>> GOOD AFTERNOON, EVERYBODY, I'M JIM DADI, AND I WOULD LIKE TO WELCOME ALL OF YOU TO THE NIH NEUROSCIENCE SERIES TODAY, AND THIS IS SEMINAR IS SPECIAL FOR NIDC, BECAUSE IT FEATURES THE FIFTH ANNUAL LECTURE WHICH HONORS THE MEMORY OF OUR FRIEND AND MEMORY OF BOB LENHOLD WHO SERVED FROM 1998-2008. I'D LIKE TO TAKE A SPECIAL OPPORTUNITY TO THANK CHRIS WENTHOLD FOR JOINING US AGAIN THIS YEAR AND WE'RE DELIGHTED TO HAVE YOU JOIN US FOR THIS SPECIAL OCCASION AND WE WOULD LIKE TO INVITE ALL OF TO YOU JOIN US FOR A LIGHT RECEPTION AT THE END OF THE LECTURE AND I WANT TO THANK THE FOUNDATION FOR T ADVANCEMENT OF EDUCATION AND SCIENCES FOR THE GENEROUS DONATION WHICH MADE THE RECEPTION POSSIBLE. NOW CATHERINE WOULD LIKE TO SAY A FEW WORDS ABOUT THE SPEAKER. >> OKAY, THANKS JIM, IT'S REALLY A PLEASURE TODAY TO BE INTRODUCING LYNN RAYMOND AS OUR SEMINAR SPEAKER AND IN PARTICULAR TO BE GIVEN THE WENTHOLD LECTURE, THIS SPRING IS ALSO A SPECIAL TIME FOR ME BECAUSE OF THIS LECTURE AND THE OPPORTUNITY TO HONOR BOB, WHO WE ALL KNOW WAS A VALUABLE ASSET TO THE NIH COMMUNITY IN MANY WAYS NOT ONLY AS A COLLEAGUE, MENTOR AND FRIEND BUT IT'S ALSOINIZE TO HAVE HIS FAMILY HERE AS WELL. IT'S PARTICULARLY PREERPT THAT LYNN WOULD GIVE THE LECTURE BECAUSE SHE STUDIES NMDA RECEPTORS AND GLUTEA MATE RECEPTORS. HER PARTICULAR ANGLE IS THE SYNAPTIC DYSFUNCTION UNDERLYING HUNTINGTON'S DISEASE. THIS IS A TOPIC THAT WAS NEAR AND DEAR TO BOB'S HEART AND HE WOULD BE HAPPY IF WE COULD BE HERE AND SEE THE DATA AS L. LYNN RECEIVED HER URPD GRADUATE DEGREE FROM THE UNIVERSITY OF ROCHESTER. SHE THEN RECEIVED MD-Ph.D. FROM EINSTEIN. AT THAT POINT SHE WENT TO HOPKINS WHERE SHE DID A RESIDENCY IN NEUROLOGY AND ALSO DID A FELLOWSHIP. POST DOCTORAL FELLOWSHIP IN RICK [INDISCERNIBLE]'S LAB AND AT THIS POINT THAT I MET LYNN, IT WAS A FUN TIME, GRADUATE STUDENT WITH RICK WHEN SHE WAS A FELLOW AND IT WAS JUST A FUN TIME WHEN LIKE BOB'S LAB, THERE WAS THIS EXPLOSION OF RESEARCH AND GLUTEA MATE RECEPTORS WITH A LOT OF ANTIBODIES DEVELOPED IN BOTH RICK'S LAB AND BOB'S LAB. AFTER THAT, LYNN WENT TO UBC AND JOINED THE PSYCHIATRY DEPARTMENT, SHE'S A NEUROLOGIST WHO CONTINUES TO HAVE A CLINICAL PRACTICE, WORKS WITH CHIEF OF THE HUNTINGTON'S CENTER THERE AND SHE ALSO RUNS THE MDPh.D. PROGRAM, BUT ALSO HEAVY EMPHASIS ON BASIC SCIENCE AND IN PARTICULAR THE SYNAPTIC MECHANISMS UNDERLYING NEURODEGENERATIVE DISEASES. SO, AS I SAID, IT'S JUST AN HONOR AND I ALSO JUST WANT TO MAKE THE REMARK THAT IT'S PARTICULARLY EXCITING NOW THAT WE HAVE THE LECTURE EVERY YEAR HERE IN PORTER BECAUSE THOSE OF YOU WHO KNEW BOB, IT WAS SUCH A DREAM OF HIS TO HAVE DEAFNESS BACK ON CAMPUS AND NIDCD IN THE BUILDING AND HONOR BOB AND HAVE THE LECTURE HERE IN PORTER IS REALLY FANTASTIC. >> THANKS SO MUCH CATHERINE FOR THE KIND INTRODUCTION AND FOR THE INVITATION. IT'S JUST A GREAT HONOR TO BE HERE AND GIVING A LECTURE IN HONOR OF BOB WENTHOLT, I DON'T HAVE TO TELL YOU, HE WAS A GREAT SCIENTIST. HE STET THE STANDARD IN THE--SET THE STANDARD IN THE FIELD FOR GLUTEA MATE BIOLOGY AND TRAFFICKING, HE ALSO TRAINED A MENTORS A NUMBER OF YOUNG SCIENTISTS WHO WENT ON TO HAVE THEIR OWN SUCCESSFUL CAREERS AND CATHERINE BEING AN EXAMPLE. AND HE WAS A GENEROUS, REALLY NICE PERSON. HE SHARED HIS EXPERTISE ANYTIME I E-MAILED HIM WITH A QUESTION, I GOT A RESPONSE IMMEDIATELY. HE SHARED HIS REAGENTS AND HE SHARED A VERY OPEN FRIENDLY, MANNER AND A WONDERFUL DRY SENSE OF HUMOR CAN AND IT WAS ALWAYS APPRECIATED, BUT I AGREE WITH CATHERINE AND WISH I CHRONIC LIVER DISEASE PRESENT THIS TALK TO BOB, HE FOCUSED A LOT OF CAREER ON NMDA TRAFFICKING AND TELL YOU TODAY ABOUT ALTERATIONS AND TRAFFICKING AND HOW THAT CAN UNDERLIE MECHANISMS IN NEURODEGENERATION, PARTICULARLY HUNTING DON DISEASE,--HUNTINGTON DISEASE IS A CARTOON CAN INDICATE FIRING OF SYNAPSIS AND THERE'S AN IDEA THAT'S GAINED ATTRACTION RECENTLY THAT'S ABERRANT STRUCTURE ORGANIZATION OR FUNCTION, AND--THIS IS IT JUST A CARTOON THAT YOU'RE NOT SUPPOSED TO BE ABLE TO READ. TO INDICATE THE COMPLEXITY OF A SYNAPSE AND THERE ARE MORE AND MORE MUTATIONS AND SYNAPTIC PROTEINS THAT ARE LINKED TO NEUROLOGIC DISEASE AND MY LAB OVER THE PAST COUPLE OF DECADES NOW HAS FOCUSED ON NMDA RECEPTORS AND THAT ARE AN EXCITATORY SYNAPSE LINKED TO SIGNALING PROTEINS INVOLVED IN ESPECIALLY IN SYNAPTIC PLASTICITY. THEY ARE COMPLEXES, FOUR SUBUNITS, TWO OF WHICH ARE GLUN-TWO, AND GLRKS LUNONE, AND WE FOCUS ON TWO A AND TWO B WHICH ARE HIGHLY EXPRESS INDEED AREAS OF BRAIN INVOLVED IN EURO DEGENERATION AND CORTEX STRIATUM AND HIPPOCAMPUS. SO THESE RECEPTORS ARE KNOWN TO BE CO SINCE DENSE DETECTORS. MAGNESIUM BLOCK IN THE PORE THAT'S VOLTAGE DEPENDENT. --ARE RESPONSIBLE FOR INDUCING PLASTIC ITS ORBED LYING LEARNING AND MEMORY, BUT OVERACTIVATION AS YOU KNOW TOO MUCH CALCIUM CAN BE DETRIMENTAL AND CAUSE NEURONAL DEATH. SO HOW DOES ONE RECEPTOR PLAY SUCH OPPOSING ROLES. ON THE ONE HAND SURVIVAL DURING SIN APT O GENESIS AND DEVELOPMENT AND PLASTICITYOT OTHER HAND CELL DEATH. SO THERE'S A MODEL THAT WAS DEVELOPED ABOUT A DECADE AGO BY JARVIS HARDING HAM AND [INDISCERNIBLE] SUGGESTING SUBCELLULAR LOCALIZATION AND NMDA RECEPTOR CALCIUM INFLUX IS PRO SURVIVAL AND INFLUX THROUGH PERSYNAPTIC RECEPTORS, SIGNAL STAFF AND THAT'S JUST DEPICTED IN THIS DIAGRAM WHERE BLUE IS THE SYNAPTIC ACTIVATION, IF IT'S LOW, FOR EXAMPLE FNEURONS AATE GETTING A LOT OF INPUT DURING DEVELOPMENT AND THERE'S LOW SYNAPTIC ACTIVITY, THAT NMDAs AND THOSE NEURONS GO THROUGH APOPTOSIS BUT AS IT INCREASES IT ONLY--IT STARTS TO PROMOTE SURVIVAL. THE EXTRA SYNAPTIC AS IT INCREASES BECOMES DEATH PROMOTING THERE MUST BE A ROLE FOR EXTRA SYNAPTIC RECEPTORS THAT WE DON'T FULLY UNDERSTAND BUT THIS IS THE MODEL AND IT'S REALLY THE BALANCE, SO, EITHER LOW SYNAPTIC ACTIVITY OR ENHANCED EXTRA SYNAPTIC ACTIVITY WILL PUSH TOWARDS CELL DEATH AND IT'S DETERMINING NEURONAL STATE. THIS IS ANOTHER CARTOON FROM ONE OF THE REVIEWS SHOWING THAT EXTRA SYNAPTIC RECEPTORS ACTIVATE CALPAIN WHICH IS ARE MOTE CRASES AND CHEW UP IMPORTANT SYNAPTIC AND OTHER NEURONAL PROTEINS AND ACTIVATE NOS, AND OXIDATIVE STRESS AS WELL AS MAP KINASES INVOLVED IN CELL DEATH, P38 IS ONE I WILL COME BACK TO. THE SYNAPTIC RECEPTORS THOUGHT TO ACTIVATE PROTEIN KINASES INVOLVED IN PLASTICITY, AND THE ERK ONE-TWO AND ALSO ACTIVATE KREB BY PHOSPHORYLATING WHICH IS A MASTER REGULATOR OF SURVIVAL TRANSCRIPTION OR GENE TRANSCRIPTION. SO, @ EXTRA SYNAPTIC SITES, IT'S THOUGHT THAT THE GLUNTB RECEPTORS PRO DOMINATE AND THE TWO-A SYNAPTIC SITE SO THERE MAY BE DIFFERENTIAL SIGNALING AND THERE ARE SOME GOOD DATA ON THAT WHERE TWO B PREFERENTIALLY ACTIVATES LTP PLASTICITY SO WITH THAT IN MIND, HOW WILL--HOW IS THIS LINKED TO HUNTINGTON DISEASE, WELL THIS GOES BACK A LONG TIME, I GUESS 40 YEARS NOW, 70S OR SO, PEOPLE ARE INJECTS NMDA RECEPTOR AGONIST OR GRIEWTA MATE INTO THE--GLUTEA MATE INTO I TRIAT UMKC AND RECAPITULATING THE MODELS AND THEY LOOK AT BRAIN TISSUE FROM PATIENTS DYING EARLY IN THE EARLY STAGE OF HUNTINGTON DISEASE AND THERE WAS A DISPROPORTIONATE LOSS OF NMDA RECEPTORS AS IF HIGH EXPRESSES WERE MORE VULNERABLE. SO WHAT IS HUNTINGTON DISEASE, I'LL GIVE A BRIEF DESCRIPTION. IT'S A CLINICAL TRI ADOF EMOTIONAL--TRIAD OF EMOTIONAL CHANGES. DEPRESSION, MOVEMENT DISORDER CHARACTERIZED BY INVOLUNTARY DANCE LIKE OR JERKY MOVEMENTS CALLED COREA, AS WELL AS DIFFICULTY MOTOR COORDINATION, BALANCE AND SPEECH. AS WELL AS EARLY ON FROM'S COGNITIVE DYSFUNCTION CHARACTERIZED BY SLOWING OF PROCESS--MENTAL PROCESSING, DIFFICULTY WITH SKILLED LEARNING, AS WELL AS ATTENTION, CONCENTRATION ORGANIZATION CALLED EXECUTIVE DYSFUNCTION. AND IT EFFECTS ROUGHLY ONE IN 10,000 SO ABOUT 40,000 U.S. AND CANADA, AVERAGE AGE OF ONSET IS QUITE EARLY FOR NEURODEGENERATIVE UNDER THE AGE OF 40 ON AVERAGE BUT THERE'S A WIDE SPREAD AND IT SLOWLY PROGRESSES, HEREDITY IS AUTOSOMAL DOMINANT SO YOU NEED A COPY OF THE MUTE ANT GENE TO CAUSE DISEASE. AND THE MUTATION IS AN EXPANSION IN THE CAG TRIPLET REPEAT AND AXON ONE OF THE HUNTINGTON GENE, MORE THAN 39 AND THAT PERSON, THAT INDIVIDUAL WILL DEVELOP HUNTINGTON DISEASE MANIFESTATION IN A LIFE SPAN, THE LONGER THE REPEAT, THE EARLIER OF AGE OFON SET, IT ENCODES A PROTEIN CALLED HUNTINGTON WHICH IS QUITE LARGE AND INTERACTING WITH MANY OTHER PROTEINS AS MANY INTERACTION DOMAINS WITH THIS EXPANDED POLYGLUTA MINE REPEAT DURING THE ED TERM NOWS, AND THIS PROTEIN IS EXPRESS INDEED MOST ORGANS OF THE BODY, IT'S ENRICH INDEED NEURONS AND THIS DISEASE IS ONE OF SELECTIVE NEURODEGENERATION, BOY THIS IS AN AUTOPSY BRAIN MATERIAL AND YOU CAN SEE THE PROFOUND LOSS OF GRAY AND WHITE MATTER IN THE CORTEX AS WELL AS THE NUCLEI, YOU CAN SEE THEM NICE AND PLUMP IN THE CONTROL AND HUNTINGTON'S BRAIN, AND YOU CAN SEE IT'S SEVERELY EFFECTED AND WITHIN THOSE STRUCTURES, IT'S THE GABAergic MEDIUM BINDS AND PROJECTION NEURONS SO THESE RECEIVE MASSIVE GLUTEA MATE INPUT FROM CORTEX AND THALAMUS AND PROJECT OUT TO OTHER BASAL GANGLIA AND NUCLEI AND ARE CRITICAL FOR CIRCUITS THAT REGULATE MOVEMENT, MOOD AS WELL AS COGNITION. SO, I GUESS, I HAVE TO TAKE A STEP BACK AND SAY WELL, WHY AM I STUDYING HUNTINGTON DISEASE, IT'S RELATIVELY RARE IF I WANT TO MAKE AN IMPACT IN NEURODEGENERATION, WHY NOT FOCUS ON THOSE DISEASES. THERE ARE MANY REASONS BUT ONE REASON IS THAT THIS IS A MORE WELL DEFINED DISEASE BECAUSE OF THE GENETICS AND WE CAN ACTUALLY IDENTIFY INDIVIDUALS THAT WILL DEVELOP--DECADES BEFORE THEY WILL DEVELOP THE DISEASE BECAUSE THEY CAN GO THROUGH PREDICTIVE GENETIC TESTING. SO WE KNOW WHO CARRIES THE GENETIC MUTATION AND WITH THAT IN MIND THERE WERE TWO LARGE PROSPECTIVE OBSERVATION OF THESE STUDIES OF THE GENE MUTATION CARRIERS TO DETERMINE PROGRESSION TOWARD DISEASE ONSET AND LOOK FOR BIOMARKERS BECAUSE THAT WOULD BE A HOME RUN IF WE HAVE AN OPPORTUNITY TO REALLY INTERVENE BEFORE THEY BECAME SICK AND I DON'T HAVE TIME TO PRESENT A LOT OF DATA THAT'S BEEN COLLECTED OVER THE YEARS. I JUST TOOK THIS FROM A REVIEW THAT KIND OF SUMMARIZING THAT WE'VE LEARNED THAT THE MOTOR DIAGNOSIS DEFINES DEDISEASE ONSET AND THAT'S A MOVEMENT DISORDER AND NEUROLOGY SPECIALIST AND I SEE ENOUGH CHANGES HERE TO SAY THIS IS UNEQUIVOCAL ONSET OF HUNTINGTON'S DISEASE BUT BEFORE THAT IF YOU USE PARTICULARLY SENSITIVE QUANTITATIVE TESTING, YOU CAN SEE SUBTLE MOTOR ABNORMALITIES AND CERTAINLY COGNITIVE DYSFUNCTION AND BOTH OF THOSE OCCUR WITH MORE THANNA 10 YEARS, USUALLY BEFORE THE MOTOR DIAGNOSIS. AT THE SAME TIME USING MRI TO STRUCTURAL IMAGING WE FOUND THAT EVEN BEFORE THAT, YOU START SEEING A LOSS OF TRIATAL VOLUME THAT MAY EVEN START 15 YEARS BEFORE THE DIAGNOSIS. SO YOU CAN SEE THAT BY THE TIME SPRN RECEIVES A DIAGNOSIS AND ENTERS THE THE PHASE OF THE DISEASE IS THEY'VE LOST 40%. WE DON'T KNOW IF THOSE ARE ALL CELLS THAT ARE 40% CELL LOSS OR DENDRITIC PROCESSES, BUT, AT ANY RATE, YOU WANT TO MOVE YOUR THERAPY BACK TO BEFORE THIS HAPPENS. IS TO TRY TO LOOK AT MECHANISMS THAT ARE HAPPENING IN THIS STAGE, A NUMBER OF MOUSE MODELS HAVE BEEN DEVELOPED AND THESE ARE JUST TIMELINES, THIS IS HUMAN DISEASE, IF THIS IS A DIAGNOSIS, THERE'S THIS PRODOMAL YELLOW PERIOD, AND A PERIOD WHERE CHANGES AND A PERIOD WHERE PEOPLE ARE PERFECTLY HEALTHY AND YOU NO CHANGES TO DETECT. BUT THIS IS WHERE YOU WANT TO INTERVENE AND WE NEED MOUSE MODELS TO STUDY IT AND THE OFFICIAL CHROMOSOME OR YAK MODEL BECAUSE IT USES THAT CHROMOSOME TO INTRODUCE THE FULL HUMAN GENOMIC DNA FOR HUNTINGTON WITH A LONG REPEAT, YOU NEED A VERY LONG REPEAT TO REPRODUCE THIS IN A MOUSE MODEL. AND IT LOOKS LIKE ADULT ONSET HUNTINGTON DISEASE AND IN TERMS OF MOTESSOR FINDING COGNITION AND MOOD. BUT THERE IS A PRETTY LONG PRODOMAL PERIOD WHERE THEY'RE RELATIVELY HEALTHY. COMPARED--AND THE TRIPLET REPEAT AND OVEREXPRESS THAT AND IT'S VERY ACCELERATED MODEL THAT DIES AT ABOUT FOUR MONTHS OF AGE. SO AGAIN WE GET BACK TO THE IDEA OF SELECTIVE DEGENERATION AND OUR LAB AND OTHERS HAVE REALLY SUGGESTED THAT FROM VERY EARLY BEFORE THERE'S ANY REAL PHENOTYPE IN THESE MICE THERE, 'S THE FUNCTION OF THE CORTICAL SYNAPSE AND THAT'S WAWE FOCUS ON AND IN PARTICULAR NMDA RECEPTORS AT THE SYNAPSE AND WE STARTED THAT WORK A LONG TIME AGO, BUT WE HAD RECENT SORT OF VALIDATION FOCUSING ON NMDA RECEPTORS FROM A STUDY THAT CAME OUT OF UCLA, THE YANG LAB WHERE THEY LOOKED AT HUNDRED HUNDREDS EVER INTERACTORS WITH THE PROTEIN PARSING THEM INTO MODULES AND THEY FOUND THAT MANY OF THE INTERACTORS WITH HUNTINGTON ARE SYNAPTIC PROTEINS AND AND THEY PARSE THEM INTO THE PRE AND POST SYNAPTIC MODULE AND THE GREEN GENE IS ONE OF THE HUB INTERACTORS FOR HUNTINGTON. >> SO I'LL START BY TELLING YOU TO LOOK AT THE SYNAPSE AS CLOSE AS WE CAN IN VIVO, WHAT WE DO IS LOOK AT MICE AT A VERY EARLY STAGES ARE JUST AFTER WEANING AND MAKE ACUTE BRAIN SLICES WHERE WE CAN RECORD USING WHOLESALE VOLTAGE CLAMP AND OTHER STIMULATING ELECTRODE TO RELEASE GLUTEA MATE AND ACTIVATE AMP O TYPE GLUTEA MATE RECEPTORS AND NORMAL RESTING POTENTIAL OR DEPOLARIZE TO RELIEVE MAGNESIUM BLOCK AND GET AN NMDA BLOCK BY AP. SO THE FIRST THING--SORRY I WANT TO MENTION THIS WORK WAS DONE BY A FORMER POST DOC AUSTEN MILNERWOOD, WHO'S MOVING ON TO HIS OWN LAB AND WORK ON PARKINSONS NOW. SO ONE OF THE FIRST THINGS AUSTEN DID WAS IF WE LOOKED AT CELLS AND FOUND MORE RECEPTORS AT THE SURFACE AND BECAUSE IF THEY WERE AT SYNAPSES THEN THEY MIGHT BE A GOOD THING AND IT WOULDN'T REALLY EXPLAIN ANY PATHOGENESIS FOR HUNTINN'S DISEASE SO THE FIRST THING IS YOU PUT AN ELECTRODE IN THE RED, GLUTEA MATE VESICLES TO FUSE AND RELEASE AND GET THESE SMALL CURRENTS CALLED SPONTANEOUS EXCITATORY CURRENTS, MOSTLY CARRIED BY AMP O BUT IN LOW MAGNESIUM AS WE DID HERE, THERE WAS A SLOWER NMDA COMPONENT WE CAN PARSE OUT PHARMACOLOGICALLY AND WE FOUND THAT USING A YAC 18 AND IT HAS THE FULL CONTROL AND THE DNA REPEATS WE FOUND THAT NMDA RECEPTOR CURRENT WAS BASICALLY THE SAME AT SYNAPSES. IF WE STAYED WITH THE ELECTRODE IN THOSE SAME CELLS AND INSTEAD STIMULATED RELEASE BY PASSING CURRENT TO ACTIVATE RELEASE FROM MANY SYNAPSES AT ONE TIME WE GET MUCH BIGGER CURRENTS, MUCH EASIER TO MEASURE, COULD I ISOLATE THE NMDA CURRENT AND AUSTIN FOUND THAT HAS HE INCREASED INTENSE ITS OF STIMULATION AND PAIRED THESE PULSES TO OPTIMIZE GLUTEA MATE RELEASE, IT BROUGHT OUT A DIFFERENCE AND WE HAD MORE CURRENT IN THE HD STRIATAL NEURONS AND THAT SUGGEST WIDE YOU RELEASE GLUTEA MATE ALL AT ONCE THAT SOME OF IT CAN SPILL OVER AND PERHAPS ACTIVATE A RECEPTOR OUTSIDE OF SYNAPSIS, TO TEST THAT MORE DIRECTLY WHAT HE DID WAS USE THE NONSPECIFIC FLUTA MATE TRANSPORT INHIBITOR, TBOA BECAUSE THE TRANSPORT ORS WHICH ARE MARELY IN THE GLEA, THE OFTROCYTES ARE PROTECTING NEURONS FROM SPILL OVER FROM THE SYNAPTIC SITES. SO WHEN WE BLOCK THAT EFFECT, WE COULD SEE A SLOWING OF THE NMDA COMPONENT AND MORE AREA UNDER THE CURVE FOR THE COLORED, TRACES AND BY BOTH METHODS THE CHARGE CARRIED BY THE NMDA RECEPTORS OR THE SLOWING OF THIS DECAY, WE FOUND A SIGNIFICANT ERNE HAINSMENT OR INCREASE IN THE ENHANCEMENT FOR YAC 128, COMPARED TO TWO CONTROLS WILD-TYPE AND YAC 18. SO PHYSIOLOGICALLY IT SEEMS THAT THERE WAS A LARGER POOL OUTSIDE THE SYNAPSE OF THE NMDA RECEPTORS COULD BE ACTIVATED WHEN SPILL OVER WAS PROMOTED. THIS WAS MEDIATED BY GLUNTB RECEPTORS SO WE INCUBATE WIDE IFENPRODIL, AND WHEN WE DID THAT THERE WAS NO SIGNIFICANT DIFFERENCE IN THE AMOUNT OF RECEPTORS BETWEEN WILD-TYPE AND 128. AS IF WAS IT JUST MORE SPECIFIC FOR GLUNTB TYPE RECEPTORS, NOW THIS GOES TO REALLY CORE TYPE OF BOB WENDTHOLT EXPERIMENT AND I TALKED TO HIM ABOUT THIS. BIOCHEMISTS DON'T BELIEVE ELECTROPHYSIOLOGYSTS BUT I NEEDED ANOTHER METHOD, PEOPLE WEREN'T GOING TO TAKE MY WORD FOR IT THAT WE WERE ACTIVA EXTRA SYNAPTIC RECEPTORS. SO WE ISOLATED SIN APT O STUDIES OF MULTIPLE ENDOCRINES AND THEN USED DIFFERENTIAL SOLUBLE AND DETERGENT RECEPTORS IN THIS POST SYNAPTIC DENSITY, THEY'RE NOT SOLUBLE, VERSES THOSE THAT WERE OUTSIDE OF WHAT WE CALL THE PSD, NOT RIGHT UNDER THE RELEASE SITE, THOSE COULD BE PRESYNAPTIC AS WELL. AND WHAT WE FOUND EESSENTIALLY FOR TWO B, SUBUNIT, THERE WAS A ROBUST INCREASE IN NUMBERS IN THE NONPSD, NONSYNAPTIC FRACTION FOR 128 TRIATAL TISSUE VERSES THE YAC 18 CONTROL MOUSE TRIAT UMKC, WE SAW SOMETHING--STRIATUM, FOR THE CORTEX. SO BY TWO METHODS WE FOUND THIS INCREASE POPULATION OF EXTRA SYNAPTIC RECEPTORS IN HD STRIATUM AND THIS WAS EARLY. THIS WAS RIGHT AT WEANING. MICE ARE NOT SYMPTOMATIC, ODOR PHENOTEEP MATTER, SO THE NEXT QUESTION WAS, WELL THAT'S GREAT BUT DOES THIS HAVE ANY IMPACT ON THE WAY THE NEURONS OR THEIR SURVIVAL IN VIVO IN THE MOUSE? SO TO LOOK AT THAT WE TURN TO A DRUG CALLED LEMMEN TEEN, IT WORKS--WILL INHIBIT FLOW THROUGH TONICALLY, LOW LEVEL ACTIVATION OF RECEPTORS, AND THOSE ARE MAINLY THE EXTRA SYNAPTIC, WHERE THE PHASIC HIGH ACTIVITY IS PROTECTED WITH LOW LEVELS, AS YOU INCREASED IT WILL BLOCK EVERYTHING AND THAT CAN PROMOTE CELL DEATH BECAUSE YOU NEED SYNAPTIC NMDA RECEPTOR ACTIVITY. SO A GRADUATE STUDENT IN MY LAB ALEX DAU, TREATED THE MICE WITH MEMANTINE OR AND WATER, FOR A PERIOD OF TWO MONTHS AND LOOKED AT LEVELS IN THE FOSTER NURSED FOCUSED ON P38 SO THAT'S ACTIVATED P38 MAP KINASE WHICH IS PART OF CELL DEATH PATHWAYS AND IT WAS INCREASE INDEED THE YAC MICE COMPARED TO WILE TYPE AND THAT WAS RESCUED WITH LOW DOSE AS WELL AS A MODERATE DOSE OF MEMANTINE, NOT AS HIGH AS I MENTIONED BEFORE THAT WOULD BLOCK SYNAPSIS, WHEN WE LOOKED AT FOSTER NURSED FOCUSED ON KREB WHICH IS PROSURVIVAL--SORRY, THAT'S THE FOSTER NURSED FOCUSED ON P38, WHENEE LOOKED AT FOSTER NURSED FOCUSED ON KREB PROSURVIVAL SIGNAL TAG WAS REDUCED IN THE 128 MICE RESCUE WIDE LOW DOSE AND INTERESTINGLY ONCE WE GOT THE DOSE UP A LITTLE BIT, THE WILD-TYPE EVEN WAS SHOWING A DECLINE IN PROSURVIVAL SIGNALING AND NO RESCUE. SO IT SEEMED AS IF IT WAS STARTING TO BLOCK SYNAPTIC RECEPTORS. SO NOW WE HAVE A DRUG THAT CAN PERHAPS REBALANCE THE PATHWAYS THAT I WAS TALKING ABOUT EARLIER, WHAT ABOUT LEARNING AND MEMORY? WELL, WE DIDN'T DO MUCH BEHAVIORIAL TESTING, IT'S NOT SOMETHING MY LAB DOES WE DID PUT THE MICE AT FOUR MONTHS OF AGE AFTER TWO MONTHS OF MEMANTINA ON AN ACCELERATOR ROD AND LOOKED AT HOW MANY TRIAL ITS TOOK FOR THEM TO NOT FALL OFF. THIS IS BASICALLY THE WILD-TYPE DOESN'T TAKE LONG. THE YAC MICE EXCEPT FOR THIS POINT TOOK ABOUT EIGHT TRIALS AND SO WE'RE SIGNIFICANTLY SLOW TORE LEARN, WHEN THEY WERE TREATED WITH MEMANTINE IN GOALS, THEY STARTED OUT POORLY BUT LEARNED QUICKLY THAN THE UNTREATED MICE. SO WE NOT ONLY REBALANCED THE--THE CELL DEATH VERSES THE SURSKIVAL PATHWAYS AND THESE ARE OTHER DOWN STREAM PROTEINS THAT HAVE BEEN SHOWN AND WORK IT FROM OUR LAB AND OTHERS TO SHOW EARLY CHANGES BUT THAT HAD AN IMPACT ON LEARN NOTHING A SKILLED MOTOR TEST. SO, WE--SO WE MIGHT BE ABLE TO TAKE MEMANTINE TO THE CLINIC, WE DON'T HAVE EARLY STADS, WE DON'T HAVE ALL THE RESULTS YET BUT ON THE OTHER HAND, IT MAY NOT BE THE MOST POTENT AND EFFICACIOUS WAY AND THERE ARE ALWAYS SOME SIDE EFFECTS OF DIRECTLY BLOCKING NMDA RECEPTORS AND WE THOUGHT IT WOULD BE INTERESTING TO NOTE, WHAT IS THE MECHANISM UNDERLYING IN INYEAS IN EXTRA SINATTIC RECEPTORS SO HOW IS THIS TRAFFICKING OCCURRING. SO FOR THAT WE TURNED BACK TO CULTURE WORK. BUT WE DECIDED WE NEEDED TO HAVE SOME EXCITATORY SYNAPSES, SO WE PUT ON THE CORACLE NEURONS DISASSOCIATING THEM AND TRANSFECTING THE STRIATUANDATONS, PLATING THEM ONE TO ONE AND THEN WE HAD A MASS CULTURE, DISASSOCIATED CULTURE BUT WE COULD IDENTIFY THE NEURONS AND THIS IS A STRIATAL NEURON CULTURE WITHOUT CORTICAL NEURONS AND WITH CORTICAL NEURONS SO CAN YOU SEE HOW DIFFERENT THE DEVELOPMENT IS WITH LONGER DENDRITIC LANES AND COMPLEXITY AND MORE SPINY PROTRUSIONS. SO WE FIRST DECIDED WE HAD--HOW EARLY CAN WE SEE THIS EFFECT WE FOUND IN THE WEANING IN THE SLICES AND CAN WE SEE IT IN A CULT SNORE SO WE USED AN END TERMINAL YFP TAGGED GLU-TWO A AND B IN THE LAB, AND USED LIVE STAINING SO WE COULD LOOK AT SURFACE RECEPTORS AND PERMEABLIZED TO LOOK AT RECEPTORS AS WELL AS GLUT-ONE WHICH IS A MARKER FOR END SYNAPTIC TERMINALS I DON'T KNOW HOW WELL IT PROJECTS BUT IT IS GREEN AND INTERNAL IS RED, B-GLUT-ONE IS BLUE, IT IS HATTER TO SEE THE BLUE HERE BASICALLY IF WE LOOK FOR COLOCALIZATION, FOR THE GLUT-ONE IT WAS IDENTICAL FOR WILD-TYPE AND YAC 128 AND SIMILAR FOR GLU-NTWOA, WHEN WE LOOK AT THE OVERALL SURFACE TO END TERMINAL RATIO IT WAS SIGNIFICANTLY INCREASED FOR GLUNTBAND FOR THE 128 MICE AND NOT SIGNIFICANTLY SO IT SEEMED TO BE FAIRLY SELECTIVE FOR TWO B, IN THE CULTURES AND APPARENT AT TWO WEEKS IN CULTURE. AND IT SUGGESTED SINCE IT WASN'T THAT SYNAPSES THAT MAYBE THERE WERE EXTRA SYNAPTIC SENSORS SO THEN WE LOOK AT FUNCTION BY PATCH CLAMPING, WHOLESALE VOLTAGE CLAMP ON NMDA AND LOOKING AT CURRENTS IN YAC WILD-TYPE MICE AND WE FOUND A SIGNIFICANT AGAIN IN THE CURRENT AND RECEPTORS OVER THE WLESALE, BUT IF WE LOOK AT SYNAPTIC RECEPTORS LOOKING AT SPONTANEOUS ACTIVITY THAT COULD BE BLOCKED BY THE ONE, THAT WAS NOT DIFFERENT BY THE TYPE, SO AGAIN SUGGESTED EXTRA SYNAPTIC COULD WE MEASURE THAT PHYSIOLOGICALLY. SO WE TURN TO METHODS THAT I THINK WERE AT FIRST MARK MAYOR AND I THINK GARY WESTBROOK USED IT TO ISOLATE EXTRA SYNAPTIC RECEPTORS, ABOUT 10 YEARS EARLIER, BUT YOU CAN PROFUSE ON NMDA AND GET THE WHOLESALE CURRENT, WASH IT OFF, ACTIVATE SINGATTICS ACTIVITY WITH A BLOCKER OF POTASSIUM CHANNELS CALLED PURA DINE AND THEN ADD MKTO ONE SO THEN IT BLOCKS CHANNELS AND IT'S USE DEPENDENT AND ONLY THE RECEPTOR HEAT SHOCK SYSTEM BE OPEN UNDER THESE CONDITIONS. SO AFTER A FEW MINUTES, YOU CAN BLOCK THE RECEPTORS, CAN YOU SEE THE FASTER DECAY COMPARED TO HERE AND WASH IT ALL OFF AND THEN AT NMDA AGAIN, THIS SHOULD BE THE REMAINING CURRENT SHOULD BE EXTRA SYNAPTIC IF YOU BLOCK EVERYTHING AND THAT WAS INCREASED FOR YAC 128. SO WE FOUND THE SAME THING IN CULTURES THAT WE'VE SEEN IN SLICES AND WE CAN NOW LOOK AT MECHANISMS. SO, AROUND THE TILE WE PUBLISH THAD THERE WAS THIS INCREASED POPULATION OF EXTRA SYNAPTIC RECEPTORS AND SLICES, RICK HUGANEERLAB, AND THE PALLETIZATION WHICH IS A POST TRANSLATIONAL MODIFICATION OF RECEPTORS OR ANY--MANY SYNAPTIC PROTEINS WHERE THE 16 CARBON IS ADDED AND CAN TARGET PROTEINS TO DIFFERENT COMPARTMENTS OR IF THEY'RE NOT INTEGRAL MEMBRANE PROTEINS. SO WE THOUGHT TO TWO-B WAS ON NTWOA, SO THREE CYSTINES NEAR THE TRANSMEMBRANE DOMAIN FOUR, THAT'S CALLED CLUSTER ONE. AND A SECOND CLUSTER SORT OF IN THE MIDDLE OF THE C-TERMINUS CALLED CLUSTER TWO WHICH HAD FIVE CYSTINES FOR GLU-NTB AND THE FIRST CLUSTER WHEN IT WAS [INDISCERNIBLE] KEPT RECEPTOR STABLE SIN ASPARITATE SIS AND REDUCE INTERNALIZATION. AND CLUSTER TWOOT OTHER HAND MOSTLY THE CONTROL WAS AT THE GOLGI AND IT REDUCED TRAFFICKING AND INCREASED SURFACE EXPRESSION. SO THIS WAS DONE IN CULTURE, WE'RE INTERESTED IN TRIATAL NEURONS AND WE DIDN'T KNOW IF THESE MECHANISMS WOULD BE THE SAME BUT WE DID KNOW THAT WHEN WE HAD DONE WORK IN TRIATAL MONOCULTURES IN THE PAST THAT THE INCREASED EXPRESS WE SAW WAS NOT DUE TO STABILITY AT THE SURFACE ONCE IT GOT THERE BUT ACCELERATION OF FORWARD TRAFFICKING SO WE'RE QUITE INTERESTED IN THE SECOND CLUSTER. THE FIRST THING WE DID IN THE LAB, TOOK DID SHE FROM ONE MONTH OR THREE MONTH OLD ANIMALS AND STRIATAL TISSUE AND USED SOMETHING CALLED THE BIOTIN EXCHANGE ASSAY TO MEASURE PALLETIZATION LEVELS ON APPROACHING SO IT BRINGS DOWN ALL THE PALITATED PROTEINS AND YOU RUN THEM OUT ON A TYPICAL SGS PAGE AND THEN BLOCK FOR THE PROTEIN OF INTEREST AND IN THIS CASE, THE GLUSPAY NTB AND THAT'S TOTAL AND YOU DAK A RATIO AND YOU CAN SEE THAT FOR THE FEBN WHICH IS THE WILD-TYPE CONTROL MOUSE, THERE'S A LOT OF PALLETIZATION THAT'S REDUCED AND YAK, 128 AT ONE MONTH OF AGE AND THAT WAS A SIGNIFICANT REDUCTION AND WE DON'T SEE THAT IN THE CORTEX, SO STRIATUM BEING MORE SENSITIVE, IT POINTED TO, WELL, MAYBE THIS IS ONE OF THE REASONS, BUT WE DIDN'T KNOW IF THAT WAS ON CLUSTER ONE OR TWO OR BOTH. AND WHETHER OR NOT, THESE CLUSTERS ACTED THE SAME WAY, IN THE STRIATAL NEURONS SO WE WENT BACK TO THE CULTURE AND USED CONSTRUCTS THAT WERE GIFTS FROM THE LAB. THE FIRST ONE WE LOOKED AT WAS CLUSTER ONE, AND AND HOW IT WAS DESCRIBED IN NEURONS, AND THE YAC FEBNOT LEFT, YAC 121 ON THE RIGHT AND ALTHOUGH WE SAW--THE THREE CS, WITH THE CONTROLS OR FOR THE YAC128. THERE WERE TRENDS BUT NOT SIGNIFICANT. SIMILARLY NO CHANGE IN SINATTIC LOCALIZATION SO IT SEEMS IN STRIATAL NEURONS WE DID NOT SO THE SAME REGULATION BY THIS CLUSTER AS THE LAB FOUND IN CORTICAL NEURONS AND NO GENOTYPE DIFFERENCES. SO IT WASN'T INVOLVED IN MODULATING SURFACES OR SYNAPTIC EXPRESSION. WHAT ABOUT CLUSTER TWO, WHEN WE LOOKED AT THAT. WE FOUND SIMILAR IN THE LAB, INCREASE IN SURFACE TO INTERNAL RATIO, FOR THE MUTE APT THAT CAN'T BE POLARIZEDDULATED ON THE AND REDUCE PALLETIZATION, REDUCE FORWARD TRAFFICKING AND WE SAW THAT DIFFERENCE FOR FEBN BUT THERE WASN'T A SIGNIFICANT DIFFERENCE FOR YAC128 AND IT ELIMINATED THE DIFFERENCE WE HAD BEFORE BETWEEN THE GENOTYPES AS IF THE EFFECT WAS THE--AND THIS WAS INCLUDED IN YOC 128 AS IF THE HUNTINGTON IS REACTING TO REDUCE THE CLUSTERRIZATION, AND ENHANCE SURFACE EXPRESSION BY THAT MECHANISM. OKAY, SO THAT SOUNDS GOOD, BUT WE'RE STILL NOT CONNECTING THAT WITH HUNTINGTON. AND TO DO THAT WE THOUGHT, THE OBVIOUS, IT WOULD BE HIP 14 SO THAT STANDS FOR HOUNDING TON ISHT ACTING PROTEIN 14, ONE OF THE FIRST FOUNDED DIRECTLY INTERACT WITH HUNTINGTON AND THE HAYDEN LAB STUDIED THIS AND KNOCKED IT OUT IN MICE AND THEY FIND A PHENOTYPE THAT'S SOMEWHAT REMINISCENT OF HUNTINGTON DISEASE WITH EARLY STRIATAL NEURONAL LOSS AND DEFICIT TO HUNTINGTON DISEASE AND MICE. IN FACT, THEY ALSO FOUND THAT THE ACTIVITY IS REDUCED, HUNTINGTON EXPRESSING MICE, SO IT'S ABILITY TO PUMMELLATE THE SUBSTRATES BUT IT HASN'T BEEN KNOWN BLOOD SAMPLE THE GLURKS NTP SUBSTRATES AND THE HINTS OF THAT IN THE MINUS MICE AND THE GLUE NTB WAS REDUCED SO WE THOUGHT WE WOULD GO TO A SIMPLE SYSTEM AND USE THE COST SEVEN NONNEURONAL CELLS TO EXPRESS FOR RECOMBINANT NMDA RECEPTORS FOR THE WILD-TYPE TWO B, AND THE 53 CS WITH ORIGINAL WITHOUT HIP, AGAIN USING THE ABE ASSAY TO LOOK AT POM ULTIMATELIATION VERSES GLUNTP, SO IN THE CELLS THERE WAS POM ULTIMATELIATIONA WITHOUT HIP 14 THAT WAS SIGNIFICANTLY ENHANCED WHEN WE OVEREXPRESSED THE HIP 14. SO IT LOOKED LIKE IT WAS INDEED A SUBSTRATE. WENT TO LOOK FURGT OR CLUSTER ONE OR TWO OR BOTH, BY LOOKING AT THE BEAT SO THIS ONE ELIMINATES THE PUMMELLATION, AND YOU CAN SEE THE PROPORTIONAL INCREASE AND NO INCREASE IF CLUSTER ONE CANNOT BE POM ULTIMATELIATED SO CLUSTER ONE BUT NOT TWO, WE THINK IS A SUBSTRATE FOR HIP 14, I CAN'T SHOW THIS OTHER DATA WE HAVE KNOCKING IT DOWN ACUTELY IN STRIATAL NEURONS IN CULTURE DOES NOT EFFECT WHOLESALE CURRENT OR DISTRIBUTION TO THE SURFACE CONSISTENT WITH THE FACT THAT THREE CS MUTE ANT. --MUTANT. SO UNFORTUNATELY IT'S AN INTERESTING STORY THAT'S DOESN'T BRING US FULL CIRC TOLL HUNTINGTON. WE HAVE CLUSTER TWO MODULATING SURFACE EXPRESS FOR NNDA RECEPTORS TARPGETTING THE SITES IN THE NEURONS, AND THAT REDUCE P A LMITOYATION, BUT NO TRAFFIC EVIDENCE FOR HIP 14 SO WE'RE LOOKING FURTHER IN MECH 95S AND HOW IT'S RELATED TO HUNTINGTON STREGZ EXPRESSION. SO JUST I HAVE--ANOTHER VERY SHORT STORY TO TELL YOU WHICH DEVIATES A BIT FROM NMDA RECEPTORS BUT, WHAT WE'VE BEEN TALKING ABOUT SO FAR IS THIS INCREASE IN EXTRA SYNAPTIC POPULATION AND THEREYA I LOT OF DEBATE AS TO WHAT ARE THOSE RECEPTORS THERE FOR AND THEY MAY BE INVOLVED IN SOME FORMS OF SYNAPTIC PLASTICITY, IN PARTICULAR GRAHAM KUHLING'S LAB SAID MAYBEIN INVOLVED IN THE HIPPOCAMPUS BUT NEURONS ARE PROTECT FRIDAY THE OVERACTIVATION OF THIS POOL BY A NUMBER OF FLUTA MATE TRANSPORTERS, EXPRESS BOTH IN NEURONS AND ASTRO CITES AND IT'S THOUGHT THAT GLT ONE, WHICH IS PREDOMINANTLY, AN ASTRO CITE MEDIATES 90% OF GLUTEA MATE UPTAKE IN THE BRAIN, OTHER VS ESTIMATED AT LEAST 50% AND THAT MAY PROTECT FROM THE LESIONS SO THE QUESTION WE HAD IS, IS THIS DYSFUNCTION OF THESE TRANSPORTERS CONTRIBUTING TO EXCESSIVE ACTIVATION AS WE SAW EVIDENCE FOR IN OUR SIGNALING STUDIES. >> IT'S JUST TO SHOW THERE ARE A LOT OF KEEPERS THROUGHOUT THAT SAY, THERE'S IMPAIRMENT GLUTEA MATE UPTAKE, AND MOST OF THEM SAY THAT OCCURS AS THE DISEASE PROGRESSES, MOST OF IT IS IN THE ACCELERATED MODEL, THE R62 MODEL AND EVEN IN THE YAC 128, THERE'S EVIDENCE FOR REDUCED--HOWEVER MOST OF IT, SO THIS IS WELL ACCEPTED VIEW, THAT YOU HAVE A COUPLING OF THIS INCREASED EXTRA SYNAPTIC WITH IMPAIRED UPTAKE THAT IS A MAJOR CONTRIBUTOR, A PUSH TOWARD CELL DEATH SIGNALING AND INTERFERING WITH PLASTICITY. AND MUCH OF THE DATA IS BIOCHEMICAL USING SIN APT O STUDIES OF MULTIPLE ENDOCRINAL PREPS, EXVIVO TO ADD TREATED EXOGENOUS GLUTEA MATE AND LOOK OVER A MINUTE TO STEADY STATE UPTAKE AND UNDER THOSE CONDITIONS MOST OF THIS IN THE R362 MODEL AND WE'VE DONE IT AS EARLY AS ONE MONTH AND FROM YAC 128 REDUCED--SO WE AGREE WITH ALL THE LITERATURE THAT IT HAPPENS EARLY AND COULD BE COULD YOU TELLING. BUT WHAT ABOUT THE REALTIME MEASUREMENT OF CLEARANCE OF SYNAPTICALLY REDUCED GLUTEA MATE. SO TO GET AT THAT, WE'VE TURNED TO A NEW OPTICAL SENSOR DEVELOPED FROM THE LUKER LAB AT THE FARMS THAT HAS CIRCULARIZED GFP. WHEN IT BINDS, IT'S VERY SPECIFIC TO BINDING GLUTEA MATE. WHEN IT BINDS AND INCREASES, GREEN FLUORESCENCE, IT ANCHORS TO CELL MEMBRANES AND THE EXTRA CELLULAR, AND IT YOU CAN TARGET IT TO NEURON EXPRESSION. NEURONAL EXPRESSION IS THE SYNAPSE PROMOTER AND THE REASON WE DID THAT IS I'M JUST GOING TO JUMP TO THIS CARTOON, SO HERE'S OUR--IT'S CALLED GLUSNFR, IT'S SITTING HERE BUT BUT IT'S A GREAT ONE. IT'S JUST TO SHOW THAT HERE'S THE STRIATAL NEURON, IT'S SITTING, IT'S NOT JUST ON THE SPINE. IT'S INTRP IN THE PLASMA MEMBRANE AND IT'S NOT ON THE ASTRO SIGHT. SO HERE WE'RE ABLE TO INTERROGATE HOW LONG DOES THE STRIATAL NEURON, HOW LONG IS IT EXPOSED AFTER GLUTEA MATE RELEASE, AND IS IT ACCURATE? DOES IT TAKE A LONGER TIME TO CLEAR THE GLUTEA MATE AS SEEN BY THE NEURONS. SO HERE'S JUST CUT OUT OF WHAT WE'RE LOOKING AT OUT OF THE WIDE FIELD MICROSCOPE, THE GFP FLUORESCENCE AND IT'S DORSAL STRAIN AND MEDIAL, THIS IS A WIDE FIELD LIGHT AND THAT WOULD BE THE CORPUS COLS ON ORDER OF MICRONS. THAT'S WHERE THE GLUSNFR IS. SO THAT'S OUR ELECTRODE SO WE WILL STIMULATE WITH CURRENT AND THEN MEASURE RELEASE AND UPTAKE OF GLUTEA MATE OR I'LL JUST SAY CLEARANCE OF GLUTEA MATE BY THIS GLUSNFR METHOD. SO THIS IS IN REALTIME. THIS IS SINGLE STIMULATION, RUNNING OVER AND OVER AGAIN AND CAN YOU SEE IT'S QUITE FAST IT RISES QUICKLY AND HERE IT IS APLOTTED AND WITHIN ABOUT 250 MILLISECONDS, THE SIGNAL IS DECAYING BACK TO BASELINE. SO WE--WE TRIED 50 PULSE TRAINS T SEE IF WE COULD CHANGE TIME COURSE OF RISE OR FALL OF THE RESPONSE SO LET'S JUST GO BACK TO SHOW YOU SLOW DOWN SIX FOLD. FOLLOWING AND COMPARED TO--SO DUMPING MORE GLUTEA MATE INTO THE STRIATUM, IT TAKES LONGER. AND THERE'S THE DIFFERENCE IN TIME COURSE, [INDISCERNIBLE] AND OTHER THINGS THAT MADE US THINK THIS IS ACCURATELY REPORTING ON TRANSPORTER FUNCTION THAT LEADS TO SOME EXTENT THAT IF WE USE THE DRUG DHK, DISCIPLINARY HYDRO--[INDISCERNIBLE] WHICH SELECTIVELY BLOCKS THONE, AND AT LOW DOSE WE COULD SLOW THE DECAY, SO IT'S INTERFERING WITH THE MEDIATED UPTAKE AND SIGNIFICANCE, SLOWING THE CLEARANCE AND IF WE DID A TRAIN OF PULSES IN THE PRESENCE--[INDISCERNIBLE] SUGGEST THANKSGIVING DOES DEPEND ON ACTION RELEASE OF GLUTEA MATE AND FURTHER MORE TRANSPORTERS ARE LIKE ENZYMES, THEY WORK BETTER AT HIGHER TEMPERATURES AND THEY WENT BLACK TO SOMETHING MORE CLOSE TO PHYSIOLOGIC 32-DEGREES. SO BY THESE MEASURES WE THOUGHT WE HAD A PRETTY GOOD REPORTER OF GLUTEA MATE CLEARANCE AND STARTED TO COMPARE STRIATAL SLICES FROM THE YAC128 AND WILD-TYPE MICE AND THEY WERE OVERLAY ASKED AND THIS IS NORMALIZING PEAKS AND THE PEAK RESPONSE IS SHOWN HERE AND THAT'S NOT SIGNIFICANTLY DIFFERENT SO THEY'RE RELEASING THE SAME AMOUNT OF GLUTEA MATE AND IT'S DECAYING--WE THEN DID PULSES WITH DIFFERENT PULSE INTERVALS AND IF ANYTHING THERE WAS A TREND TOWARDS ACTUALLY SHORTER DECAY OR FASTER CLEARANCE. TAKEN--THEY'S THE PEAK RESPONSE, AND THE KEEP TIME CONSTANT WAS NOT CHANGED, SO WE THOUGHT WELL, MAYBE WE JUST DUMP GLUTEA MATE SO WE WENT TO THE HALF SECOND TRAIN AND AGAIN, WHY INCREASED THE AMOUNT OF FLUTA MATE RELEASE BUT WHEN WE NORMALIZE TO THE END OF THE STIMULATION AND LOOK AT THE K, IT WAS PRETTY IDENTICAL FOR BOTH PEAK AND THE AND TRENDING TOWARDS A LITTLE FASTER AND THAT TREND WAS EVEN MORE PROMINENT AND 32-DEGREES. THE DECAY TIME CONSTANT SHORTER, THE CLEARANCE IS FASTER, NOT SLOWER. THESE ARE PR SYMPTOMATIC MICE BUT AGAIN, ACTUALLY A MONTH OLDER THAN WHEN WE SAW A DEFICIENCY OF GLUTEA MATE UPTAKE BY THE PREP, SO THEN WE THOUGHT MAYBE WE JUST HAVE TO GO AND LOOK AT MICE THAT ARE REALLY SICK, WE WENT TO 15 MONTHS AND SINGLE PULSES THAT ARE COMPLETELY OVERLAID AND THERE'S NO DIFFERENCE IN TIME CONSTANTS WITH THE TRAIN OF PULSES WHAT CAME OUT OBVIOUSLY WAS LESS GLUTEA MATE WAS RELEASED AND THE AREA UNDER THE CURVE FOR THE AMOUNT OF GLUTEA MATE AND AMOUNT OF STIMULATION WAS SIGNIFICANTLY LOWER FOR THE JAC 128, BUT THE TIME CONSTANT FOR THE DECAY WAS THE SAME. SO EVEN IN THE REALLY ILL MICE, THERE WAS NO IMPAIRMENT OF GLUE UTRA MATE UPTAKE SO EVEN THOUGH WE HAD DONE THIS AND DONE THE SAME THING, THE RSIX-TWO MODEL THERE'S DATA MORE IN THE LITERATURE, MAYBE WE NEED TO LOOK AT THAT MODEL. SO WE GOT SMART AND COMPARED THEM TO THE LITTER MATE CONTROLS, AT A TIME WHEN THEY WERE SICK AND FOUND THE RED, WAS SIGNIFICANTLY FASTER IN TERMS OF GLUTEA MATE CLEARANCE BASED ON THIS REPORTER ASSAY. AND THAT WAS--WITH DIFFERENT PULSE INTENSITIES, DIFFERENT NUMBERS OF PULSES, THE DECAY TILE IS FASTER, YOU ALSO NOTICE THAT THE PEAK RESPONSE WAS SMALLER AND THAT'S KNOWN THAT THEY LOSE SYNAPSES EARLY AND THERE WAS NO CORRELATION--THE PEAK AND THE DECAY TILE WHEN WE LOOKED AT THAT. NOW THIS IS A FLUORESCENT REPORTER AND PEOPLE LIKE JEFF DIAMOND HAVE OTHER WAYS TO LOOK AT THIS WHICH ARE QUITE ELEGANT AND WE THOUGHT WE WOULD GIVE THAT A TRY WHICH IS TO LOOK MORE DIRECTLY AT GLT-ONE FUNCTION, WE DID WHOLESALE PATCH CLAMP ON ASTRO SITES BECAUSE THAT'S ELECTROGEN GENIC TRANSPORTER AND WHEN YOU GET GLUTEA MATE UPTAKE THROUGH THE TRANSPORTER YOU GET INWARD CURRENT THAT HAS REALLY TWO COMPONENTS, HA THE SORT OF STEADY STATE THAT'SA NOT THE TRANSPORTER CURRENT, THAT'S STILL ARE REMAINS AFTER THE BLOCK OF TRANSPORTERS AND CAN ISOLATE THE SYNAPTIC TRANSIENT CURRENT AND COMPARE THAT BETWEEN THE BETWEEN THE SMALL CURRENT AND IN FACT WE SAW THE SAME THING, THE DECAY CONSTANCE FOR THIS CURRENT WERE 10 FOLD FASTER THAN WHAT WE SAW WITH THE GL, SNFR, REPORTER BUT THEY WERE FASTER FOR OUR SIX, TOO. SO BY ALL MEASURES INSED OF IMPAIRED GLUTEA MATE UPTAKE, WE THINK WE HAVE PERHAPS AN ATTEMPT TOWARDS COMPENSATION FOR THE FACT THERE WERE MORE EXTRA SYNAPTIC RECEPTORS AND ENHANCED WITH SYNAPTIC RELEASE AND WE STILL DON'T HAVE A REASON WHY YOU WOULD SEE SOMETHING DIFFERENT IF YOU USE CHRONIC EXOGENOUS EXPOSE TOWER GLUTEA MATE. BUT, THIS IS THE KIND OF CARTOON YOU WILL FIND IN MOST REVIEWS OF THE SYNAPTIC MECHANISMS OF HUNTINGTON'S DISEASE AND I HIGHLIGHTED THIS. IT'S KNOWN THERE'S INCREASED STIMULATION OF THE RECEPTORS AND THAT REUPTAKE OF GLUTEA MATE IS DIMINISHED BY THE ENHANCED SUSCEPTIBILITY AND METABOLIC TOXIC EFFECTS, SO WE THINK THAT WE HAVE TO REVISE THAT AND THAT UNDERCONDITIONS THAT ARE MORE INVITO WITH THE SYNAPSES AND LOOKING AT SYNAPTIC RELEASE, WE REALLY DON'T HAVE ANY EVIDENCE FOR THIS AND THE IMANT TAKE HOME POINTS FOR THAT IS THAT, IT MAY NOT BE WORTH WHILE TARGETING ENHANCEMENT OF UPTAKE TO PROTECT NEURONS FROM THESE EXTRA SYNAPTIC RECEPTORS BECAUSE PROBABLY THEY'RE DOING SOMETHING IMPORTANT, CERTAINLY IN OTHER PARTS OF THE GRAIN AND IF THEY'RE NOT DOWN REGULATED THEN, UPREGULATING THEM MAY NOT BE THE BEST APPROACH. SO I WILL SUMMARIZE AND JUST GO OVER EVERYTHING I TOLD YOU, SYNAPTIC AND EXTRA SYNAPTIC BCE FOR NMDA RECEPTORS FUNCTION AND SIGNAL IS ALTERED EARLY, AT GLUTEA MITTERGIC SYNAPSESOT MOUSE MODELS, THEY'RE CONSEQUENCES WE CAN DETECT, USING BIOCHEMISTRY AND THE TISSUE, DOWN STREAM SIGNALING, THAT WE SEEM TO HAVE REDUCED CELL SURVIVAL AND FACILITATION OF SITE PATHWAYS THIS WOULD HAVE POSED PLASTICITY AND WE'RE STARTING TO LOOK AT THAT. MEMANTINE AND SIMILAR DRUGS THAT TARGET RECEPTORS COULD BE USEFUL FOR SWELLING PROGRESSION BUT PROBABLY YOU NEED TO START QUITE EARLY AND NOW WE KNOW ABOUT THE PMAL STAGE WE HAVE BIOMARKERS WE CAN USE IT FOR MEASURES THAT MAY BE POSSIBLE BUT MAYBE WE CAN FIND MORE EFFECTIVE AND WE'RE STILL GOING TO LOOK AT MECHANISM AND WE DON'T THINK THAT ENHANCING GLUTEA MATE UPTAKE IS GOING TO BE USEFUL STRATEGY AND I WANT TO GIVE YOU THE TAKE HOME MESSAGE THAT HUNTINGTON DISEASE CAN BE A MODEL FOR STUDYING THE IMPACT OF EARLY INTERVENTION TO MAINTAIN SYNAPTIC INFLUENCE. THANKS. [ APPLAUSE ] >> I SHOULD SAY SOMETHING SINCE I DIDN'T DO ANY OF THESE EXPERIMENTS. HIPICTURES OF PEOPLE FROM THE LAB BUT I THINK I FORGOT TO MENTION THAT PARSON DID THE iGLUSNFR PROJECT WITH MATT AND [INDISCERNIBLE]. SO THAT'S A CLOSE COLLABORATION AND WE'VE BEEN COLLABORATING WITH THE HAYDEN LAB. AND THIS IS THE FUNDING. >> [INAUDIBLE QUESTION FROM AUDAIENCE ] >> OKAY, SO, ANOTHER OBSERVATION THAT MAY BE LISTENED TO THAT IS MATT FOUND THAT HE NEEDED STRONGER SIMULATION TO MEASURE THOSE EXPERIMENTS SO I'LL ASK YOU LATER BUT THEY'RE SMALL AND HE NEEDED TO STIMULATE A LOT OF GLUTEA MATE RELEASE TO MEASURE THE CURRENT. MAYBE THEY'RE JUST NOT VERY SENSITIVE? MAYBE YOU NEED A HIGH LEVEL OF GLUTEA MATE TO MEASURE THAT CURRENT AND AS IT FALLS BELOW THAT LEVEL, YOU CAN'T DETECT IT ANYMORE AND YOU CAN DETECT IT WITH MORE SENSITIVE FLUORESCENCE. THE GLUSNFR BINDS GLUTEA MATE AND IS SORT OF A BUFFER, SO ANY BUFFER, WHETHER IT'S CALCIUM INDICATOR IN A CELL WILL CHANGE THE TIME COURSE OF THE THING YOU'RE MEASURING. SO THAT'S DEFINITELY GOING TO SLOW THINGS DOWN. SO WE--I THINK THOSE TWO THINGS TOGETHER MAY EXPLAIN IT BUT I'M HAPPY TO HERE WHAT YOU THINK. >> TELL ME LATER. OKAY. >> WELL, ALZHEIMER'S DISEASE, SO STEWART LIFTON AND OTHERS ARE VERY EXCITED ABOUT DTHEY'RE WORKING OF A BETTER VERSION OF IT. AND THEY'RE HAVING A COUPLE OF TRIALS IN HUNTINGTON'S DISEASE THAT HAVE NOT BEEN PUBLISHED YET, THEY'VE START WIDE MIDSTAGE DISEASE WHERE PEOPLE HAVE A GROSS COGNITIVE IMPAIRMENT, MEASURED BY THE MANY MENTAL STATE EXAMINE WHICH IS NOT SENSITIVE. YEAH, I THINK IF WE ARE GOING TO DO THIS, THAT'S THE TARGET THAT WE NEED TO--YES YES? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> IT WON'T BE IN THE ASTROCYTES BUT WE DID REPEAT THESE EXPERIMENTS I'M NOT SHOWING THEM WITH THE PROMOTER AND WE GOT THE SAME