAIN DELIVERED BY DR. RICHARD HUGANIR. HE HAS MADE MAJ ORCONTRIBUTIONS TO OUR UNDERSTANDING OF SYNAPTIC NEUROTRANSMISSION, SOME OF WHICH I THINK HE IS GOING TO SHARE WITH US TODAY. SO PLEASE JOIN US AFTER THE LECTURE FOR A LIGHT REACCEPTION AT THE CONCLUSION OF--RECEPTION AT 9 CONCLUSION OF THE LECTURE AND I WANT TO THANK THE ADVANCED DEPARTMENT OF EDUCATION AND SCIENCES FOR G O THEIR GENEROUS DONATION OF FUNDS FOR THIS LIGHT RECEPTION AND WITHOUT FURTHER AH --ADIEU, RICK WOULD YOU JOIN US AT THE PODIUM, PLEASE. >> I'M ROBBIE AND I WANT TO TAKE THIS OPPORTUNITY TO INTRODUCE DR. HUGANIR, I'VE KNOWN HIM FOR MANY YEARING AND I USED TO BE AT THE DEPARTMENT OF NEUROLOGY IN THE DEPARTMENT OF NEUROSCIENCE AND I COULD SEE FIRST HAND HOW WELL HE RAN THE PROJECT PROGRAM AT THAT TIME AS WELL AS THE SUPERB RESEARCH STUDY STUDY AND HE WAS VERY KIND TO SHARE VARIOUS REAGENTS WE NEEDED FOR OUR OWN WORK IN THOSE DAYS. DR. HUGANIR, IS THE PROFESSOR AND DIRECTOR OF THE DEPARTMENT OF NEUROSCIENCE WITH THE CROSS APPOINTMENT IN PHARMACOLOGY, HE'S ALSO THE CO DIRECTOR OF THE BRAIN SCIENCE INSTITUTE AND AN INVESTIGATOR OF THE HARVARD INSTITUTE AS L. HE RECEIVED HIS Ph.D. FROM CORNELL UNIVERSITY IN 1982 AND FOLLOWING WHICH HE DID A POST DOCTORAL FELLOWSHIP WITH PAUL GREEN GARD AT YALE AND MOVED WITH HIM TO ROCKEFELLER. AT ROCKEFELLER HE BECAME AN ASSISTANT PROFESSOR AND THEN IN 1988 O HE MOVED TO JOHNS HOPKINS UNIVERSITY AS ASSOCIATE PROFESSOR. IN 2006 BECAME A DIRECTOR FOR THE DEPARTMENT OF NEUROSCIENCE. HE RECEIVED NUMEROUS PRESTIGIOUS AWARDS INCLUDING THE MEMBERSHIP TO THE NATIONAL ACADEMY OF SCIENCES IN 2004 AND IS ALSO A MEMBER OF THE INSTITUTE OF MEDICINE THAT SINCE 2011. HE WAS PREVIOUSLY A MEMBER OF THE NINDS BOARD OF SCIENTIFIC COUNSELORS AS WELL AS CHAIR FROM 2003-2006. HE'S ALSO CURRENTLY THE ASSOCIATE EDITOR OF NEURONS AND SAT ON OTHER PRESTIGIOUS JOURNALS IN THE EDITORIAL BOARD. HE CONTRIBUTED IMMENSELY TO THE UNDERSTANDING OF GLUTEA MATE RECEPTORS AS WELL AS PLASTICITY AND TODAY HIS TALK WILL BE ABOUT REGULATION OF GLUTEA MATE RECEPTORS AND SYNAPTIC PLASTICITY IN THE BRAIN. >> THANK YOU. >> THIS IS REALLY TRULY AN HONOR. I KNEW BOB VERY WELL AND WE BEGAN WHEN I STARTED--WE BOTH STARTED WORKING ON GLUTEA MATE RECEPTORS EXACTLILET SAME TIME AND THAT WAS IN THE LATE 80S AND 90S. BOB, I WAS TRAINED AS A BIOCHEMIST AND BOB WAS A--ULTIMATE BIOCHEMIST. REALLY KNEW HIS BIOCHEMISTRY AND I THINK CONTINUED DOING IT, YOU KNOW UP TO THE VERY END OF HIS CAREER. HE WAS A VERY HANDS ON LAB MANAGER AND HE--WE ACTUALLY WERE FRIENDLY COMPETITORS IN MY LAB AND HIS LAB, I KNOW TRENT BLACK STONE RIGHT HERE IS HELPING DEVELOP THE RIGHT ANTIBODIES, BUT WE WERE WORKING ON THE EXACT SAME ANTIBODIES IN 2 DIFFERENT LABORATORIES AND USED THEM TO CHARACTERIZE THE DISTRIBUTION IN THE CHEMISTRY OF THE RECEPTORS AT THAT TIME. CRAIG WAS TRYING TO PURE PIE THE--PURIFY THE LAB AND BOB WAS DOING IN HIS LAB USING CHROMATOGRAPHY AND BOTH OF US GOT SCOOPED BY STEVE HINEMAN WHO CLONED THE FIRST GLUTEA MATE RECEPTOR. SO WE WERE COLLEAGUES AND FRIENDLY COMPETITORS AND WE PUBLISHED THAT, BOB AND I PUBLISHED 2 PAPERS TOGETHER, 1 WAS A SELL PAPER AND 1 WAS A NATURE NEUROSCIENCE PAPER SO THEY WERE PRETTY GOOD PAPERS AND SO IT'S A REAL HONOR FOR ME TO BE HERE TODAY. SO, BUT I'M GOING TO DO IS TELL YOU ABOUT SOME OF OUR RECENT WORK ON ARE REGULATION OF GLUTEA MATE RECEPTORS AND PLASTICITY IN THE BRAIN AND SO THIS HAS BEEN AN INTEREST OF MINE FOR MANY, MANY YEARS TO STUDY WHAT HAPPENS IN YOUR BRAIN WHEN YOU LEARN SOMETHING, IT COMES FROM THE INTEREST IN LEARNING IN MEMORY SO I'VE BEEN TRYING TO UNDERSTAND, HOW MEMORIES ARE ENCODED IN YOUR BRAIN, WHAT MOLECULAR CHANGES OCCUR WHEN YOU LEARN SOMETHING AND HOW YOU CAN MAINTAIN THOSE MEMORIES FOR YEARS AND DECADES. SO WE'VE BEEN CHARACTERIDING THIS AT A MOLECULAR LEVEL, AND TRYING TO UNDERSTAND WHAT MOLECULES CHANGE AND WHAT CIRCUITS CHANGE AND HOW THIS ENCODES MEMORY. SO JUST A LITTLE PRIMER FOR JUST VERY QUICK--BREAK DOWN AS WE ALL KNOW THE BRAIN IS A NETWORK OF BILLIONS OF NEURONS SO THE HUMAN BRAIN HAS MAYBE A HUNDRED OR 200 BILLION NEURONS AND EACH OF THESE NEURONS MAKE CONNECTIONS WITH THE AVERAGE OF ABOUT 10,000 OTHER NEURONS, SO THEY'RE BILLIONS AND QUADRILLIONS OF SYNAPSE IN YOUR BRAINS, AND CONNECTIONS, THESE CONNECT NECKSS BETWEEN THE CELLS AND THE BRAINS AND NEURONAL CIRCUITS, AND THESE CIRCUITS THAT COME TO RELY ON OUR BEHAVIOR AND ENCODE INFORMATION AND PROVIDE US WITH THE COMPLEXITY THAT GIVES US EMOTIONS AND ENABLES US TO LEARN THINGS AND REMEMBER THINGS. SO THE PREVAILING THEOR SETHAT DURING LEARNING AND MEMORY IS THAT YOU STRENGTHEN CONNECTIONS AND WEAKEN IN OTHER CONNECTIONS AND THIS FORMS A NEW CIRCUIT WHICH ACTUALLY PHYSICALLY ENCODES THE MEMORY. SO WHEN YOU RECALL THE MEMORY, YOU'RE ENGAGING THAT CIRCUIT AND THAT'S WHAT BRINGS TO MIND THE MEMORY. SO WE'VE BEEN STUDYING THE SYNAPTIC TRANSMISSION AND TRY TO UNDERSTAND HOW SYNAPSE SYSTEMS FOD MID AND LEARNING MODIFIED DURING LEARNING AND MEMORY AND MECHANISMS NEAR INCREASING AND DECREASING STRENGTH THAT MAY UNDERLIE PLASTICITY. SO, OF COURSE WHAT HAPPENS AT A SYNAPSE IS YOU HAVE A PRESYNAPTIC NERVE TERMINAL SHOWN HERE AND IT'S A GAP BETWEEN THE PRESYNAPTIC TERMINAL AND THE POST INAPTIC NEURON AND THE TRANSMITTER COMES DOWN, THE ACTION POTENTIAL COMES DOWN, THE PRESYNAPTIC TERMINAL ALLOWS THE RELEASE OF NEUROTRANSMITTERS WITHIN THE DIFFUSE ACROSS THE BIBBED AND RECEPTORS ON THE POST INAPTIC SIDE AND THIS PROCESS OF SYNAPTIC TRANSMISSION CONVERTING ELECTRICAL SIGNAL AND CHEMICAL SIGNAL AND BACK INTO THE ELECTRICAL SIGNAL, BY THESE RECEPTORS. SO THESE RECEPTORS HAVE BEEN THE FOCUS OF MY WORK FOR OVER 30 YEARS NOW, TRYING TO UNDERSTAND HOW THESE RECEPTORS WORK AND HOW THEY'RE MODUMENTED AND HOW MODULATION OF RECEPTORS MAY BE INVOLVED IN PLASTICITY AND LEARNING AND MEMORY. SO, NOW, THIS PROCESS AS A MENTIONED IS NOT STATIC, IT'S VERY DYNAMIC, IT CAN BE STRENGTHENED AND WEAKENED RAPIDLY AND LONG LASTINGLY AND 1 OF THE KEY PARADIGMS THAT HAS BEEN STUDIED FOR DECADES NOW, IS A FORM OF PLASTICITY IN THE HIPPOCAMPUS WHICH MANY OF YOU MAY KNOW IS CALLED LONG-TERM POTENTIATION. THIS IS ILLUSTRATED HERE, SO THIS IS JUST A CARTOON OF A--A BRAIN SLICE OF OF REGIONS OF THE BRAIN CALLED THE HIPPOCAMPUS AND OF COURSE THIS IS A CRITICAL REGION OF THE BRAIN, IMPORTANT FOR LEARNING MEMORY, THE CLASSIC STAYS OF THE PATIENT HM WHO LOST THIS HIPPOCAMPUS DUE TO SURGERY, HAS SHOWN THAT THE--THIS IS CRITICAL FOR LEARNING NEW MEMORY, SO HN, THIS PATIENT WAS NOT ABLE TO LEARN ANYTHING NEW. YOU REMEMBER HIS CHILDHOOD, HE REMEMBERED HIS PAST BUT HE COULD NEVER LEARN ANYTHING NEW AND EACH DAY HE WOULD REINTRODUCE HIMSELF TO HIS DOCTORS, THE DOCTORS WOULD HAVE TO REINTRODUCE THEMSELVES TO THEM. THIS IS CRITICAL FOR LEARNING AND IN THE 70S, THERE WAS DISCOVERED A UNIQUE FORM OF PLASTICITY WHICH IS A COMMON PLACE AND THAT IS THIS FORM OF PLASTICITY. AND YOU STIMULATE AND ACTIVATE A SET OF NEURONS AND RECORD FROM THE POST SYNAPTIC CELLS FROM THIS REGION CALLED THE CA REGION 1 OF THE HIPPOCAMPUS AND YOU CAN GET A STABLE BASELINE SHOWN HERE SO THIS IS MEASURING AND THEN IF YOU GIVE A HIGH FREQUENCY STIMULATION, JUST A BRIEF, YOU COULD GET AN INCREASE IN THE STRENGTH OF SYNAPTIC TRANSMISSION AND THIS IS VERY LONG LAST 8ING AND HENS THE NAME LONG-TERM POTENTIATION. SO INVITRO IN THE SLICE PREPARATION, THIS CAN LAST FOR SEVERAL HOURS BUT IN THE ATTACK BRAIN INCREASE IN SYNAPTIC STRAIN, CAN LAST FOR DAYS, WEEKS AND EVEN A YEAR. SO THIS IS REALLY A REMARKABLE BIOLOGICAL PROCESS, SO BRIEF STIMULATION CAUSES THIS LONG LASTING STRENGTH IN SYNAPTIC TRANSMISSION WHICH IS VERY, VERY, STABLE. NOW IN THE SAME REGION OF THE BRAIN, CAN YOU GET LONG-TERM DEPRESSION, SO THE OPPOSITE OF THIS MECHANISM, AND IN THIS CASE, AGAIN, CAN YOU GET A BASELINE TRANSMISSION SHOWN HERE, AND INSTEAD OF GIVING A HIGH FREQUENCY STIMULATION, GIVE A LOW FREQUENCY STIMULATION, THIS RESULTS IN A LONG-TERM DEPRESSION AND WEAKENING OF THE SYNAPTIC CONNECTIONS IN THIS AREA. SO IT'S THOUGHT THAT THESE 2 TYPES OF MECHANISMS, LONG-TERM POTENTIATION AND DEPRESSION ARE BOTH TO SCULPT THESE CIRCUITS AND HOW THIS PROCESS OCCURS SO THIS IS A GOAL, MANY, MANY, SCIENTISTS FOR MANY YEARS AND TRY TO UNDERSTAND WHAT ARE THE MOLECULAR MECHANISMS THAT CAN UPREGULATE AND DOWN REGULATE TRANSMISSION AND DOES THIS SORT OF PLASTICITY UNDERLIE LEARNING AND MEMORY--OR ADD OR SUBTRACT THE NUMBER OF RECEPTORS TO INCREASE OR DECREASE SYNAPTIC STRENGTH. SO I DECIDED LONG TIME AGO TO REALLY FOCUS IN ON THIS POST INAPTIC SIDE AND TRY TO UNDERSTAND THE MECHANISMS OF REGULATION OF RECEPTORS AND SEE IF THEY COULD BE INVOLVED IN SYNAPTIC PLASTICITY SUCH AS LTD AND LTP. SO 1 WAY TO MODIFY PROTEIN STRUCTURE OF COURSE, AND FUNCTION IS BY PROTEIN PHOSPHORYLATION, SO PROTEIN PHOSPHORYLATION IS 1 OF THE CALMSTIMISMS OF MOST TRANSLATION MODIFICATION THAT'S KNOWN TO REGULATE FUNCTION, SO OVER THE YEARS WE HAVE PHOSPHORYLATION BY PROTEIN KINASEIS, THESE KINASEIS IS PHOSPHORYLATE MANY, MANY, SITES OVER 40 DIFFERENT SITES WE THINK IN THE AMPORECEPTORS, AND THEY ARE--THESE PHOSPHORYLATION EVENTS OCCUR BY DIFFERENT PROTEIN KINASE AND I GUESS DIFFERENT SIGNALING PATHWAYS, AS WELL AS DEPHOSPHORYLATION BY A VARIETY OF PROTEIN PHOS FOCUSED ON--PHOSPHORYLATION AS WELL. NOW THIS IS RAPID. IT CAN OCCUR WITHIN MILLI SECONDS TO SECONDS BUT IT ALSO IS VERY STABLE. IT CAN LAST FOR THE LIFE OF THE PROTEIN BECAUSE IT'S CHEMICALLY VERY STABLE. HOWEVER, IT'S--IT CAN BE RAPIDLY REVERSED BY PROTEIN PHOS TO TAS WHICH CAN REVERSE DEPHOSPHORYLATE THE RECEPTORS AND BRING THEM BACK TO BASAL STATE. SO THIS IS A DYNAMIC WAY TO REGULATION RECEPTOR FUNCTION BUT AS WE'LL TALK ABOUT LATER, RECEPTORS TURNOVER ABOUT EVERY 30 TO 40 HOURS AND THIS CAN ONLY MODIFY PROTEIN FUNCTION FOR SEVERAL DAYS AND THIS WILL NOT ANSWER THE QUESTION OF WHAT HAPPENS, YOU KNOW HOW DO YOU STABILIZE SYNAPTIC TRANSMISSION FOR WEEKS OR FOR EVEN DECADES. NOW THE OTHER THING WE'VE BEEN INTERESTED IN IS MEMBRANE TRAFFICKING OF OF THESE RECEPTORS, SO THESE RECEPTORS ARE HIGHLY CONCENTRATED AT THE SYNAPSE SIS, THIS IS ILLUSTRATED HERE, WE HAVE 2 MAJOR TYPES OF SYNAPSES IN THE BRAIN, EXCITATORY SIN ASPARITATE SIS AND INHIBITORY SIN ASPARITATE SIS AND THESE ARE MEDIATED BY DIFFERENT TRANSMITTERS, THE MAJORITY OF EXCITATORY TRANSMISSION SYSTEM BY GLUTEA MATE IN THE CENTRAL NERVOUS SYSTEM, THE MAJORITY OF INHIBITORY TRANSMISSION MEDIATED BY GAMMA IMMUNO BUT TERRIC ACID OR GABA. AND THESE OCCUR IN THE REAGENTS ALONG THE DENDRITE AND YOU CAN SEE THAT THE RECEPTORS, GABBA RECEPTORS KNOW WHERE TO CLUSTER AND RIGHT OPPOSITE AND TERMINAL RELEASE OF GABBA WHERE THE GLUTAMATE RECEPTORS CLUSTER OPPOSITE, THE NERVE TERMINALS RELEASE IN GLUTEA MATE. THIS IS PRETTY INTERESTING PHENOMENA, AGAIN, THESE RECEPTORS ARE SYNTHESIZED IN THE ER GOLGI OF THE CELL, BUT THEN THEN THEY HAVE TO ARE SORTED AND INSERTED INTO PLASMA MEMBRANE AND STABILIZE THAT SYNAPSE SIS TO MAINTAIN THIS TIGHT CONCENTRATION OF THE RECEPTORS AT THE APPROPRIATE POINT OF CONTACT. SO, WE'VE BEEN INTERESTED IN THE MECHANISMS INVOMPLE INDEED IN TARGETING AND OVER THE LAST 20 YEARS OR SO, WE FOUND THIS IS NOT VERY STATIC PROCESS AGAIN, IT'S INCREDIBLY DYNAMIC SO THESE RECEPTORS ARE RAPIDLY ENDO SIGNIFYITOSED AND RECYCLED BACK TO THE PLASMA MEMBRANE. BUT EVENTUALLY THEY WILL BE ENDOSIGNIFYITOSED AND DEGRADED BY BOTH PROTEIN COMPLEX STUDIES OF MULTIPLE ENDOCRINAL AND LYSOSOMAL PATHWAYS. SO WE'VE BEEN TRYING TO UNDERSTAND EACH OF THESE STEPS AND TRY TO--THE EVENTUAL GOAL IS TO UNDERSTAND WHAT REGULATES THE STEADY STATE LEVELS OF RECEPTORS IN THE SYNAPSE AND THEREFORE THE STRENGTH OF SYNAPTIC TRANSMISSION, SO IN THEORY IF YOU REGULATE THE STEPS THERE THEY'RE RATE LIMITING AND PRODUCE AN INCREASE OR DECREASE IN THE LEVELS OF SEREPRESENTORS OF SYNAPSE SIS. --SO WE STUDIED MANY DIFFERENT RECEPTOR SYSTEMS OVER THE YEARS NICK O 10IC RECEPTORS GABBA A-RECEPTORS THE LAST 15 YEARS OR SO, WE REALLY HAVE BEEN EXCLUSIVELY LOOKING AT GLUTEA MATE RECEPTORS PRIMARILY AMPORECEPTORS AND NMDA RECEPTORS. AND AS MANY OF YOU KNOW, AMPOE RECEPTORS MEDIATE THE MAJORITY OF THE TRANSMISSION IN THE BRAIN. AND ABOUT 70% OF IT WHERE NMDA PLAY IMPORTANT ROLES IN SOME BRAIN REGIONS BUT THEY PLAY A CRITICAL ROLE IN INDUCING LTP AND LDB, PRIMARILY BECAUSE OF CALCIUM PERMEABILITY OF THESE TYPE OF RECEPTORS. NOW TODAY I'M GOING TO FOCUS ON THIS AND THE REASON WE FOCUS ON AMPORECEPTORS BECAUSE THEY REALLY DO MEDIATE THE MAJORITY OF THE EXCITATORY TRANSMISSIONS. SO IF YOU MODIFY THE TRANSMISSION, YOU WILL MODIFY THE SYNAPTIC STRENGTH. SO, A LITTLE PRIMARY REVIEWERRER ALL AND AMPOE RECEPTOR STRUCTURE AND AGAIN, BOB WENTHOLD CONTRIBUTED A LOT TO THIS CHARACTERIZATION. THE RECEPTOR, THE CORE SUBUNITS OF THE RECEPTORS ARE SHOWN HERE, THERE ARE 4 DIFFERENT GENE PRODUCTS FLUTE A 1 THROUGH 4, THEY COMBINE AS DIMERS OF DIMER IN MOST CASES IN A TETRARINE. SURROUND THE REGION AND THEY HAVE GLUTEA MATE OUT HERE AND THERE'S A TRANSMEMBRANE DOMAIN AND A TERMINAL REGION AND THE BRAIN SHOWED HERE AND WAS DEFINED BY BOB, SO HE PUBLISHED A PAPER WHICH IS INCREDIBLY HIGHLY CITED DEMONSTRATING THE MAJORITY OF THE RECEPTOR TYPES IN THE BRAIN CONSISTENT FLUTE A 2 AND 1. GLUTEA 2 ASK 3, AND A SMALL%AGE CONTAIN COMBINATIONS OF GLUTEA 1. NOW, SINCE THAT TIME, WE NOW KNOW THAT THERE ARE ALSO ACCESSORY PROTEINS, CALLED TARPS AND CORNISHONS AND IT SEEMS LIKE OFFER MONTH THERE'S A TRAN MEMBRANE ASSOCIATED PROTEIN. SO IT'S MUCH MORE COMPLEX THAN WHAT WE THOUGHT. BUT THESE ARE THE CORE RECEPTOR SUBUNITS AND I WILL WILL FOCUS ON THESE TODAY. SO AS I SAID, WE'VE CHARACTERIZED PHOSPHORYLATION OF THESE RECEPTORS BY MULTIPLE PROTEIN KINASES BUT WE'VE ALSO CHARACTERIZED PROTEINS THAT BIND TO THE RECEPTORS AND THESE ARE THE IDEA BEHIND THIS IS THAT FOR THE RECEPTORS TO BE STABILIZED AT SYNAPSES, YOU HAVE TO CROSS LINK THEM TO THE CYTOSKELETON TO INCREASE STABILITY OF SYNAPSE SIS BUT ALSO TO DYNAMICALLY REGULATE THEM YOU HAVE TO LINK THEM TO THE MACHINERY OR TO EXIT THE MACHINERY OR PROMOTE INSERTION OR PROMOTE REMOVAL OF RECEPTORS FROM THE PLASMA MEMBRANE. SO WE AND SEVERAL PEOPLE OVER THE YEARS HAVE BEGAN TO CHARACTERIZE THIS AND HERE, IS JUST AN EXAMPLE OF SOME OF THE PHOSPHORYLATION SITES WE CHARACTERIZED, AT LEAST THE PUBLISHED 1S. AND SO YOU CAN SEE THAT THERE ARE MULTIPLE POSFORALATION SITES ON THE C-TERMINAL REGION ON ALL THE DIFFERENT SUBUNITS, THERE'S ALSO A MIDDLE SITES SHOWN HERE, SO OTHER POST TRANSLATIONAL MODIFICATIONS AND ALSO WE'VE CHARACTERIZED A VARIETY OF THE UCIBOL WINNENNATION SITES IN THE REGIONS AS WELL. NOW I MUST NOTE THAT ACTUALLY ANOTHER GRADUATE STUDENT OF MINE, CATHERINE IS HERE, NINDS, LAB RIGHT NEXT TO CRAIG, CHARACTERIZE SOME OF THE FIRST PHOSPHORYLATION SITES ON THE THIS RECEPTOR AND ALSO HAS BEEN WORKING ON UBIQUITIENATION OF THE RECEPTOR, BUT SHE ALSO WAS A POST DOC WITH BOB, SO THE CONNECTIONS WITH BOB GO ON AND ON. SO, SO WE CHARACTERIZE THESE PHOSPHORYLATION SITES AND HAVE SHOWN THEY'RE INVOLVED IN MODULATION OF RECEPTOR FUNCTION, BUT IN ADDITION WE'VE BEEN CHARACTERIZING INTERACTING PROTEINS AND SO, WE AND OTHERS OVER THE LAST 15 YEARS OR SO HAVE CHARACTERIZED LARGE COMPLEX OF PROTEINS THAT ARE INVOLVED THAT BIND DIRECTLY OR INCORRECTLY TO AMPORECEPTORS AND REGULATION OF THEIR FUNCTION. SO TODAY I WILL TALK ABOUT A RELATIVELY NEW 1, A PROTEIN CALLED KEBRA AND HOW KEBRA IS REGULATING DISPUNKZ PLASTICITY IN THE BRAIN. NOW THAL WORK IT'S BECOME VERY CLEAR THAT THESE RECEPTORS BIND MANY PROTEINS AND WE MAPPED A VARIETY OF THE BINDING SITES SHOWN HERE, SO WE KNOW WHERE A LOT OF THESE PROTEINS BIND EITHER TO C-TERMINAL REGIONS OR THIS MEMBRANE PROXIMAL REGION BUT ALSO EXTRA CELLULAR REGIONS OF PROTEINS SUCH AS NARP, SECRETED PROTEIN BIND OF THE EXTRA CELLULAR DO MAINS OF AMPORECEPTORS. SO OVER THE YEARS OUR LAB AND MANY LABS ACROSS THE WORLD HAVE SHOWN THAT REGULATION RECEPTOR AND ITS INVOLVEMENT OF SYNAPTIC PLASTICITY IS INVOLVED IN ALL BRAIN PROCESS, LEARNING MEMORY AND FEAR, ANXIETY, REWARD BUT IMPORTANTLY IN ALMOST BASICALLY EVERY BRAIN DISEASE YOU CAN THINK OF, FROM DEPRESSION, CHRONIC PAIN, SCHIZOPHRENIA AND DOWN TO AUTISM. SO I'M GOING TO TRY TO TELL 3 QUICK STORIES TODAY, THE FIRST 1 IS ABOUT THIS PROTEIN CALLED KEBRA, AND SO WE BEGAN TO WORK ON KEBRA BEFORE IT HAD A NAME, IT WAS A NUMBER. AND WE GOT IT BECAUSE IT BOPPED TO THIS PROTEIN, WE CHARACTERIZED FOR SEVERAL YEARS CALLED PIC 1, PIC 1 BINDS TO AMPORECEPTORS DIRECTLY THROUGH THE DOMAIN AND WE STARTED SEARCHING FOR OTHER PROTEINS THAT MAY INTERACT WITH PIC 1, USING YEAST 2 HYBRID SYSTEM AT THE TIME AND WE PULLED OUT A PROTEIN, BACK IN 1999, THAT JUST HAD A NUMBER, CALLED KEO A1969, AND WE PULL IT A LOT OF DIFFERENT INTERACTING PROTEINS AND DIDN'T THINK ABOUT IT AND THEN, ABOUT 2006, THE SERIES OF PAPERS STARTED COMING OUT AND--BY THIS TIME, THE GENE HAD HAD IDENTIFIED AND BEEN GIVEN A NAME CALLED KEBRA, BECAUSE IT'S HIGHLY ENRICHED IN KIDNEY AND BRAIN AND IT WAS MAPPED USING SNP ANALYSIS TO HUMAN MEMORY PERFORMANCE. SO--SO THIS PROTEIN, THERE ARE 2 DIFFERENT SNPS, THAT ARE COMMON, 30% OF YOU HAVE 1 SNP WHICH WE MIGHT WANT THE TO CALL SMART SNP. AND ABOUT THE OTHER 70% HAVE THE OTHER SNP WHICH IS THE LESS SMART SNP. BUT LUCKILY IT'S ONLY--IT'S A PRETTY SUBTLE DIFFERENCE. IN THIS PAPER, THEY MEASURED HOW MANY WORDS YOU COULD REMEMBER, SO PEOPLE WHO HAD THE SMART SNP, COULD REMEMBER UP TO 8 OR 9 WORDS WHERE PEOPLE WITH THE LESS SMART SNP COULD ONLY LEARN 6 OR 7 WORDS. SO IT'S PRETTY SUBTLE DIFFERENCE BUT IT'S BEEN REPRODUCED IN SEVERAL POPULATIONS AND SO THIS SEEMS TO BE INVOLVED IN WORKING MEMORY IN SOME WAY TO ENHANCE THIS SNP, BUT ENHANCE YOUR ABILITY TO GET--TO REMEMBER OR RECALL. NOW IT'S BEEN REPRODUCED IN OTHER POPULATIONS BUT ALSO HAD HAS BEEN SHOWN TO BE THE SMART SNP IS PROTECTIVE AGAINST LATE ONSET ALZHEIMER'S DISEASE AS L. SO THESE PAPERS MADE US GO BACK AND WE REALIZED THIS WAS THE GENE WE ISOLATE INDEED 1999. THE SO WE WENT BACK TO STUDY THIS AND TRY TO HAPPENED HOW THIS KIBRA IS DOING THIS, IS IT REGULATING SYNAPTIC TRANSMISSION, DOES IT REGULATE RECEPTOR TRAFFICKING. SEE BY THIS TIME HAD BEEN SHOWN TO INTERACT WITH VARIETY OF INTERESTING PROTEINS, SEVERAL THAT ARE--WERE THOUGHT TO BE IMPORTANT FOR SYNAPTIC PLASTICITY, NOTABLY, DATA WHICH I'LL COME TO AT THE END OF THE TALK WHICH HAD BEEN THOUGHT TO BE ROLELY CRITICAL MORE MAINTENANCE OF PLASTICITY, LTP, AND THE MAINTENANCE OF MEMORIES. SO WE ADDED PICK INTO THIS COMPLEX, AND STARTED TO STUDY IT IN MORE DETAIL. SO HERE'S EARLY EXPERIMENTS WHERE WE TAKE PICK 1 AND WE EXPRESS IT IN HETEROGENEOUSROLOGYST CELLS, HEK CELLS AND IT'S A RELATIVELY DIFFUSE DISTRIBUTION, WE EXPRESSED IT BY ITSELF, WE FORM THESE THAT ARE SHOWN IN THESE HHK CELLS. AND CO-TRANSFECT PICK 1 AND KIBRA, WILL RECRUIT PICK1 INTO THESE CLUSTERS SHOWN HERE. I HOPE YOU CAN SEE THEM. NOW, INVITRO, YOU CAN ALSO FROM THESE CELLS, YOU CAN IP, IF YOU IP, THE KEBRA, WITH A MIC TAG, YOU PULL DOWN VUBSLY KIBRA, BUT YOU ALSO IP, PICK SO THERE'S INTERACT IN THE YEAST THROUGH HYBRID, IN THIS CELL SYSTEM, AND YOU CAN DETECT THAT USING BIOCHEMICAL TECHNIQUES. SO WE WENT ON AND THIS WORK WAS DONE BY LAUREN, A STUDENT IN MY LABORATORY AND SHE COULD PULL I. P. KIBRA FROM BRAIN, AND COIAND PICK 1 IF SHE IPED KIBRA FROM IT, THEE ALSO PULL TODAY DOWN, TOO, AND CAN'T DETECT THEM ALL, BUT OTHER AMPORECEPTOR INTERACTING PROTEINS, INCLUDING NSF, BRICK 1 AND SEC, 8, SO KEBRA SEEMS TO BE IN THIS COMPLEX WITH AMPORECEPTORS AMPORECEPTORS A ND WITH PICK 1 AND IS POISED TO REGULATE RECEPTOR FUNCTION AND RECEPTOR, THIS TIME WE WERE FOLLOWING UP PICK1'S TRAFFICKING OF AMPORECEPTORS. SO EAR IF YOU OVEREXPRESS KIBRA IN NEURONS AND CULTURE, WE CURRENTLY DON'T HAVE REALLY GOOD ANTIBODIES SO WE LOOK AT OVEREXPRESSION, AND WE CAN SEE THAT KIBRA, WILL LOCALIZE WITH RAB5 PROTEIN INVOLVED IN EARLY PROTEIN ENDOSTUDIES OF MULTIPLE ENDOCRINES AND ANOTHER PROTEIN THAT'S INVOLVED IN THE EMPCYST AND MEMBRANE TRAFFICKING, SO WE THINK THIS IS REFLECTING A PATHWAY FOR RECEPTOR RECYCLING AND REINSERTION, AND THAT KIBRA IS INVOLVED IN THIS PATHWAY SIMILAR TO WHAT WE THINK IS GOING ON WITH PIC 1 AND SOME OF THESE OTHER AMPA RECEPTOR ASSOCIATED PROTEINS. SO 1 APPROACH WAS TAKEN TO OVEREXPRESS KIBRA, SO IF WE OVEREXPRESS KIBRA FOR STAINS IN THE LEVEL SHOWN HERE, YOU GET A SIGNIFICANT DECREASE IN THE AMOUNT OF SURFACE RECEPTOR BY OVEREXPRESSING KIBRA, THIS IS ACTUALLY VERY SIMILAR TO WHAT WE SEE WITH OVEREXPRESSION OF PIC 1, SO WE THINK THAT THE KIBRA ARE INVOLVED IN EITHER PROMOTING ENDOCYTOSIS OR PREVENT TGF FROM RECYCLING BACK TO THE SURFACE OF MEMBRANE. AND A BELIEVER IN GENETIC TECHNIQUES SO WE WENT ON TO MAKE A MUTATION IN THE KIBRA GENE AND WE GENERATED A KIBRA CONDITIONAL KNOCK OUT AS SHOWN HERE, AND SO, JUST BASICALLY KNOCKING OUT, 1 OF THE CRITICAL DOMAINS OF KIBRA, SO KIBRA HAS A WW-DOMAIN AND A C2 DOMAIN. THESE ARE CONSERVED DOMAINS WITHIN THE FAMILY OF PROTEINS AND WE KNOCK THAT OUT AND PUT IT OUT OF FRAME AND HERE'S THE TARGETING, WE GOT APPROPRIATE TARGETS OF THE GENE SHOWN HERE AND YOU CAN SEE THAT WE GET TOTAL ELIMINATION OF PICK1 OF THE CONSIT AND USING A VARIETY OF DIFFERENT ANTIBODIES WE DO NOT DETECT KIBRA AT ALL. THE LEVEL OF OTHER PROTEINS IS NOT CHANGED AS SHOWN HERE AND WHEN WE ANALYZE THIS IN MORE DETAIL AS SHOWN HERE, CAN YOU SEE THAT THERE'S REALLY NO CHANGE OF SYNAPTIC PROTEINS BUT INTERESTINGLY, THERE'S A CHANGE IN A PROTEIN CALLED WWC2. NOW KIBRA'S REAL GENETIC NAME IS WWC1. SO THIS IS--[LAUGHTER] --THIS IS A HOME LOG MUCH KIBRASO IT'S HIGHLY HOMOLOGOUS TO WW--C1, AND YOU CAN SEE THAT IN EARLY ANIMALS, THIS GENE IS UPREGULATED AND SOMEHOW PROBABLY COMPENSATING FOR THE LOSS OF KIBRA, THIS COMPENSATION GOES AWAY IN THE ADULT, AND YOU COME BACK TO BASAL LEVELS OF WWC2. SO THESE MOUSE ARE ACTUALLY OKAY, THEY SEEM TO HAVE NO OVERT BEHAVIORIAL PHENOTYPE, THE MORPHOLOGY OF THE BRAIN APPEARS TO BE FINE. SO WE WEPT ON TO CHARACTERIZE THE PLASTICITY IN MORE DETAIL, AND SO, IF YOU LOOK AT SYNAPTIC TRANSMISSION IN THESE MICE, THE--THE INPUT OUTPUT CURVES ARE THE SAME, SHOWING THAT THERE'S NO--DRAMATIC INCREASE IN THE SELF-OF SYNAPTIC AMPA RECEPTORS AND BASAL CONDITIONS. AND USING THE FACILITATION, THIS IS A MEASURE TO LOOK AT PRESYNAPTIC FUNCTION. YOU SEE THERE'S NORMAL PRESYNAPTIC FUNCTION IN THE KIBRA KNOCK OUT COMPARED TO WILD-TYPE. NOW IN CONTRAST WHAT WE FIND IS THAT IN THE KIBRA KNOCK OUT YOU GET A SIGNIFICANT DECREASE IN THE MAGNITUDE OF LBP. AND IN LTD. SO, THIS IS ACTUALLY VERY SIMILAR TO WHAT WE SEE WITH THE PICK 1 KNOCK OUT, THERE'S A DEFICIENCY IN PLASTICITY BOTH IN LPD AND LTD. SO THIS DATA SUGGESTS THAT KIBRA IS INVOLVED IN AMPA RECEPTOR FUNCTION AND INVOLVED IN SYNAPTIC PLASTICITY BOTH POTENTIATION AND DEPRESSION. NOW, THE NICE THING ABOUT DOING GENETICS IS OF COURSE YOU CAN DO BEHAVIOR, AND WE CAN LOOK AT LEARNING AND MEMORY IN THESE MICE, AND ANALYZE DIFFERENT FORMS OF MEMORY AND IN THIS CASE, WE USED A TRACE CONDITIONING MODEL AS SHOWN HERE AND IN THIS CASE, WHAT YOU DO IS YOU PRESENT A TONE AND THEN GIVE A SMALL INTERVAL AND THEN YOU GIVE A SHOCK AND OF COURSE WHAT THEY--WHAT THEY LEARN TO ASSOCIATE THE TONE WITH THE SHOCK AND THEY WILL START--THEY WILL FREEZE WHEN THEY LEARN THIS BEHAVIOR. THE THIS TRACE ACTUALLY MAKES THIS FORM OF MEMORY BOTH DEPENDENT ON HIPPOCAMPUS AND THE AMYGDALA SO IT'S A NICE WAY TO TEST A HIPPOCAMPAL FUNCTION SO HERE'S TRAINING SESSIONS, CAN YOU SEE THE WILD-TYPE LEARN VERY QUICKLY AND CAN FREEZE IN RESPONSE TO THE TONE VERY QUICKLY. KIBRA ACTUALLY SHOWED A SLOTTER RATE OF LEARNING, BUT THEY DID REACH THE FINAL STINT, THE FINAL EXTENT OF THE WILD-TYPE IS SHOWN HERE, HOWEVER IF YOU COME BACK THE NEXT DAY AND TEST THEM FOR CONTEXTURAL FEAR, YOU PUT THEM BACK IN THE SAME CHAMBER THAT THEY WERE TRAIN INDEED AND YOU CAN SEE WITHOUT A TONE, THE WILD-TYPE MICE WILL FREEZE, THAT THE KEYBOARD RAMICE DO NOT. YOU PUT THEM IN A DIFFERENT CONTEXT IN A DIFFERENT CONTEXT, THEY SHOW NO FREEZING AT ALL AND THIS IS JUST A CONTROL. NOW IF YOU TAKE THEM IN THE NOVEL CONTEXT AND THEN GIVE THEM PRESENT THE TONE THIS SUGGESTS KIBRA IS INVOLVED IN PLASTICITY AND AMPA RECEPTOR FUNCTION BUT ALSO IN LEARNING AND MEMORY. AND SO WE'RE FURTHER LOOKING AT THE HUMAN GENETICS OF THIS, THE--THE SNIPS ARE THE INITIAL SNP WAS DISCOVERED IN THE ENTRON AND WASN'T CLEAR WHAT IT WAS DOING AT KIBRA FUNCTION BUT WE FOUND OTHER SNPS, THAT MAY BE CORRELATING WITH HIPPOCAMPAL ACTIVATION AND LEARNING AND SO WE'RE--WE'RE LOOKING AT HOW THESE SNPS, AFFECT KIBRA PROTEIN STRUCTURE AND FUNCTION AND EVENTUALLY HOPEFULLY WILL MAKE A KNOCK OUT OR KNOCK IN MOUSE TO ENGINEER THESE HUMAN ALLELES INTO A MOUSE TO SEE IF IT AFFECTS LEARNING AND MEMORY. SO THAT'S THE STORY OF KIBRA, BUT BEFORE I LEAVE TI WANT IT POINT OUT THAT IT'S INVOLVED IN THE HIPPO PATH PATHWAY AND IT WAS INENTIALLY DISCOVER INDEED DROSOPHILA AND IT'S INVOLVED IN THE GROWTH OF EPITHELIAL CELLS SHOWN HERE AND IT'S A COMPLEX PATHWAY WHERE KIBRA WILL ACTIVATE A CASCADE INCLUDING HIPPO KINASE THAT PROMOTES CELL PROLIFERATION. THIS IS AN INTERESTING DISCOVERY BECAUSE MANY GENES FOUND IN DROSOPHILA THAT AFFECT CELL GROWTH ACTUALLY HAVE BEEN SHOWN LATE TORE BE INVOLVED IN SYNAPTIC TRANSMISSION AND PLASTICITY, AND INVERTEBRATES. SO WE THINK KIBRA IS PLAYING A ROLE IN DEVELOPMENT BUT ALSO LATER IN THE MATURE NERVOUS SYSTEM TO REGULATE SYNAPTIC PLASTICITY. SO THE OTHER APPROACH WE'VE TAKEN IS ACTUALLY TO DO A THE LAW OF IMAGING SO TO DYNAMICALLY IMAGE RECEPTORS AS THEY TRAFFIC IN NEURONS AND WE'VE USED A VARIETY OF TECHNIQUES IN CULTURE SO WE CULTURE NEURONS IN THIS COVER SLIPS AND THEN WE TRANSFECT THEM WITH GFP TAG FOR RECEPTORS AND IN THIS CASE, A Ph SENSITIVE FLUORINE TAGGED RECEPTOR AND IN THIS WAY, WE CAN WATCH THE RECEPTORS AS THEY MOVE FROM THE PLASMA MEMBRANE INTO ENDOSIDIC VESICLES WHICH ARE HIGHLY AX SIDIC AND SO, UNDER THESE CONDITIONS THE RECEPTORS GET QUENCHED WHEN THEY'RE EXPOSED TO THE CAM PARTMENTS IN THE ENDOSTUDIES OF MULTIPLE ENDOCRINE, SO CAN YOU LOOK AT THIS TO LOOK THE ENDOCYTOSIS SHOWN HERE OR CAN YOU USE IT TO LOOK AT INSERS OF RECEPTORS INTO THE PLASMA MEMBRANE, AND USE THIS TECHNIQUE TO CHARACTERIZE WHAT REGULATES INSERTION AND ENDOCYTOSIS OF AMPORECEPTORS AMPORECEPTORS AND THEREBY AFFECT EVENTUALLY THE LEVELS OF SYNAPTIC AMPORECEPTORS. SO 1 EXAMPLE SHOWN HERE THIS, IS A--USING A TURF MICROSCOPE SO THIS IS TOTAL TURNOVER REFLECTION IN MICROSCOPY, SO WE'RE LOOKING AT THE BOTTOM SURFACE OF THE CELL AND CONTACT WITH THE COVER SLIP, AND HERE, CAN YOU SEE THAT THE--THIS SHOWS A TAG RECEPTORS AT SYNAPSIS, THIS IS THE BRIGHT SPOTS HERE AND WE CAN ACTUALLY BLEACH THAT SO THESE WILL HIGH POWER LASER AND WE BLEACH IT SO WE GET A VERY NICE BACKGROUND AND NOW WHAT WE CAN DO IS WATCH AS THE RECEPTORS THAT ARE COMING FROM THE ENDOSTUDIES OF MULTIPLE ENDOCRINES ARE INSERTED INTO THE PLASMA MEMBRANE AND CAN YOU SEE THESE INSERTION EVENTS. THESE ARE INDIVIDUAL ENDOSEDDIC VESICLES THAT ARE INSERTING INTO THE PLASMA MEMBRANE. NOW CAN YOU LOOK AT THIS HIGHER RESOLUTION HERE, THIS IS POINT OF INSERTION AND THE RECEPTORS LATERALLY DIFFUSE INTO THE DENDRITIC MEMBRANE. AND LOOK AT TRAFFICKING RECEPTORS AND MOLECULES WHAT PHOSPHORYLATION REGULATE THE PROPERTIES OF THESE INSERTION EVENTS, TOO, TO EVENTUALLY REGULATE SYNAPTIC LEVELS. HOWEVER, RECENTLY WE'VE BEEN TRYING TO DO THIS ACTUALLY IN VIVO, SO WE'VE IN SCALING THIS UP TO BEYOND THE COVER SLIP BEYOND THE CULTURE DISH TO DO THIS IN ATTACKED ANIMALS, AND IN FACT, IN AWAKE BEHAVING INTACT ANIMALS. SO WE STARTED OUT, WE'RE MAKING TRANSGENICS AND KNOCK IN FLOURINE TAG RECEPTORS BUT WE'RE ALSO DOING INUTER O ELECTROPRORATION, AND YOU ARE FAMILIAR WITH THIS TECHNIQUE. YOU TAKE THE EMBRYOS IN THIS CASE, MICE, OUT OF THE MOTHERED EMBRYONIC DAY 15 OR SO, AND YOU INJECT DNA INTO THE BRAIN, INTO THE VENTRICLE AND THEN YOU PUT ELECTRODES ACROSS THE BRAIN AND YOU DRIVE THE DNA INTO THE VENTRICULAR WALL AND WE DO AT TIMES SO WE'RE GOING TO LAYER, 5 OR 2-3 CELLS IN THE CORTEX. AND THEN YOU PUT--THIS IS AMAZING, I DON'T DO THIS, MY POTE DOC DID THIS, BUT YOU PUT THE BABIES BACK IN AND LET THE MOTHER GIVE BIRTH TO THEM, AND AFTER BIRTH YOU CAN IMAGE THESE FLUORESCENT, GREEN FLUORESCENT RECEPTORS IN THE BRAIN. SO THIS IS A SLICE OF CORTEX, THIS IS JUST AN ACUTE SLICE LOOKING AT THE TAG OF THE AMPORECEPTORS GLUTEA MATE 1 AND WE ALSO CO TRANSFECT WITH DSRED, SO WE CAN LOOK AT THE TOTAL STRUCTURE OF NEURONS SHOWN HERE. HERE WE'RE DOING P16 AND 88. YOU CAN ZOOM IN AND GIVE FINE STRUCTURE AND YOU SEE THE SPINES AND THE SYNAPSE SIS SHOWN HERE, BUT WHAT WE REALLY WANT TO DO IS DO THIS IN VIVO, WHERE WE HAVE CRANEIO WINDOWS, WHERE IT'S NOW A COMMON TECHNIQUE, WHERE YOU TAKE AWAY, A PORTION OF THE SKULL AND PUT IN A COVER SLIP WITH DENTAL CEMENT AND CAN YOU IMAGE DIRECTLY INTO ABOUT A FEW HUNDRED MICRONS, INTO THE BRAIN SURFACE. NOW INITIALLY WE STARTED ON THE BARRELL CORTEX BUT WE'RE NOW SPREADING AND LOOKINGA THIS IN MOTOR CORTEX AND VISUAL CORTEX, SO YOU TAKE THIS MOUSE AND YOU IMMOBILIZE IT ON A 2-PHOTON MICROSCOPE, AND IN THIS CASE, WE'RE ANESTHETIZING THE MICE AND AS I MENTIONED, I'LL TELL YOU A BIT IN A MINUTE, WE'RE WORKING UP TO A POINT WHERE THEY'RE ACTUALLY AWAKE AND BEHAVING MICE. SO YOU FIX THEIR HEAD, SO IT'S--YOU GET STABILITY FOR THE IMAGING, AND THIS HAS BEEN SUCCESS ENVELOPE MANY LABORATORIES NOW. SO FIRST WE GO IN AND WE LOOK AT THE SOPHISTICATED MAT O SENSORY CORTEX AND WE MAP THE INPUTS USING INTRINSIC IMAGING SO HERE YOU CAN STIMULATE AS MANY OF YOU KNOW, SINGLE WHISKERS WILL INNOVATE LARGE PORTIONS OF CORTEX SHOWN HERE AND IF WE WANT TO ACTUALLY MAP THE REGION OF THE BRAIN THAT'S INVOLVED IN SAY STIMULATING THIS WHISKER, YOU CAN STIMULATE AND THEN RECORD THIS INTRINSIC IMAGING, THIS IS JUST A MEASURE OF BLOOD FLOW, INDIRECTLY MEASURE A BLOOD FLOW IN THE BRAIN AND THEN YOU CAN STIMULATE A DIFFERENT REGION AND MAP OUT WHERE THE DIFFERENT WHISKERS, MAP ON TO THE CORTEX AND CAN YOU SUES THE VASCULATURE TO REALLY USE AS LANDMARKS TO FIND THOSE REGIONS. SO WHAT HAPPENS? THEN WE CAN ZOOM INTO THAT REGION AND USE THE 2-PHOTON MICROSCOPY TO IMAGE THE RECEPTORS, SO HERE WE'RE GOING TO START AT THE TOP OF THE BRAIN AND GO DO A Z-STACK THROUGH, ABOUT 250-MICRONS, AND THE GREEN IS THE AMPORECEPTORS AND THE MAGENTA IS THE DS RED, SO CAN YOU SEE AS WE GO DOWN, YOU CAN SEE ALL THE PROCESSES AND FINALLY WE HIT THE CELL LAYER, 2--LAYER 2-3 CELLS AT THE , AND THEN CAN YOU RECONSTRUCT THIS, AS SHOWN HERE. NOW WE CAN CONTROL THE AMOUNT OF LABELING EITHER USE A LOT OF DNA, A LITTLE DNA OR REUSE TRICKS WITH CREE LOCK SYSTEM TO GET MORE SPARSE LABELING BUT YOU CAN THEN ZOOM IN AND ACTUALLY LOOK AT RECEMENTORS IN THE INTACT BRAIN SO HERE THIS IS A DENDRITE SHOWING THESE BEAUTIFUL AMPORECEPTORS AT THESE SPINES, SHOWN HERE. AND WE'RE LOOKING AT AMPORECEPTORS IN VIVO IN THE ATTACK BRAIN AND WHAT WE'VE FOUND IMMEDIATELY IS THAT YOU SEE A WIDE VARIETY OF EXPRESSION OF RECEPTORS AT SYNAPSES, SO THIS SPINE AS LAT OF AMPORECEPTORS THIS, 1 DOES NOT SO THIS IS A VERY STRONG SYNAPSE SIS THIS, IS A VERY WEAK SYNAPSE SO THIS IS EXACTLY WAWE'RE INTERESTED IN, HOW DOES THE CELL DO THIS IT? HOW DOES IT DIFFERENTIALLY SORT AND TARGET THESE RECEPTORS TO THIS SYNAPSE AND NOT THIS SYNAPSE WHICH WAS ONLY ABOUT 2 OR 3-MICRONS AWAY FROM EACH OTHER. NOW WHAT WE FOUND IS I THINK EVEN MORE AMAZING IS THAT THIS CAN BE STABLE FOR A MONTH. SO WHAT--CAN YOU GO, YOU CAN IMAGE A MOUSE, COME BACK 4 DAYS LATER AND IMAGE IT AGAIN, FLY EXACTLY THE SAME SYNAPSE, AND LOOK AT IT--IT'S AMPORECEPTOR CONNECTICUT TENTS AND SO HERE'S A SPINE THAT'S VERY STABLE AND ALSO HAS A LOT OF AMP O RECEPTORS, HERE'S A SPINE THAT HAS VERY LITTLE AMPA RECEPTORS, AND THE SPINE IS STRUCTURALLY STABLE OVER 30 DAYS. BUT THERE'S NO AMPORECEPTORS. SO AGAIN, IT MAINTAINS A DIFFERENTIAL, CONTENT OF AMPORECEPTORS, ALTHOUGH, REMEMBER, THESE RECEPTORS TURNOVER EVERY 30-40 HOURS. SO THE RECEPTORS AT THE END ARE TOTALLY DIFFERENT AT THE BEGINNING OF THE RECORDING SESSION. SO THERE'S GOT TO BE SOMETHING THAT'S GOING TO TAG THIS SYNAPSE, TO SAY I'M A POETIC AT THE PRESENT TIMEIATED SYNAPSE, I'M NOT AND YOU HAVE TO DELIVER RECEPTORS. SO THIS IS REALLY WHAT WE WANT TO UNDERSTAND IS HOW, SOMETHING CAN BE MAINTAINED FOR WEEKS TO MONTHS TO IN THEORY DECADES. SO, WE'VE BEGUN TO LOOK AT THE EFFECT OF AC50 ON THIS, SO WE CAN ACTUALLY USE ACUTE STIMULATION OF THE WHISKER OR DO SENSORY DEPRIVATION, THERE'S A LOT OF PROTOCOLS IN THE SOPHISTICATED MAT O SENSORY CORTEX, SO IF YOU LOOK AT CONTROL, LOOK AT CONTROL MOUSE WITH NO STIMULATION, YOU GET A VERY STABLE EXPRESSION OF AMPORECEPTORS IN SPINES, IF YOU START DEFLECTING SO HERE WE'RE JUST DOING--ACTUALLY A PREY HIGH FREQUENCY STIMEULOGISTS OF THE WHISKER OVER A PERIOD OF COUPLE HOURS, AND YOU CAN SEE THAT AMPORECEPTOR CONTENTS GO UP, YOU START RECRUITING AMPORECEPTORS TO THESE SPINES AND THIS OCCURS WITHIN AN HOUR AND IS STABLE FOR--I'LL SHOW YOU, UP TO 72 HOURS. HERE'S A COUPLE OTHER EXAMPLES WHERE YOU HAVE RELATIVELY SMALL SPINE, A LARGER SPINE AND THAT END UP RECRUITING AMPA RECEPTORS DURING THE ACUTE STIMULATION. SO, WHEN YOU START LOOKING AT THIS, IT TURNS OUT, THAT STIMULATING THE WHISKERS IN GENERAL, PRODUCES MUCH MORE DYNAMIC ACTIVITY OR AMPORECEPTOR MOVEMENT IN THE SPINE. IN FACT, MOST OF IT IS AN INCREASE, SO YOU SEE AN AVERAGE INREECE IN THE AMPA RECEPTORS OF ALL SPINES, IF YOU LOOK AT ALL SPINES, BUT AS I SHOW YOU, YOU SEE SIGNIFICANT SPINES GOING DOWN AND A LOT OF THEM ARE NOT CHANGING AT ALL. SO FOR EXAMPLE, IF YOU JUST LOOK AT 1 BRANCH OF A DENDRITE, YOU SEE THIS WHOLE BRANCH WILL GO UP IN CONCERT, WHERE IF YOU LOOK AT OTHER BRANCHES, THIS IS THE BIGGEST 1 WE'VE EVER SEEN AND YOU SEE VERY DRAMATIC INCREASE AMPORECEPTOR CONTENT, SOME ARE SMALLER AND THIS GOES DENDRITE BY DENDRITE, THEY SEEM TO CLUSTER WITHIN DENDRITIC REGIONS. HOWEVER SOME, WE SEE FOE CHANGE AT ALL, AND SOME WE SEE DECREASES. SO WE'RE NOW ANALYZE THANKSGIVING DATA IN DETAIL, WHAT'S DIFFERENT ABOUT THESE DENDRITES OR ARE THERE CLUSTERING OF POTENTIATED SYNAPSES, NEIGHBOR ANALYSIS AND INTERROGATING THE DATA TO TRY TO UNDERSTAND WHAT EXACTLY IS GOING ON: NOW WE ALSO SEE, A SMALL INCREASE IN SPINE VOLUME AS L. SO SPINE VOLUME IN GENERAL SEEMS TO BE INCREASING WHEN YOU ADD AMPORECEPTORS TO A SPINE, WE SEE THE SPINE VOLUME IS INCREASES. NOW IN INCREASE IN THE AMPORECEPTOR COMPACT IS BLOCKED BY NMDA RECEPTOR ANTAGONIST, SUCH AS CPP. AND SO, IN FACT WE SEE A SLIGHT DECREASE IN THE LEVEL OF AMPORECEPTOR CONTENT IF YOU INJECT CBP BEFORE STIMMULING THE WHISKERS, SO WE ARGUE THAT THIS IS SOME FORM OF NMDA PLASTICITY THAT'S RECURRING THAT'S RECRUITING RECEPTORS TO THE INAPSEIS. SOPHISTICATED NOW THE NICE THING IS THAT THIS IS LONG LASTING SO YOU STIMULATE FOR AN HOUR AND THEN YOU COME BACK 72 HOURS LATER AND THE AMPORECEPTOR CONTACT FILL UP SO WE THEN IS A CORRELATE OF LTP THAT WE CAN OBSERVE IN VIVO AND IN THEORY WE CAN GO OUT, A WEEK, 2 WEEKS, 3 WEEKS, AND REALLY TRY TO UNDERSTAND WHAT'S REGULATING THIS PROCESS. REMEMBER, WE'RE INJECTING DNA SO WE CAN MUTATE THE RECEPTORS, WE CAN MAKE MUTATIONS IN THE C-TERMINAL DOMAINS AND ANYTHING, WE CAN INJECT THIS INTO ANY KNOCK OUT, WE HAVE 40 OR 50 KNOCK OUTS OR INS IN THE LAB, SO WE CAN INJECT THIS AND LOOK AT THE DYNAMICS, THE DEHAIR OF THE RECEPTORS IN SAY A PFD 95 KNOCK OUT SYNAPTIC PROTEIN KNOCK OUT. SO FINALLY WE CAN ALSO DO MORE LONG-TERM CHRONIC MANIPULATIONS SUCH AS SENSORY DEPRIVATION SO HERE YOU GET IN THIS CASE, WE TRIM THE WHISKERS SO THEY HAVE NO SENSORY STIMULATION INTO THE BARRELL FIELDS AND YOU SEE THAT WITHIN 3 DAYS AFTER TRIMMING, YOU SEE A DECREASE AND THE LEVEL OF AMPEE RECEPTOR CONTENT OF SYNAPSE SIS, IF YOU KEEP TRIMMING, STAY DOWN, YOU LET THE WHISKERS GROW BACK, IT COMES BACK, AND FROM'S A SLIGHT OVERSHOOT IN THIS CASE. SO WE CAN LOOK AT MANIPULATIONS OF SENSORY INPUT AND MORE CHRONIC SENSORY DEPRIVATION PARADIGMS. NOW, I MENTIONED WE'RE NOW--WE'RE NOW SWITCHING TO THE MOTOR CORTEX AND THIS IS A FUN PROJECT STUDENT VS STARTED AND HE IS TEACHING MICE TO--FOR A REACHING TASK WHERE THEY LEARN VERY WELL HOW TO PICK UP A PELLET OF FOOD AND YOU CAN TRAIN THEM AND MAKE IT AWKWARD FOR THEM TO PICK IT UP AND YOU CAN TRAIN THEM TO DO THIS WITHIN 1-2 DAYS AND SO WHAT HE WILL BE DOING IS LOOK IN THE MOTOR CORTEX BEFORE AND AFTER THIS TRAINING PERIOD AND THEN WE ARE ALSO TRYING TO SET IT UP SO WE CAN DO THIS WHILE THEY'RE IMMOBILIZED. THEY CAN LEARN IT WHILE THEY'RE AWAKE AND BEHAVING AND WE CAN IMAGE THEM AND WE WATCH ON A TRIAL, THE TRIAL BASIS WHAT'S HAPPENING AS AT SINATTIC LEVEL, WHAT'S HAPPENING TO THASMA RECEPTORS SO I THINK THIS WILL BE A VERY FUN PROJECT. ALL RIGHT AND SO, THE LAST STORY I WANT TO TELL IS RECENT PAPER WE HAD ON ENZYME PK N, DATA I MENTIONED THAT IN THE BEGINNING AND THIS IS REALLY INTRIGUING FORM OF PKC, IT'S A--MANY OF YOU MAY BE FAMILIAR WITH IT, IT'S--SO PKCs HAVE A CATALYTIC DOMAIN AND A REGULATORY DOMAIN SHOWN HERE AND PKN IS A FORM OF TRUNCATED DATA, IT'S ALTERNATIVE START SITE AND IT'S UNREGULATED KINASE THAT'S ACTIVE AND WORK MOSTLY FROM FACTORS LABORATORY OVER THE LAST 10 YEARS HAS SUGGEST THAD THIS IS REALLY KEY FOR THE MAINTENANCE OF LTP AND FOR SYNAPTIC PLASTICITY. AS WELL AS LEARNING AND MEMORY. THIS TRUNCATED FORM IS PRIMARILY EXPRESSED IN BRAIN TISSUES SHOWN HERE. NOW IN FIELD AND TODD HAS RELIED A SYNTHETIC PEPTIDE THIS, IS BASED ON THE PSEUDOSUBSTRATE DOMAIN WITHIN PKM'S DATA SHOWN HERE AND SO THIS IS LOOKS LIKE A SUBSTRATE FOR DATA AND IT'S ACTUALLY A VERY POTENT POTENT INHIBITOR AND WHAT TODD AND OTHERS HAVE SHOWN OVER THE LAST 10 YEARS IS IF YOU ADD THIS TO LTP, POTENTIATED SLICES, POTENTIATED SYNAPSIS, IT REVERSES THE POTENTIATION SHOWN HERE. SO THIS IS IN SLICE PERPERATION, IN VIVO HE HE SHOW FEDERAL YOU INFUSE, IT INTO THE HIPPOCAMPUS, AGAIN YOU GET A DEPRESSION NOW IT ERATIONS MORE MEMORY, SO IF YOU TRAIN A MOUSE IN MORE PARADIGMS, IF IT'S FEAR CONDITIONING, YOU CAN TRAIN THE MOUSE TO AVOID THIS AREA, SHOWN HERE, SO HERE'S THE TRACING, THIS IS A RAT, RUNNING BACK AND FORTH AND AVOIDING THIS REGION WHERE HE GETS SHOCKED AND SOY THIS IS A WELL LEARNED PARADIGM, HOWEVER IF YOU INFUSE THIS, THIS PEPTIDE, AFTER THE MOUSE AND LEARNED, 44 HOURS LATER IT TOTALLY FORGETS THE AREA AND STARTS GOTTING IN THIS AREA, CAN YOU DO THIS AFTER 3 DAYS AFTER OR 7 DAYS AFTER OR 25 DAYS AFTER SO IF YOU INFUSE THE PEPTIDE, IT ACTUALLY REVERSES OR ERASE THIS MEMORY. NOW THIS IS LED OVER THE LAST 10 YEARS TO A SLEW OF PAPERS USING THIS INHIBITOR PEPTIDE, SO YOU KNOW THIS IS CATHLY WHAT WE'RE INTERESTED IN AND HOW DOES PROTEIN KINASEIS, HOW DO THEY REGULATE SYNAPTIC TRANSMISSION AND SO ABOUT 4 YEARS OOPING, WE STARTED WORKING ON PKMs DATA TO IDENTIFY WHAT ARE THE SUBSTRATES FOR PKMs DATA AND DOES IT FORPHOSALATE AMPORECEPTORS AND PHOSPHORYLATE THE SCAFFOLDING PROTEINS, ET CETERA. SO IF WE TAKE A MORE GENERAL APPROACH, WE USE THESE ENZYMES BUT WE ALSO USE A KNOCK OUT OF PKMs DATA AND THIS IS WHAT WE SHOW HERE SO WE DELETE THE CADDA LYTIC DOMAIN AND WE SHOW IT HERE, WE GET THE CORRECT TARGETS AND WE DETECT NO PKCDATA IN THE CONSITTATIVE KNOCK OUT SHOWN SHEER EITHER WITH END TERMINAL OR C-TERMINAL ANTIBODIES OTHER FORMS ARE NOT ANY DIFFERENT CROSS THE HETEROZYGOTE OR WILD-TYPES SHOWN HERE. THERE'S NO APPARENT COMPENSATION OCCURRING IN THIS KNOCK OUT. NOW, THE FIRST SURPRISE FOR US WAS FIRST OF ALL THERE'S NO EFFECT ON BASAL SYNAPTIC TRANSMISSION, SO THESE HAVE NORMAL INPUT, OUTPUT CURVES AS I MENTIONED BEFORE, NORMAL SYNAPTIC STRENGTH, NORMAL PRESYNAPTIC FUNCTION, POST FACILITATION, EXACTLY THE SAME, BUT THE BIG SURPRISE IS THAT THERE WAS NO EFFECT ON LONG-TERM POTENTIATION, SO, WE WOULD HAVE PREDICTED THAT THESE MICE WOULD HAVE A--AN UNSTABLE LPP, AND THAT I--IT SHOULD DECAY THE BASELINE WITHIN ABOUT 60 MINUTES BUT THESE KNOCKOUT MICE HAVE--YOU KNOW FINE LTP THAT LASTS FOR SEVERAL HOURS, AS SHOWN HERE, USING 2 DIFFERENT PROTOCOLS, THAT INDUCE THIS FORM PF OF PKC, PKM-ZETA PRESUMABLY, THE PKM-ZETA OF LTP. NOW, 1 COULD ARGUE THAT THERE'S SOME CONSITTATIVE COMPENSATION THAT YOU KNOCK OUT THE GENE FROM BIRTH, AND SO, MAYBE SOMETHING'S HAPPENING TO COMPENSATE FOR THIS. SO WHAT WE DID AND WE DID A CONDITIONAL KNOCK OUT, SO WE TAKE THESE MICE AND THESE ARE--WE USE A CAM KINASE 2 CREE ER AND THIS IS A MOUSE WHERE THE CREE, THE ENZYME THAT WILL KNOCK OUT THE GENE IS EXPRESSED ONLY IN PATHWAY GIVES RAMTAL CELLS, IN PRINCE PALE CELLS THROUGHOUT THE BRAIN AND ALSO SENSITIVE TO TAMOXIFEN, SO IT'S INACTIVE UNTIL YOU INJECT THE MICE WITH TAMOXIFEN. SO WE CAN WAIT UNTIL THEY'RE ABOUT 10 WEEKS OLD AND THEN INJECT THEM WITH TAMOXIFEN AND WAIT A COUPLE WEEKS WHERE THE PROTEIN DECAY AND GET KNOCKED OUT, AND YOU CAN SEE IN THE CA 1 REGION OF THE HIPPOCAMPUS REASON YOGEE, THE REASON WE MEASURE FUNCTION, WE GET ABOUT 85% KNOCK DOWN. ON AVERAGE. SO THIS--HAVE YOU A VERY RAPID KNOCK DOWN AND IT TURNS OUT, AGAIN, THERE'S NO EFFECT ON SYNAPTIC TRANSMISSION, EITHER INPUT OR OUTPUT OR PRESYNAPTIC FUNCTION BUT IMPORTANTLY AGAIN, WE SEE NO EFFECT ON MAINTENANCE OF LTP IN HIPPOCAMPUS. THIS IS SURPRISING BUT THE KILLER EXPERIMENT WAS THAT ZIP STILL HAS THE SAME EFFECT IN THE KNOCK OUT AS IT DOES IN THE WILD-TYPE ANIMAL. SO HERE WE'RE LOOKING AT THE PKC KNOCK OUT, HERE'S THE WILD-TYPE, YOU SEE WE GET A ZIP, INHIBITS LTP SHOWN HERE AND ALSO HAS A LITTLE BIT EFFECT ON THE BASELINE BUT IN THE KNOCK OUT, IT HAS EXACTLY THE SAME EFFECT. SO OUR CONCLUSION FROM THIS IS TAKEN--THEY ACTUALLY PKMs DATA IS NOT THE TARGETED ZIP AND THAT THERE'S SOMETHING ELSE THAT ZIP IS ENGAGING TO REVERSE PLASTICITY, AND ACTUALLY DO ERASE MEMORIES. SO IN ADDITION, THESE ARE THE KNOCK OUTS AND AGAIN WE LOOK AT THE TRACE HERE, CONDITIONING SHOWN HERE AND THE WILD-TYPE AND MUTANT LEARN, ALMOST IDENTICALLY AND THE CONTESTURAL FEAR AND THE TRACE MEMORY IS ALSO TOTALLY--SIMILAR IN BOTH WILD-TYPE AND KNOCK OUT. SO THIS IS TRACE FEAR CONDITIONING, WE ALSO LOOKED AT THE WATER MAZE AND AGAIN THE ANIMALS LEARNED VERY WELL AND USING AS MANY OF YOU KNOW, THEY LOOK AT RETENTION AND LOOK AT PROBE TRIALS AND YOU SEE THAT WE SEE THE WILD-TYPE AND THE KNOCK OUT HAVE SIMILAR RETENTION OF THIS MEMORY, EVEN 72 HOURS AFTER TRAINING. SO, AGAIN, PPKM DATA HAS NO EFFECT ON LEARNING IN THESE MICE. SO WE'RE NOW LOOKING FOR THE REAL TARGET OF ZIP. WE'RE DOING BIOCHEMISTRY AS A AN AFFINITY LIAISON GAPPED. THERE IS A POSSIBILITY IT COULD BE ANOTHER PKC ISOFORM ALTHOUGH IT WOULD BE A VERY DIFFERENT MECHANISM, BECAUSE THE OTHER PKCs DON'T HAVE THIS TRUNCATED FORM THAT WE ARE NOW ACTUALLY, THIS WEEK WE'RE DOING PKC IOTA EXPERIMENTS WHERE WE KNOCKED OUT PKC IOTA TO SEE IF IT AFFECTS PLASTICITY AND WHETHER THEY'RE STILL SENSITIVE TO THE ZIP TITTED. --TIDE. SO JUST IN SUMMARY, I TOLD YOU 3 STORIES TODAY, WE'VE BEEN LOOKING AT PHOSPHORYLATION OF RECEPTORS AND HOW THEY'RE REGULATED BY A VARIETY OF PROTEIN CAIN ACES AND--KINASES AND THERE ARE MANY DIFFERENT PROTEIN KINASES THAT AFFECT RECEPTOR FUNCTION AND INTERESTINGLY, NEUROMODDULATORS PLAY A EELLY IMPORTANT ROLE IN ACTIT--REALLY IMPORTANT ROLE IN ACTIVATING RECEPTOR FUNCTION. IN FACT, DRUGS OF ABUSE SUCH AS COCAINE AND THERAPEUTIC DRUGS SUCH AS PROZAC, WILL REGULATE THESE PATHWAYS AND REGULATE RECEPTOR TRAFFICKING. AND THEN FINALLY WE CHARACTERIZED A COMPLEX OF PROTEINS INCLUDE THANKSGIVING PROTEIN KIBRA I TOLD YOU ABOUT WHICH WE THINK ARE INVOLVED IN REGULATING RECEPTOR TRAFFICKING AND ALSO FUNCTION TO MODULATE RECEPTOR FUNCTION DURING PLASTICITY AND TO ENCODE, HELP ENCODE LEARNING AND MEMORY AND AFFECT OTHER HIGHER BRAIN PROCESSES. SO, THE OTHER THING I WANT TO POINT OUT IS THAT A LOT OF THESE GENES HAVE BEEN IDENTIFIED AS--KIBRA HAS BEEN ASSOCIATE WIDE MEMORY PERFORMANCE IN HUMANS BUT WE'VE ALSO SEEN THAT A LOTE OF OUR GENES WE CHARACTERIZE SUCH AS KRIP 1, HAVE THE ASSOCIATED VARIETY OF DISEASES SUCH AS AUTISM AND SCHIZOPHRENIA AND INTELLECTUAL DISABILITY, SO IT BE AFFECTING THE FUNCTION OF THIS COMPLEX AND RECEPTOR FUNCTION AND PLASTICITY WILL AFFECT COGNITIVE ABILITY IN HUMANS. SO, I HAVE A GREAT GROUP OF PEOPLE REALLY, FANTASTIC GROUP OF STUDENTS AND POST DOCS AND LAUREN Mc KUCH, IS A POST DOCTORAL FELLOW AT M. I.T. YOUNG ZANG, DID THE PIONEERING IN VIVO IN THE CRANIAL WINDOW AND ALL THE IN UTERO PREPARATION. JULIA INITIATED THE PKM DATA PROG EXPECT COLLABORATE WIDE LENORA AND POST DOCTORAL FELLOW AND LABORATORY, AND THEN I HAVE A LOT OF OTHER PEOPLE DOING INTERESTING STUFF CURRENTLY IN THE LAB AND FINALLY I WANT TO THANK, SUPPORT, FOR THE RESEARCH FROM HOWARD HUGHES MEDICAL INSTITUTE AND NINDS AND NIMH. AND I'LL BE HAPPY TO TAKE QUESTIONS. [ APPLAUSE ] >> I HAVE A COUPLE OF QUESTIONS ABOUT THE KIBRA. IN THE KNOCKOUT MICE YOU'VE SHOWN THAT SEVERAL OF THE SYNAPTIC PROTEINS LEVELS DON'T CHANGE. HAVE YOU LOOKED AT THE PHOSPHORYLATION STATUS OF THESE PROTEINS. >> SO WE VICTORY LOOKED AT PHOSPHORYLATION SITES, SO, YOU KNOW, I DIDN'T GO INTO--BUT PREVIOUS STUDIES, YOU KNOW THE PHOSPHORYLATION DOES IN AFFECT THESE COMPLEXES, THIS SITES INHIBIT ASSOCIATION WITH PROTEINS AND OTHER PHOSPHORYLATION SITES INCREASE THE ASSOCIATION WITH SOME OF THE PROTEINS. BUT WE HAVEN'T LOOKED AT THAT, I'M SORRY. >> ANY INSIGHT ABOUT THE KIBRA AND PICK IN THE FORMAL MICE WITH HUMAN DEVELOPMENT, HOW IS IT REGULATED? >> WELL WE HAVEN'T--SO FAR WE HAVEN'T FOND ANY HUMAN IMITATION THIS IS PICK 1. NOW THE KIBRA STORY IS INTERESTING, SO WE'RE COLLABORATING WITH PEOPLE AT THE LIBRA INSTITUTE AT JOHNS HOPKINS AND SO THEY HAVE, HUNDREDS OF PATIENTS WHERE THEY'VE GENOTYPED KIBRA AND ALSO LOOKED AT HIPPOCAMPAL ACTIVATION AND LEARNING AND SO WE'RE LOOKING AT THIS IN GREAT DETAIL BECAUSE THESE OTHER SNPSs AND THESE OTHERS AFFECT THE STRUCTURAL COMPONENTS FOR EXAMPLE, THE C2 DOMAIN OF KIBRA AND THESE MUTATIONS, THESE SNPS, AFFECT LIPID BINDING OF THE C2 DOMAIN, SO THAT'S VERY INTERESTING TO FOLLOW THAT UP IN MOUSE MODELS BUT, SO FAR, WE DON'T KNOW--THERE'S NO, YOU HADITATIONS IN PIC THAT WE KNOW OF IN HUMANS. >> ANOTHER THING, YOU MENTIONED THAT THE SMART SNP OF KIBRA HAS A REDICKIVE ROLE IN THE DIMER'S DISEASE, COULD YOU ELABORATE ON THAT. >> SO, WHAT WE FOUND IS ACTUALLY--THAT WAS PUBLISHED RESULT, SO IT'S FOUND THAT THE PEOPLE LOOKING AT SNP POPULATIONS, THE PEOPLE WITH THE SMARTER SNP, HAVE LESS LATE ONSET ALZHEIMER PATIENT HYMER'S DISEASE, COMPARED TO OTHERS, IN FACT WHAT WE FOUND IS IMAGING STUDYING WITH THE INSTITUTE IS THAT AS HUMANS AGE, THE 1S WITH THE SMARTER SNP, THEIR HIPPOCAMPAL ACTIVATION IS HIGHER, IT'S MAINTAINED WHERE 1S WITH THE OTHER LESS SMART SNP, THEY'RE HIPPOCAMPAL ACTIVATION DECREASES WITH AGE BUT THAT'S CONE CYSTENT WITH THE IDEA THAT-- CONSISTENT WITH AN AGE DEPEND END EFFECT ON HIPPOCAMPAL FUNCTION. >> THANK YOU FOR A GREAT SEMINAR, DO WE HAVE TIME FOR QUESTION SYMPATHETIC NERVOUS SYSTEM. >> SO I WAS INTRIGUED BY THAT UPREGULATION IN THE WWC HOMOLOGUE IN THE KNOCK OUT BUT NOT FOR A MONTH, AND I'M WONDERING WHAT THE RELATIVE TEMPERRAL EXPRESSION PROFILES OF THOSE 2 GENES ARE AND WHAT YOU THINK THERE'S A GENETIC INTERACTION GOING ON THERE? >> SO WE'RE AGAIN SEEING THAT IN MUSEUM HANS,---HUMANS, BUT IN MICE THEY DEVELOPMENTALLY GO UP AND GO DOWN. THEY'RE PRESINENT ADULTS AND INTERESTINGLY, WE SEE THESE PLASTICITY PHENOTYPES ONLY IN THE ADULTS AND NOT IN THE YOUNGER ANIMALS SO SUGGESTING THAT WWC 2 IS COMPENSATING IN THE YOUNGER ANIMALS. NOW, WE HAVE DOUBLE KNOCK OUTS OF WC1 AND C2 IF THEY'RE LETHAL SO WE'RE NOW MAKING CONDITIONAL DOUBLE KNOCK OUTS TO SEE FOR EXAMPLE, DO THEY HAVE MORE SEVERE BEHAVIORIAL DEFECTS, FA WE KNOW WWC 1 AND C2 INTERACT, THEY FORM HETEROGENEOUS RARINE.S, SO THEY ARE FUNCTIONALLY ACTING TOGETHER BUT THAT'S ABOUT ALL WE KNOW RIGHT NOW. >> THERE ARE OTHER MEMBERS OF THE GENE FAMILY? >> IN MICE THERE'S ONLY 2, IN HUMANS THERE'S 3, WWC1, 2, AND 3. >> THANK YOU. >> THESE ARE TUMOR SUPPRESSORS AND IN THE DROSOPHILA SYSTEM SO MANY PEOPLE ARE STUDYING THESE IN HUMAN CANCERS AS WELL. >> YEAH, BRIEFLY I WAS WONDERING, YOU MENTIONED 4 OR 5 YEARS AGO, FOR SUBSTRATE TO PKM, DO YOU FIND SUBSTRATES AND IF SO DID THAT AFFECT THE FUNCTIONS OF ANY OF THOSE PROTEINS? >> YES WE DID, WE FOUND--WE TOOK A VARIETY OF APPROACHES, WE DID AN INVITRO CANDIDATE APPROACH WHERE WE MIXED OUR FAVORITE PROTEINS WITH PKMDATA, WE FOUND FORFORALATION, EVENTS, WE FOUND FUNCTIONAL EFFECTS AND WE WE'VE ALSO DONE PROTEOMIC, FOSSIL PROTEOMICS ON THE KNOCK OUT COMPARED TO WILD-TYPE SO WE ISOLATED SYNAPSIS FOR WILD-TYPE KNOCK OUT AND FUNCTIONAL PROTEOMICS. BUT THAT'S ABOUT THE TIME YOU GOT THE DATA THAT KNOCKS OUT PKM DATA THAT HAD NO FUNCTIONAL EFFECT. SO WE START FOCUSING ON THAT AND SO, I'M NOT SAYING PKMs DATA IS NOT IMPORTANT, IT MAY BE INVOLVED IN PLASTICITY AND RECEPTOR FUNCTION, IT'S DEFINITELY THERE, AND IT'S REGULATE BIDE LTP SO THE EXPRESSION OF IT IS REGULATED BY LTP. BUT BY OUR DATA I WOULD ARGUE THAT IT'S NOT THE KEY MOLECULE FOR MEMORY RETENTION AND MAY NOT BE THE REAL TARGET. >> [INDISCERNIBLE] >> SO THAT'S REALLY INTERESTING, WE WANT TO DO THAT ON THE PICK2, PICKBINDS TO PKMs DATA SO WE HAVEN'T DONE LATE PHASE LTP, NOW OF COURSE, YOU SEE SIGNIFICANT EVENTS ON THE EARLY PHASE, BUT YOU MIGHT HAVE A DIFFERENTIAL EFFECT ON THE LATE PHASE. >> WELL, THANK YOU AGAIN FOR A REALLY INFORMATIVE DISCUSSION. KD--SALLY KD--SALLY [ APPLAUSE ]