>> HI, EVERYBODY. I GUESS IT'S TIME THAT WE CAN GO AHEAD AND START. MY NAME IS ROD, I'M A PROGRAM DIRECTOR WITH THE NINDS AND IT'S MY PLEASURE TO INTRODUCE THE SEMINAR TODAY ON BEHALF OF THE GSIG. THE ELDERLY, AS YOU KNOW, COMPRISE THE FASTEST GROWING POPULATION, SEGMENT OF OUR POPULATION, AND AGING ITSELF IS THE LARGEST SINGLE RISK FACTOR IN MOST CHRONIC DISEASES. CANNINGLYACCORDINGLY, THE TRANSNIH GSIG INTEREST GROUP WAS FOUNDED IN 2011 TO BOTH RAISE AWARENESS AND ESTABLISH A COLLABORATIVE FRAMEWORK AND PROMOTE AND FURTHER RESEARCH AND DISCOVERIES OF COMMON RISKS AND MECHANISMS IN CHRONIC DISEASES OF AGING. ONE WAY THAT THE GSIG RAISES AWARENESS IS THROUGH HIGH PROFILE SEMINARS THAT ARE PUBLICLY BROADCAST, AS THE SEMINAR THAT WE'RE HAVING TODAY. PREVIOUS SPEAKERS HAVE INCLUDED DR. FRANCIS COLLINS, DR. JAMES KIRKLAND AND DR. RICHARD MILLER. TODAY WE HAVE THE PLEASURE OF WELCOMING DR. JIE SHEN, A PROFESSOR IN THE DEPARTMENT OF NEUROLOGY AT THE HARVARD MEDICAL SCHOOL. DR. SHEN WAS TRAINED IN BIOCHEMISTRY AS AN UNDERGRADUATE AT EAST NORMAL UNIVERSITY IN SHANGHAI AND RECEIVED HER PH.D. IN 1994 FROM THE UNIVERSITY OF VIRGINIA. THE BODY OF WORK THAT SHE'LL TALK ABOUT TODAY IN MANY WAYS STARTED WHEN SHE WAS A POST DOCK POST-DOCTORAL ASSOCIATE AND PUBLISHED A PAPER IN WHICH SHE KNOCKED OUT PRESENILIN 1. THIS WAS A MAJOR STEP FORWARD FOR ALZHEIMER'S DISEASE AND ALSO IN OTHER WAYS, AND HAS BEEN CITED MORE THAN 500 TIMES AND HAS BEEN A BUILDING BLOCK THAT SHE HAS BUILT UPON IN ASSEMBLING AN IMPORTANT AND INFLUENTIAL BODY OF WORK. AS HER CAREER HAS MOVED FORWARD, SHE'S BEEN RIGOROUS, AN ORIGINAL THINKER, AND A LEADER, AND THIS HAS BEEN IN THE FIELD OF AGE-DEPENDENT NEURODEGENERATION. PLEASE JOIN ME IN WELCOMING DR. SHEN. [APPLAUSE] >> I WANT TO THANK ROD FOR THE NICE INTRODUCTION, AND ALSO FOR INVITING ME TO COME HERE TO SHARE WITH YOU OUR WORK, AND SO INITIALLY, I GUESS THAT'S SORT FROM NOT READING EMAILS CAREFULLY AND JUST REALIZED A FEW MINUTES AGO, THIS WAS GOING TO BE BROADCASTED, SO I HAVE TO TAKE OUT ALL THE UNPUBLISHED DATA AND I FELT LIKE I HOPE, YOU KNOW, THE SEMINAR WILL STILL BE EXCITING AS ALL OF US KNOW, WE'RE ALWAYS MORE EXCITED ABOUT UNPUBLISHED WORK. ONCE IT'S PUBLISHED, IT'S KIND OF -- OH, IT'S DONE. SO IN A SENSE KIND OF GOOD, I WON'T BE SO RUSHING TO, YOU KNOW, TO THE LAST SLIDE AND WE'LL HAVE MORE TIME FOR DISCUSSION, AND ALTHOUGH THE TITLE I INITIALLY GAVE WAS GOING TO BE ON ALZHEIMER'S AND PARKINSON'S BECAUSE MY LAB ACTUALLY DIVIDED HALF AND HALF. HALF THE LAB WORKS ON ALZHEIMER'S DISEASE AND HALF WORKS ON PARKINSON'S DISEASE BECAUSE WE FELT LIKE THERE WAS -- THERE ARE A LOT OF SYNERGY BETWEEN THE TWO DISEASES AND ALSO WE USE VERY SIMILAR METHODOLOGIES TO STUDY THE DISEASE MECHANISMS. HOWEVER, I HAVE TO ADMIT, I TELL MY POST DOCS I'M SOMEWHAT BIASED BECAUSE ALZHEIMER'S IS SO MUCH MORE PREVALENT, AND IF WE HAVE TO CHOOSE ONE DISEASE TO AVOID, I WOULD DEFINITELY TAKE ALZHEIMER'S DISEASE. THERE'S NOT A SINGLE DRUG, WHEREAS PARK SOB PARKINSON'S DISEASE HAS A DECENT DRUG THAT CAN CONTROL THE SYMPTOMS REASONABLY WELL. SO FOR ALL THESE REASONS, AND I HAVE ALWAYS BEEN SORT OF LIKE MORE PASSIONATE ABOUT UNDERSTANDING THE BASICS OF ALZHEIMER'S DISEASE, AND, THEREFORE, THE TALK I PUT TOGETHER LAST NIGHT WAS GOING TO BE FOCUSING ON ALZHEIMER'S DISEASE, WITH ONLY ONE SUMMARY SLIDE ON PD AND SHOWING THE SYNERGY BETWEEN THE TWO DISEASES. SO THIS IS SORT OF MY BRIEF INTRODUCTION. THIS FIRST SLIDE SUMMARIZES THE APPROACH WE USE TO STUDY THE ALZHEIMER'S PARKINSON'S DISEASES. AS ROD HAD INTRODUCED, I WAS TRAINED AS A MOLECULAR GENETICIST STUDYING ALTERNATIVE SPLICING, AND SO I ALWAYS FIND THAT TO BE KIND OF TOO ABSTRACT FOR ME, ALTHOUGH I ENJOYED THE BASIC SCIENCE AND I FOUND THAT I WANTED TO STUDY SOMETHING THAT'S MORE RELEVANT TO HUMAN HEALTH, SO WHEN I STARTED MY POST DOC AND I HAD CHOICES OF CONTINUING SORT OF BASIC SCIENCE, IMMUNOLOGY, WITH A VERY SORT OF FUNDAMENTAL QUESTION, OR SORT OF A APPLY MY BACKGROUND IN BIOCHEMISTRY AND MOLECULAR GENETICS TO STUDY THE DISEASE MECHANISM. SO IT DID TAKE ME SEVERAL DAYS TO CONVINCE SUSAN TO ALLOW ME TO WORK ON DEMENTIA IN THE LAB, LARGELY DEDICATED TO THE BASIC MECHANISM, MEMORY, SO I FELT LIKE THESE TWO SIDES ARE REALLY TWO SIDES OF ONE COIN, THEY'RE VERY, VERY RELATED, AND WITH THE IDENTIFICATION OF PR PRESENILIN 1 AND 2 IN 1995 AND I FEEL THE BASIS IS RIGHT FOR A MOLECULAR BIOLOGIST TO USE MY GENETIC BACKGROUND TO STUDY THE DISEASE MECHANISMS. SO THAT'S WHAT WE DO. THE REASON WHY I PICKED ALZHEIMER'S AND PARKINSON'S DISEASE IS BECAUSE THE MUTATIONS AND IDENTIFYING THESE GENES CAUSE THE DISEASE AT A VERY HIGH PENETRANCE. SO THAT BASICALLY ALLOWS US TO USE GENETIC APPROACHES TO IDENTIFY THE FUNCTION AND THE DYSFUNCTION OF THESE DISEASE-LINKED GENES IN ALZHEIMER'S OR PARKINSON'S DISEASES, AND WE GENERATE GENETIC MODELS, AND THEN USE ALL SORTS OF NEUROBIOLOGY TO APPROACH IT TO STUDY THE PATHOPHYSIOLOGY IN THE RELEVANT NEUROCIRCUIT. NEWENEURAL CIRCUIT. I DON'T THINK THIS AWD YEBS NEEDS MUCH INTRODUCTION FOR ALZHEIMER'S DISEASE, AND I STILL WANT TO HIGHLIGHT THE PER VAI LANCE OF THE DISEASE, CONSIDERING THE FACT THAT THE POPULATION IS LIVING LONGER AND LONGER, AND FOR MANY OF US, THE CHANCE OF DEVELOPING ALZHEIMER'S DISEASE LATER IN LIFE IS QUITE REAL, BECAUSE IT'S 50%. NOT ONLY -- THIS IS KNOWN OBVIOUSLY AS SORT OF A MEMORY DISEASE, BUT ACTUALLY GOES FAR BEYOND THAT, ONCE A LATER STAGE PATIENT DEVELOPS SEVERE DEMENTIA, THAT REALLY SORT OF LIKE ALLOW THEM NOT TO BE ABLE TO TAKE CARE OF THEMSELVES AT EVEN THE MOST BASIC DAILY FUNCTIONS, AND NEUROPATHOLOGICALLY, IT WAS DEFINED BY THE LOSS OF SYNOPSIS IN THE NEURONS AND THE SHRINKAGE OF THE WHITE MATTER AND GRAY MATTER AS WELL AS ENLARGEMENT OF THE LATERAL VENTRICLES, AND ALSO THE OTHER KEY FEATURES ARE THE INTRACELLULAR NEURO -- THE EXTRA CELLULAR AMYLOID PLAQUE. FOR MANY YEARS, AND WE HAVE KNOWN THAT THE AMYLOID PLAQUES IS THE POOREST QUALITY OF THE DEMENTIA. THE AMYLOID PRECURSOR PROTEIN WAS LINKED TO ALZHEIMER'S DISEASE ABOUT 20 YEARS AGO, AND THE MUTATIONS WOULD PRESENT ABOUT 10% OF ALL OF THE FAD MUTATIONS AND THE PR PRESENILIN FAMILY, THEY SHARE SEQUENCE HOMOLOGY AND MORE THAN 200 MUTATIONS HAVE BEEN IDENTIFIED IN THIS GENE FAMILY. SO OF COURSE AS WE ALL KNOW, THE PREVAILING HYPOTHESIS IN THE FIELD HAS BEEN THE -- ESPECIALLY THE LONGER 42 IS NECESSARY AND SUFFICIENT TO INITIATE A DISEASE PROCESS. THE KEY EVIDENCE IN SUPPORT OF THIS HYPOTHESIS INCLUDES THOSE THREE, AND FOR EXAMPLE, AMYLOID PEPTIDES ARE THE MAJOR COMPONENTS OF AMYLOID PLAQUE, AND I THINK THE MOST -- THE STRONGEST EVIDENCE IS ACTUALLY THE FACT THAT MUTATIONS IN ATP ARE CAUSAL AND ARE -- THEY CAUSE DISEASE AT A VERY HIGH PEN CHENS, SO DEFINITELY A BETA AND APP HAVE SOMETHING TO DO WITH THE PATHOGEN CYST OF ALZHEIMER'S DISEASE, AND FAD MUTATIONS IN PRESENILINS, A LOT OF THEM DO CAUSE SELECTIVE INCREASE OF THE LONGER PEPTIDES. HOWEVER, THERE ARE A FEW EXPERIMENTAL EVIDENCE THAT ARE NOT PARTICULARLY SORT OF LIKE AT ODDS WITH THE HYPOTHESIS. FOR EXAMPLE, FROM MANY YEARS, PEOPLE HAVE KNOWN THAT THE AM EMPLOYED PLAQUE ARE NOT THE BEST CORRELATE FOR THE SEVERITY OF COGNITIVE IMPAIRMENT IN AD, AND ALSO MANY, MANY LINES OF TRANSJE NICK MICTRANSGENIC MICE HAVE BEEN PRODUCED, PRODUCING LOTS OF ABB OR FORMS OF A BETA AND MOST OF THEM, IF NOT ALL, DO NOT PRODUCE SIGNIFICANT NEURODEGENERATION. THE EXPLANATION HAS BEEN THAT MAYBE THE MOUSE BRAIN IS MORE RESISTANT TO DEVELOP AGE-DEPENDENT NEURODEGENERATION. LASTLY, ALL OF THE AMYLOID BASICALLY SO FAR HAVE FAILED TO MEET THE END POINTS IN TERMS OF IMPROVING COGNITIVE AND ALSO DAILY FUNCTION IN AD PATIENTS. SO WHAT CAUGHT MY ATTENTION, AND FROM THE VERY BEGINNING, WAS ACTUALLY THE DISTRIBUTION OF THE MUTATION, AND EVEN IN THE ORIGINAL PAPER, THE FINE MUTATIONS ARE SCATTERED THROUGHOUT THE CODING SEQUENCE, AND AS YOU CAN SEE HERE, THE RED DOTS REPRESENT THE -- MUTATIONS IN PRESENILIN 1. THERE ARE MANY, AND ALSO SOME OF THESE AMINO ACIDS HAVE MULTIPLE MUTATIONS AND THEY SCATTER THROUGHOUT THE CODING SEQUENCE, UNLIKE APP MUTATIONS TEND TO CLUSTER AROUND A CLEAVAGE SITE, PRESENILIN MUTATIONS ARE VERY BROADLY DISTRIBUTED, AND THERE IS A BIOLOGIST, WHEN I LOOK AT -- AS A BIOLOGIST, WHEN I LOOK AT THE SLIDE, MY SORT OF GUT FEELING IS THAT THIS PROTEIN IS SO IMPORTANT IN THE CENTRAL PROCESSES OF THE DISEASE, THEREFORE, A SINGLE NUCLEOTIDE CHANGE RESULTING IN A SINGLE AMINO ACID RESIDUE ALTERATION IS SUFFICIENT TO CAUSE THE DISEASE AT ALMOST 100% CERTAINTY. AND THE OTHER IS THAT THE MUTATIONS, TO ME, ARE LIKELY TO BE LACK OF FUNCTION MUTATIONS BECAUSE THEY DON'T CLUSTER AROUND ANY FUNCTIONAL DOMAINS, AND THE DIVERSE DISTRIBUTION WOULDN'T BE CONSISTENT WITH THE MUTATION, SOMEHOW DESTABILIZES THE PROTEIN STRUCTURE, CAUSE A GENERAL IMPAIRMENT OF THE FUNCTION OF THE PROTEIN. SO WITH THIS IN MIND, AND SO I DECIDED TO USE GENETIC APPROACH TO STUDY THE NORMAL FUNCTION OF PRESENILINS IN THE BRAIN. SO THIS SLIDE SUMMARIZES THE EARLIER WORK WE DID, AND BECAUSE THE PS1 KNOCKOUT DIED AT -- THAT CLEE PREE CLEUDED ANALYSIS OF FUNCTION IN THE CEREBRAL CORTEX, HOWEVER, WE DIDN'T DO A NUMBER OF STUDIES TO IDENTIFY PRESENILIN FUNCTION IN THE DEVELOPING CORTEX, AND THAT ALLOWED US TO ACTUALLY GOT QUITE A LOT OF INFORMATION, USEFUL INFORMATION THAT AIDED OUR LATER STUDIES. FOR EXAMPLE, PRESENILINS ARE EXPRESSED IN ALL CELL TYPES IN THE DEVELOPING CEREBRAL CORTEX, HOWEVER, THEIR FUNCTION, INDIVIDUAL CELL TYPES, ARE VERY DIFFERENT. AND I WILL NOW GO INTO THE DETAILS, BUT I THINK THAT'S A VERY IMPORTANT MESSAGE, IS THAT FOR US TO TAKE INTO A STUDY, IS BECAUSE OF THE CELL SPECIFICITY OF ITS FUNCTION, WE OUGHT TO STUDY ITS FUNCTION IN THE RELEVANT CELL TYPE IN THE ADULT BRAIN. THE OTHER TAKE-HOME MESSAGE IS THAT FROM ALL THE GENETIC STUDIES, WE FOUND THAT NOTCH IS REALLY THE KEY MEDIATOR IN THE DEVELOPING CEREBRAL CORTEX. WITH THAT IN MIND, AND WHEN WE WANT TO DEVELOP MOCK MODELS TO STUDY PRESENILIN FUNCTION IN THE ADULT BRAIN, WE SPECIFICALLY LOOK AT THE G EXPRESSION PATTERN. BECAUSE OF NOT A SINGLE GOOD ANTIBODY THAT IS VERY GOOD, AND FOR IMMUNOHISTO CHEMISTRY, TO STILL RELYING ON HYBRIDIZATION TO KNOW WHERE PRESENILIN IS EXPRESSED VERY ABUNDANTLY. THIS SHOWS THAT PRESENILIN 1, FOR EXAMPLE, IS EXPRESSED VERY HIGHLY OF THE HIPPOCAMPUS AND ALSO THE REST OF THE CORTEX. SO WE TOOK ADVANTAGE OF SPECIFIC PROMOTER THE ALPHA CHAIN OF THE CAM KINASE 2 PROMOTER EXPRESSED EXACTLY IN THOSE CELL TYPES OF THE ADULT CEREBRAL CORTEX STARTING FROM THE POSTNATAL WEEK, LIKE THE THIRD WEEK. AND SO WE USED THE CRELOX TECHNOLOGY BY INTRODUCING TWO INTO PS1 INTERRON 1 AND 3-RBGS AND WE KNOW -- 2 AND 3 WILL CAUSE NO ALLEY OH, SO NORMALLY THIS MOUSE IS COMPLETELY NORMAL BECAUSE INTRODUCTION OF THESE TWO VERY SHORT SITES DOES NOT AFFECT TRANSDESCRIPTION, SPLICING OR TRANSLATION. HOWEVER, WHEN WE CROSS TO THIS SPECIFIC, WE CALL IT THE POSTNATAL FOREBRAIN BECAUSE THAT'S VERY BROAD, AND ONCE YOU CROSS THESE TWO MICE TOGETHER, IN THOSE CELLS THAT NORMALLY EXPRESS HIGH LEVEL OF PS1, CRE WILL MEDIATE SITE-SPECIFIC -- TO REMOVE PS1 ESSENTIAL XONS RESULTING IN NO ALEO, ONLY IN -- CELL TYPES AND IT STARTS LIKE POSTNATAL DAY 18 AND BY THE TIME THE MICE ARE ABOUT FOUR WEEKS OLD, AND THE GENE IS INACTIVATED IN MOST IF NOT ALL OF THESE NEURONS. BECAUSE PS2, NO MICE HARDLY HAS ANY DETECTABLE PHENOTYPE, SO WE'RE ABLE TO CROSS THE PS1 SINGLE -- TO PS2 NO BACKGROUND TO GENERATE -- WES WE ALSO CALL IT CONDITIONAL DOUBLE -- MICE. SO FIRST WE WANT TO KNOW WHETHER PRESENILINS ARE IMPORTANT FOR COGNITION IN THE ADULT BRAIN, AND WE SUBJECT THOSE MICE TO TWO HIPPO CAMPO DEPENDENT -- LIKE -- WHAT WE DISCOVERED WAS THAT THERE IS A GENE DOSAGE EFFECT, AND THE DOUBLE -- MICE EXHIBIT MORE SEVERE LEARNING MEMORY DEFICIT THAN THE SINGLE CKO MICE, AND ALSO THERE IS A VERY STRONG AGE DEPENDENT EFFECT, AND SO AS THE MICE GET OLDER, AND THE PS CDKO MICE COMPLETELY FAIL TO LEARN THE WATERMAZE OR THE CONDITIONING, INDICATING THAT AND THERE IS REALLY VERY STRONG AGE-DEPENDENT EFFECT LINKED TO LOSS OF PRESENILINS, AND THIS SHOWS THE SUBTLE BUT SIGNIFICANT MEMORY IMPAIRMENT, AND SO BECAUSE IN THIS PARTICULAR PARADIGM, WE GAVE THE MICE SIX TRIALS A DAY, AND THIS IS LIKE A PRETTY INTENSIVE TRAINING PROTOCOL, THEREFORE, THE SUBTLE -- MORE SUBTLE PS1 SINGLE CKO MICE ELIMINATED. I ALWAYS LIKE TO USE THE ANALOGY, ONE MAY BE BRIGHTER, BUT IF THE ONE WHO IS A LITTLE BIT LESS BRIGHTER AND TH STUDIES REALLY HARD FOR EASY TASK, THEY PROBABLY WILL DO SIMILARLY WELL. THIS IS REALLY KIND OF WHAT WE FOUND WITH THESE MICE, AND IF WE GAVE THE MICE SIX TRIALS A DAY FOR FIVE DAYS, THEN -- AND THE PS1 CKO AND THE PS2 -- MICE PERFORMED AS WELL AT THE CONTROL MICE, WHEREAS THE PS CDKO DIDN'T SHOW SIGNIFICANT LONGER LATENCY, LATENCY MEANS THE TIME IT TAKES FOR THEM TO FIND THE HIDDEN PATH, AND ALSO WE SAW A SIGNIFICANT REDUCTION IN TERMS OF THE QUADRANT OCCUPANCY, AND THE PATH ON PLATFORM LOCALIZATION. IF WE SUBJECT THE MICE TO ONLY FOUR TRIALS A DAY, THEN WE COULD SEE SIGNIFICANT LONGER LATENCY BY THE PS1 SINGLE KO MICE INDICATING LOSS OF PS1 ALONE IS ENOUGH TO PRODUCE COGNITIVE DEFICIT IN THESE MICE. SO SUBSEQUENTLY, WE WANT TO UNDERSTAND THE CELLULAR BASIS FOR THE LEARNING MEMORY DEFICITS. WE DID A LOT OF ELECTROPHYSIOLOGY, AND IN THIS LINE OF DOUBLE CKO MICE, WE FOCUSED ON THE HIPPOCAMPUS AND PARTICULARLY THE COLLATERAL PATHWAY, THE CA3, CA1 SYNAPSE, AND WE WANT TO FIND AGE-DEPENDENT DEFECTS, SO WE PACEICALLY MEASURED A NUMBER OF PRESNAP TICK AND POST SNAP TICK PARAMETERS, WE STARTED ANALYZING THESE MICE AT FOUR WEEKS OF AGE POST NATELY, BECAUSE THIS IS WHEN PR PRESENILIN IS ACTIVATED. THEN WE DID FIVE WEEKS, SIX WEEKS, SEVEN AND EIGHT. THE FIRST DEFICITS WE SAW WAS THE FREQUENCY FACILITATION, AND THEN BY THE TIME THE MICE ARE SIX WEEKS OLD, WE SAW POST -- DEFECTS, FOR EXAMPLE, THE NMDA RECEPTOR MEDIA RESPONSES. SO WE GENERATED TWO ADDITIONAL LINES OF DOUBLE CKO MICE, AND ONE OF IS ENACT SERRATED I ENACT INACTIVAT ED, THIS IS SHOWN IN THE LAST OF PS1 -- IN WHICH IT IS ELIMINATED IN MOST OF THE C1 NEURONS AND OF COURSE WE CONTINUES TO ANALYZE THESE MICE IN ADDITION TO THE FOUR BRAIN CKO MICE. OF COURSE A VERY IMPORTANT CONTROL EXPERIMENT IS TO MAKE SURE THAT THERE WAS NO LOSS OF PRESENILIN IN THE POST SYNAPTIC NEURONS OR PRESYNAPTIC NEURONS -- SO WE CROSSED TO THE ROSA 26 REPORTER LINE WHICH WILL BASICALLY TELL US WHERE CRE IS EXPRESSED, IN A VERY SENSITIVE MANNER BECAUSE EVEN IF YOU HAVE A FEW CELLS THAT EXPRESS CRE IN REGIONS, FOR EXAMPLE, WE WILL BE ABLE TO SEE THOSE BLUE CELLS, AND THE SAME IS TRUE FOR THIS, SO BASED ON THIS ANALYSIS, WE KNOW THAT IF WE ANALYZE THE MICE AT TWO MONTHS OF AGE, THESE ARE REALLY, REALLY HIGHLY RESTRICTIVE. FOR CA3CKO MICE, RECOMBINATION ONLY OCCURS IN -- AND FOR CA1, RECOMBINATION ONLY OCCURS IN CA1 NEURONS. THE RESULTS, AS USUAL, SURPRISED US. WE THOUGHT BASED ON THE FACT THAT RECEPTOR FUNCTION IS REPAIRED IN FOUR BRAIN CKO MICE, WE WOULD SEE LTP DEFICITS IN THE CA1 DOUBLE CKO MICE, BUT THE NATURE TOLD US THAT THAT'S NOT THE CASE. ALTHOUGH THE RECORDING WAS DONE IN CA1 AND DATE OF BIRTH INDUCED LTP WAS ACTUALLY NORMAL WHEN WE KNOCK OUT PRESENILIN IN CA1 NEURONS ALONE. SURPRISINGLY, IF WE KNOCK OUT PRESENILIN IN THE PRESYNAPTIC NEURONS, WE SAW A PRETTY DRAMATIC REDUCTIONS IN TBS-INDUCED LTP. WE FURTHER LOOKED INTO -- THIS IS KNOWN TO BE THE BASIS OF LTP, AND SO THIS IS THE SLIDE, AND IT SHOWS THAT IN FOUR BRAIN DOUBLE KO MICE, MEASURED INTRACELLULARLY USING INHIBITORS BLOCKED -- RECEPTOR RESPONSE, BUT IF WE KNOCKED OUT EITHER IN PRE OTHER POST SYNAPTIC NEURONS AND WE DO NOT SEE ANY IMPAIRMENT OF NMD RECEPTOR FUNCTION, SO WE ALSO EXAMINED PRESYNAPTIC FUNCTION IN ALL THREE LINES OF THESE DKO MICE, WHAT WE FIND IS THAT FOR EXAMPLE, PAIR POST FACILITATION IS IMPAIRED IN THIS PRESYNAPTIC SPECIFIC DCKO MICE AND ALSO FREQUENCY FACILITATION IN THESE MICE IN A FREQUENCY DEPENDENT MANNER, AND LOOKING TO FURTHER WHETHER THIS PRESYNAPTIC FUNCTION MAY ANALYZE LTP DEFICIT, WE USED THE FIRST -- OF DPS TO INDUCE FREQUENCY FACILITATION, WHAT WE SAW IS THAT THE WILD TYPE SLICES SHOWS NICE FACILITATION AS EXPECTED, HOWEVER, INACTIVATION FAILED SO SHOW ANY FACILITATION. WE ONLY SAW THE DEPRESSION. SO TO MEASURE MORE SPECIFICALLY NEUROTRANSMITTER RELEASE AND BECAUSE IN THE CA3DKO MICE RECEPTOR FUNCTION IS NORMAL SO WE COULD HAVE USED THE OPEN CHANNEL BLOCKER MKO1 TO MEASURE -- SORT OF DIRECTLY, AND THE MKO1, WHAT IT DOES IS THAT IT WILL IRREVERSIBLY BIND TO NMD RECEPTORS SO WHEN AN NMD RECEPTOR IS ACTIVATED BY THE RELEASE OF NEUROTRANSMITTER AND BASICALLY IT WILL BE BOUND BY MKO1 AND PERMANENTLY INACTIVATED. SO WE SAW THIS DECAY, AND IN CONTROL SLICES, AND IN THE KNOCKOUT, WE SAW A DELAY IN THE SYNOPTIC DETAIL INDICATING THAT FEWER GLUCONATE IS RELEASED, SO WE ALSO USED ANOTHER WAY TO MEASURE NEUROTRANSMITTER RELEASE PROBABILITY, AND IN THE FOREBRAIN DOUBLE CONDITIONAL KNOCKOUT MICE, BECAUSE IN THESE MICE, NMD RECEPTOR FUNCTION IS ALREADY IMPAIRED SO WE CAN'T REALLY USE THIS OPEN CHANNEL BLOCKER METHOD TO EVALUATE PROBABILITY OF RELEASE, SO WE USED MINIMAL STIMULATION KIND OF PROTOCOL, AND WE ALSO SAW THAT AND THE USUAL RESPONSE IS IMPAIRED IN FOUR BRAIN DKO MICE. THEN I DIDN'T SHOW THE DATA BUT WE ALSO FOUND THAT FREQUENCY FACILITATION DEFECT IS DEPENDENT ON EXTERNAL CONCENTRATION SO THAT KIND OF LED US TO WANTING TO LOOK AT WHETHER CALCIUM INFLUX OR EFLUX IS AFFECTED IN THE ABSENCE OF PRESENILINS. SO WE MEASURED CALCIUM INFLUX BY MEASURING THE FUNCTION OF CALCIUM CHANNEL WHICH MEDIATED CALCIUM INFLUX FROM THE EXTRACELLULAR SPACE, AND THAT IS NORMAL, AND THEN WE ALSO LOOK AT CALCIUM EFLUX FROM INTRACELLULAR STORE, SUCH AS ER, USING -- IT BASICALLY INHIBITS -- INVOLVED IN THE REFILLING OF ER CALCIUM, SO IF YOU BASICALLY REMOVE THE MECHANISM OF CALCIUM REFILLING INTO THE ER, WHAT YOU FIND IS THAT CALCIUM WILL BE DEPLETED THROUGH THE LEAK CHANNEL AND THEN WHAT WE FOUND IS THAT WHEN WE TREAT THE CONTROL SLICES, THEY KIND OF MIMIC THE FACT OF THE KNOCKOUT. FOR EXAMPLE I SHOW YOU HERE, AT 20 HERTZ, YOU SEE THE BIGGEST DIFFERENCE IS THAT THE IMPAIRMENT OF SYNAPTIC FACILITATION IS VERY SIMILAR TO THE KNOCKOUT SLICE, WITHOUT ANY TREATMENT, AND ONCE WE TREAT KNOCKOUT SLICES WITH -- WE SEE THE FACT THAT BASICALLY THE SLICE DOES NOT SORT OF, LIKE, SHOW ANY MORE FACILITATION. SO INDICATING THAT CALCIUM EFLUX UNDERLIES THESE FACILITY -- IN THESE MICE, AND BECAUSE ER CALCIUM RELEASE IS MEDIATED THROUGH TWO TYPES OF RECEPTORS, ONE IS THE -- AND THE OTHER IS IP3 RECEPTORS. SO WE USED TWO DIFFERENT RECEPTOR BLOCKERS, AT A HIGH CONCENTRATION OR ANOTHER SPECIFICALLY ANTAGONIST, DANTROLENE, AND WE FOUND THAT TREATMENT OF EITHER OF THESE DRUGS MIMICS THE -- TREATMENT, MEANING THAT BASICALLY IT CAN MIMIC THE KNOCKOUT EFFECT AND ALSO IN THE KNOCKOUT, THE TREATMENT -- ANY MORE EFFECT. SO INDICATING THAT RYANODINE MEDIA RECEPTOR RELEASE IS INVOLVED IN THE PROCESS, THE RECEPTOR BLOCKER DOESN'T HAVE ANY EFFECT. SO THEN WE WANT TO LOOK AT CALCIUM RELEASE MORE DIRECTLY, AND WE DERIVED THE HIPPOCAMPAL -- PRESENILINS ARE VERY IMPORTANT FOR NEURODEVELOPMENT IN MANY ASPECTS, SO WE CAN REALLY USE THE -- KNOCKOUT TO DERIVE NEURONAL CULTURE BECAUSE THE CULTURE IS ESSENTIALLY A MESS AND YOU CAN'T REALLY INTERPRET THE DATA, IN MY VIEW, SO WE BASICALLY USED THE FLOCKED MICE TO -- FROM THE POSTNATAL -- THEN WE INTRODUCED CRE CARRYING EITHER A FUNCTIONAL CRE -- OR A DEFECTIVE CRE RECOME BY NAIS, AND WE FOUND THAT AFTER THE NEURONAL CULTURE, WE WILL INTRODUCE THE VIRUS AND ANALYZE THESE MICE AND THIS CULTURE AT A DIV14, OF COURSE WE ALSO LOOK AT THE INACTIVATION PATTERN OF PRESENILINS, WE SAW A SLIGHT REDUCTION OF DIV2 AND MORE AND MORE WITH MORE DAYS IN CULTURE, BY DIV7 AND THE PRESENILIN ACTIVATION IS COMPLETE. SO AT 14, WE DID NOT SEE ANY MORPHOLOGICAL DIFFERENCES, MEANING THAT CULTURE IS VERY HEALTHY AND WE DON'T SEE ANY LOSS OF NEURONS OR SNAP CYST, AND WE CONFIRMED AGAIN INDEED THE INFECTION IS COMPLETE AND IT'S ALMOST 100%, AND WE DON'T SEE ANY RESIDUAL AMOUNT OF PRESENILINS, SO ALSO WE WENT FURTHER TO SEE THAT, AND THIS CULTURE, HIPPOCAMPAL CULTURE ACTUALLY CAN'T RECAPITULATE TO HAVE SLICE SO IS THIS IS REALLY A GOOD SYSTEM TO STUDY THE MECHANISM, AND INDEED THAT'S THE CASE, THESE CULTURE RECAPITULATE BASICALLY THE PAIRED PULSE FACILITATION DEFECT AND THE CULTURE ALLOWED US TO STUDY WHY AND WHETHER THE IMPAIRMENT IN NEUROTRANSMITTER RELEASE IS DUE TO A SMALLER REASONABLE -- SO WE MEASURED -- USING HIGH CON SEB TRAITION OCONCENTRATION OF SUCROSE AND WE FOUND THAT'S ACTUALLY NOT AFFECTED. WE ALSO DID THE MKO1 EXPERIMENT, FOUND THAT PROBABILITY OF RELEASE IS ALSO SIMILARLY IMPAIRED IN THOSE CULTURE COMPARED TO SLICES FROM THE DCKO MICE. SO FINALLY FROM ALL THE CONTROL WE CAN LOOK AT CALCIUM RELEASE MORE DIRECTLY, AND THE KIND OF APPROACH WE USE IS THAT WE USE HIGH CONCENTRATION OF POTASSIUM -- TO STIMULATE CALCIUM RELEASE, AND BOTH CALCIUM INFLUX AND THE CALCIUM EFLUX, AND WHAT WE'RE MEASURING IS THE RISE OF CALCIUM, THAT'S A COMPOSITE OF THE CALCIUM INFLUX AND ALSO CALCIUM EFLUX, AND WHAT WE FOUND IS THAT THE TREATMENT OF EITHER AT A HIGH CONCENTRATION MIMIC THE EFFECT OF THE KNOCKOUT BASICALLY WE SEE A REDUCTION OF CALCIUM RISE IN -- AND LOOKS LIKE THIS RISE IS REALLY DUE TO IMPAIRMENT OF RYANDINE CALCIUM RELEASE, AND IN THE KNOCKOUT CULTURE, IF WE TREAT WITH RYANIDINE AT HIGH CONCENTRATION AND DID NOT SEE FURTHER REDUCTION OF CALCIUM, INDICATING THAT THIS IS REALLY THE CALCIUM INFLUX AND THIS IS THE CALCIUM EFLUX, THIS PART IS SELECTIVELY AFFECTED, AND I SEE THREE RECEPTOR BLOCKADE DOES NOT AFFECT THE -- IN EITHER GEE GENOTYPES. SO TO SUMMARIZE ALL THIS, BASICALLY WHAT WE DISCOVERED IS THAT PRESENILIN CAN REGULATE LPT THROUGH TWO DIFFERENT MECHANISMS. ONE IS PURELY THROUGH A PRESYNAPTIC MECHANISM, AND SO FOR EXAMPLE, IF YOU MOVE PRESENILINS IN THE PRESYNAPSE NEURON, WHAT YOU YOU SEE IS CALCIUM REDUCED CALCIUM RELEASE FROM THE RYANADINE RECEPTOR IS IMPAIRED AND THIS LEADS TO IMPAIRMENT OF PRESYNAPTIC FUNCTIONS MEASURED EITHER BY NEUROTRANSMITTER RELEASE PROBABILITY, PAIR POST FACILITATION, OR FREQUENCY FACILITATION. THAT ALONE IS SUFFICIENT TO CAUSE LTP IMPAIRMENT IN THE ABSENCE OF ANY CHANGES OF NMD RECEPTOR FUNCTION. HOWEVER, IF WE REMOVE PRESENILIN IN BOTH PRESYNAPTIC AND POST SYNAPTIC NEURONS, AND WE ALSO SAW IMPAIRMENT, A LARGE IMPAIRMENT OF NMD RECEPTOR FUNCTION, AND THIS ITSELF CAN CAUSE LPT DEFICIT AS WELL. AND OF COURSE IT'S ALL INTERESTING TO STUDY PRESENILIN FUNCTION IN THE SYNAPSE, AND SORT OF BECAUSE OF MY INTEREST IN THE SYNAPTIC FUNCTION, BUT ULTIMATELY OUR INTEREST IN PRESENILIN IS ITS ROLE IN THE PATHOGEN CYST OF ALZHEIMER'S DISEASE AND OUR BEHAVIORAL ANALYSIS ALREADY HINTED THERE WAS A VERY BIG AGE DEPENDENT EFFECT IN THESE MICE. SO WE LOOK AT THESE MICE AS THEY GET OLDER, AND THESE ARE THE MICE AT NINE MONTHS OF AGE, WHAT WE SAW IS THAT THE AN DRI TICK SPINES ARE MOSTLY GONE AND ALSO THE DA DRI TICK COMPLASSITY OF THE SURVIVING NEURONS IS VERY MUCH COMPROMISED. YOU SEE THIS HUGE ENLARGEMENT OF LATERAL VENTRICLES AND THIS IS DUE TO LOSS OF CORTICAL VOLUME AND ALSO CORTICAL NEURONS. SO BASICALLY THESE MICE ACTUALLY RECAPITULATE THE NEUROPATHOLOGY OF AD VERY WELL, EXCEPT THE AMYLOID PLAQUE, BECAUSE PRESENILIN IS PART OF THE GAMMA SESECRETASE, WITHOUT THE KEY COMPONENT, PRESENILIN, DEGENERATION IS REDUCED. ALSO THESE MICE DEVELOP AGE-DEPENDENT INFLAMMATORY RESPONSES LIKE -- UPPER REGULATION OF MANY INFLAMMATORY MARKERS, AND THE TAU IS ALSO HYPERPHOSPHOR ATED IN THESE MICE. SO THAT PROMPTED US TO THINK THAT MAY LOSS OF ESSENTIAL NEURONAL FUNCTION OF PRESENILINS IN THE ADULT BRAIN MAY UNDERLIE DEGENERATION AND DEMENTIA IN AD. SO WE ANALYZED FURTHER OF THESE NEURODEGENERATION FEATURES IN THESE MICE, AND FOUND A FEW VERY INTERESTING FEATURES. WE ANALYZED MICE FROM TWO MONTHS OF AGE AND AT TWO MONTHS, AND THESE MICE DO NOT HAVE ANY SIGNIFICANT LOSS OF NEURONS OR SYNAPSES. AS THEY GET OLDER, THEY PROGRESSIVELY LOSE MORE CORTICAL NEURONS, SO OUR AGE-DEPENDENT ANALYSIS REVEALED THAT AT TWO MONTHS OF AGE, ALTHOUGH THE TOTAL NUMBER OF SYNAPSES AND NEURONS IS NOT SIGNIFICANT LAY LI REDUCED BUT NEURODEGENERATION IS ALREADY BEGINNING TO OCCUR IN A VERY SMALL PERCENTAGE OF NEURONS, SO WE CALCULATE THAT AT THAT TIME -- PERCENT OF NEURONS UNDERGO TI PO PI PTOSIS. SO YOU SEE MORE AT TWO MONTHS OF AGE IN THE DCKO MICE, SO YOU SEE A FORWARD INCREASE IN APOPTOSIS. THIS IS BECAUSE IN THE ADULT BRAIN, THERE IS VERY LITTLE APOPTOSIS GOING ON IN THESE MICE. SO YOU SEE A HUGE INCREASE, BUT THIS INCREASE ONLY REPRESENTS A TINY PERCENTAGE OF THE TOTAL NUMBER OF CORTICAL NEURONS. SO THAT'S KIND OF -- PRESENILIN IS INACTIVATED IN MOST IF NOT ALL OF THESE CORTICAL NEURONS, BUT CELL DEATH ONLY OCCURS IN VERY SMALL PERCENTAGE OF CELLS. OF COURSE THAT MAKES IT EXTREMELY DIFFICULT TO USE BIOCHEMICAL APPROACHES TO STUDY THE DOWNSTREAM TARGET OF PRESENILIN IN PROMOTION OF ACTIVATION OF APOPTOSIS. THE OTHER INTERESTING FEATURE IS THAT THERE IS A SIGNIFICANT DELAY IN ACT VASE OF APOPTOSIS, AT SIX WEEKS OF AGE, WE STILL DID NOT SEE A SIGNIFICANT INCREASE IN APOPTOSIS. ONLY FOUR WEEKS LATER, WE SAW THIS HUGE INCREASE. HOWEVER, THIS TINY OCCURRENCE OF APOPPTOSIS OVER TIME WOULD CUMULATIVELY HAVE MORE AN MORE CORTICAL NEURONAL LOSS AND THE BEHAVIORAL DATA I SHOW YOU IS AT SIX MONTHS OF AGE, BY THE TIME THEY LOSE 18% OF CORTICAL NEURONS, THESE MICE BASICALLY COMPLETELY FAIL TO LEARN ANY BEHAVIORAL PARADIGMS. WE ACTUALLY DID ADDITIONAL OTHER BEHAVIOR TASKS ALSO, BASICALLY THEY CAN'T DO ANYTHING. SO THAT'S REALLY MIMICKING THE DEVEER DEMENTIA WELL. JUST AS I MENTIONED, PRESENILIN IS PART OF THE GAMMA SE SECRETASE COMPLEX, WE WANTED TO ASK WHETHER DEGENERATION CAUSED BY LOSS OF PRESENILIN -- GAMMA SEE SECRETASE -- WE DECIDED TO USE A GENETIC APPROACH BECAUSE MORE THAN 50 GHANA SECRETASE SUBSTRATES HAVE BEEN REPORTED ALTHOUGH IT'S NOT CLEAR HOW MANY OF THEM ARE PHYSIOLOGICAL. BECAUSE MOST OF THEM WERE IDENTIFIED IN EXOGENOUS CELL LINES WITH OVEREXPRESSION SYSTEMS. SO WE SIMILARLY, USING THE SAME CRE LINE TO GENERATE -- CKO MICE, SO THEN WE COMPARED THE DEVELOPMENT OF THE PHENOTYPES IN THE -- WITH THE PRESENILIN CKO MICE, THEY ARE VERY SIMILAR, ALTHOUGH THE MORE RECENT ELECTROPHYSIOLOGY ANALYSIS INDICATED THERE WAS SOME DIFFERENCES BECAUSE OF DIFFERENCES IN PRESENILIN ALLYL VERSES -- ALLYL AND THERE WAS SOME DIFFERENCES IN THEIR CONCOURSE OF GENE ACTIVATION. BUT IF WE LOOK BY THE MONTH, THEY ARE VERY, VERY SIMILAR. YOU SEE AGE-DEPENDENT NEURODEGENERATION, ALSO AGE-DEPENDENT PROGRESSION OF MEMORY IMPAIRMENT, AND WHEN -- IS INACTIVATED, SO THESE DATA SUGGEST THAT PART OF THE STORY IS THAT LOOKS LIKE MOUSE BRAIN IS NOT NECESSARILY MORE RESISTANT TO NEURODEGENERATION BECAUSE APPARENTLY GAMMA SECRETASE IS EXTREMELY IMPORTANT FOR CORTICAL NEURON SURVIVAL IN AN AGE-DEPENDENT MANNER BECAUSE IF YOU ELIMINATE EITHER OF THE GAMMA SECRETASE COMPLEX IN A HIGHLY SELECTIVE MANNER, VERY LATE IN LIFE, AND EITHER OF THESE CASES, YOU CAN BASICALLY CAUSE AGE-DEPENDENT NEURODEGENERATION, ACTUALLY QUITE PROFOUND NEURODEGENERATION IN THE MOUSE BRAIN, AND AS I MENTIONED THERE ARE SO MANY GAMMA SECRETASE SUBSTRATES, BUT NOT AN APP ARE CLEARLY AGREED UPON PHYSIOLOGICAL SUBSTRATES SO WE WANT TO KNOW IF GAMMA SECRETASE ACTIVITY IS SO IMPORTANT FOR NEURONAL SURVIVAL, WHETHER IT'S DUE TO EITHER OF THE PHYSIOLOGICAL SUBSTRATES, SO WE MADE NOTCH 1, NOTCH 2 SINGLE AND A DOUBLE CONDITIONAL KO MICE, MORE RECENTLY WE DEVELOPED APP, APP/APLP1/APLP2 SINGLE KO MICE TO ADDRESS THIS QUESTION, OF COURSE THIS PARTICULAR EXPERIMENT HAS EVEN MORE PROFOUND IMPLICATION BECAUSE AFTER 20-PLUS YEARS OF IDENTIFICATION OF APP AS A VERY IMPORTANT GENE INVOLVING AP PATHOGENESIS, WE STILL DON'T KNOW APP NORMAL FUNCTION AND THE INTERPRETATION OF MANY OF THE INSIGHTS WE HAVE OBTAINED FOR AD PATHOGEN CYSESIS -- WE TYPICALLY ATTRIBUTE ALL THE PHENOTYPES IDENTIFYING THESE MICE TO INCREASE THE PRODUCTION OF A BETA. HOWEVER, THEORETICALLY, IT'S POSSIBLE THAT OVEREXPRESSION OF APP ITSELF COULD CAUSE THESE PHENOTYPES BECAUSE TYPICALLY THESE STUDIES WERE NOT DONE USING APP WILD TYPE TRANSJE NICK MICE AS CONTROLS, SO WE FELT THAT FOR US IN THE FIELD, IT'S REALLY IMPORTANT TO UNDERSTAND THE NORMAL FUNCTION OF APP, AS I SHOW YOU FROM OUR STUDY OF PRESENILIN, OBVIOUSLY IT'S EXTREMELY IMPORTANT TO KNOCK OUT ALL GENE FAMILY MEMBERS, IN OTHER WORDS, WE HAVE TO MAKE A KNOWN MUTATION IN ORDER TO REVEAL -- FUNCTION. SO I WILL ONLY SHOW YOU THE PUBLISHED DATA ON THE NOTCH 1, NOTCH 2, AND I GUESS THAT'S THE FUN PART OF SCIENCE, ALWAYS SURPRISES. WE DID NOT FIND BASICALLY ANYTHING IN THESE MICE. DURING DEVOLVEMENT, IF YOU KNOCK OUT NOTCH CONDITIONALLY OR IN -- OR IF YOU OVERPRODUCE THE INTRACELLULAR DOMAIN, YOU HAVE STRIKING PHENOTYPES. BUT IN THE ADULT, FOR EXAMPLE, WHEN WE OVEREXPRESS NICD AND UNDER THE PROMOTER, WE COULD OVERPREX! PRESS A HUGE ACT OF, WE COULD NOT FIND ANY EFFECT. SIMILARLY, IF WE KNOCK OUT NOTCH 1/NOTCH 2, THESE ARE THE TWO NOTCHES EXPRESSED IN NEURONS OF THE ADULT BRAIN, SUPPOSEDLY, AND WE BASICALLY FAILED TO DETECT ANY NEURODEGENERATION, EVEN WHILE WE KNOCK OUT BOTH OF THEM. SO GOING BACK TO WHAT DOES IT MEAN, THE FACT THAT INACTIVATION OF PRESENILIN MIMIC THE MAJORITY OF HE CENTRAL FEATURES OF AD, AND SO WE FOUND THAT LOSS OF PRESENILIN FUNCTION IS VERY IMPORTANT FOR NEURODEGENERATION IN AD, BECAUSE WE THINK THE SEVERE DEMENTIA IS CAUSED BY LOSS OF NEURONS, THIS IS WHY THE DISEASE IS PART OF -- IS THE MOST COMMON NEURODEGENERATIVE DISEASE, AND WE BASICALLY LOOK AT ALL THE EVIDENCE, AND FOR EXAMPLE, LIKE I SAID, FROM THE VERY BEGINNING, THE LARGE NUMBER AND ALSO THE DIFFUSE DISTRIBUTION OF THE MUTATIONS IN PS1/2 ARE CONSISTENT WITH A PARTIAL LOSS OF FUNCTION MECHANISM. AND ALSO THE FACT THAT INACTIVATION OF PRESENILIN IN THE ADULT MOUSE BRAIN PHENOCOPIES, THE KEY FEATURES OF AD ALSO IS SUGGESTIVE THAT LOSS OF PRESENILIN MUNG FUNCTION MIGHT BE IMPORTANT FOR AD PATHOGENESIS, AND THERE ARE A LOT OF PAPERS IN LITERATURE SHOWING THAT WHEN YOU USE INCREASING AMOUNTS OF GAMMA SECRETASE INHIBITORS, WHEN USING MIMICKING INHIBITORS, THAT'S OBVIOUSLY MIMICKING THE ENZYME FUNCTION, AND THEY ACTUALLY VERY MUCH MIMICKED THE FAD MUTATIONS. SO THIS IS THE WORK PUBLISHED BY ANOTHER GROUP, AND NOT US, AND SO IT'S ACTUALLY NOT ONE PAPER. THERE IS LIKE 10-PLUS PAPERS IN LITERATURE SHOWING THIS VERY CURIOUS FEE SURE I FEATURE IS THAT WHEN YO U USE INCREASING AMOUNTS OF GAMMA SECRETASE INHIBITORS AND THAT YOU HAVE THIS BELL CURVE IN TERMS OF THE EFFECT ON A BETA 42 GENERATION, WHEREAS A BETA 40 IS VERY SIMPLE, WHEN YOU USE MORE INHIBITOR, ENZYME ACTIVITY IS IMPAIRED AND YOU INHIBIT THE PRODUCT GENERATION. BUT 42 INITIALLY AT A LOW CONCENTRATION, YOU SEE INCREASED PRODUCTION OF A BETA 42, SO OF COURSE AT THIS RANGE, YOU WILL SEE INCREASE 42-40 RATIO, THAT'S VERY SIMILAR TO A LOT OF FAD MUTATIONS. AND SO THE OTHER IS THAT THE PRESENILIN MUTATION IMPAIRS THE GAMMA SECRETASE ACTIVITY, SO I WILL GO THROUGH SOME OF OUR DATA AND ALSO THE DATA IN LITERATURE. THIS IS ACTUALLY WHAT'S PUBLISHED AND VERY, VERY EARLY ON. A FEW MONTHS, COUPLE MONTHS AFTER THE INITIAL IDENTIFICATION OF PS1 AS A KEY GENE IN ALZHEIMER'S DISEASE, OUR GROUP LINKED PS1 TO THE NOTCH PATHWAY, BECAUSE WHAT SHE FOUND IS THAT A SUPPRESSOR OF LIN 12, A SUPPRESSOR OF LIN 12 IS BASICALLY -- THEY LABEL CELL 12, WHICH IS BASICALLY A SUPPRESSOR OF LIN 12, AND THAT SHEER SEQUENCE HOMOLOGY TO PS1. ALSO THIS SLIDE SHOW -- THEY ALSO SHOW FUNCTIONAL HOMOLOGY BECAUSE IT CAN RESCUE THE AGGRAVATING DEFECTS OF CELL 12 VERY WELL, BUT IF YOU USE FAD MUTATIONS AND IT SHOWS REDUCED ACTIVITY TO RESCUE THE CELL 12 LOSS OF FUNCTION MUTANT PHENOTYPE INDICATING THAT THE FAD MUTATION CAUSED REDUCED BIOLOGICAL ACTIVITY. BUT IN THE -- SYSTEM, BECAUSE FAD MUTATION INCREASES 42 GENERATION IN MOST INSTANCES, THAT WAS ALWAYS INTERPRETED AS SORT OF A TOXIC GAIN OF FUNCTION MECHANISM, BUT WE FIGURED THAT BECAUSE IF IT'S A LOSS OF FUNCTION, IF YOU DON'T DO EXPERIMENTS VERY CAREFULLY, IF YOU OVEREXPRESS WITHOUT CARING HOW MUCH YOU'RE OVEREXPRESSING, THAT COULD HAVE VERY WELL COMPENSATE FOR THE LOSS OF FUNCTION. AND ALSO IF YOU DO THE EXPERIMENT IN THE PRESENCE OF INDODGENDOGENOUS PRESENILIN, THAT'S EVEN MESSIER. SO WE DECIDED TO USE PS -- CELLS IN ININT DUESIN INTRODUCING TINY AMOUNT, W E DID A DOSE CURVE TO MAKE SURE THAT THE AMOUNT OF PRESENILIN WE INTRODUCE IS IN THE LINEAR RANGE FOR THE EXAMINATION OF GAMMA SECRETASE ACTIVITY, WHAT WE FOUND REALLY IS THAT INDEED THE MUTATIONS FOUND LOSS OF GAMMA IS A KREE TASTSECRETASE GENERATION, WE BASICALLY LOOK AT THESE TWO GAMMA SECRETASE CLEAVAGE AND ALSO AS YOU WOULD IMAGINE, THE EFFECT ON THE INHI BAITION OF GAMMA SECRETASE ACTIVITY WOULD HAVE BEEN DEPENDENT ON THE SPECIFIC LOCATION OF THE ENZYME ENZYME -- OF THE MUTATION IN THE PROTEIN, AND INDEED THAT'S THE CASE, SOME OF THEM LIKE LO LIKE THE DELTA X ON 9 -- HAS THE MOST SUBTLE EFFECT ON GAMMA SECRETASE ACTIVITY, AND SOME OF THESE MUTATIONS, FOR EXAMPLE, THE L435F HAS THE MOST SEVERE MUTATION, AND SO WE BASICALLY DECIDED TO TEST THE EFFECT OF THESE MUTATIONS IN VIVO, WE GENERATED THESE TWO -- MOUSE TO LOOK AT EFFECT OF EACH OF THESE MUTATIONS IN VIVO. UNFORTUNATELY THAT'S NOT PUBLISHED. I WAS VERY EAGER TO SHARE WITH YOU THESE RESULTS BUT I CAN'T. SO INSTEAD I WILL SHOW YOU THIS SLIDE, AND SHOWING YOU THAT THE LOCATION OF THIS L435F MUTATION IS VERY CLOSE TO THE SITE OF GAMMA SECRETASE, ACTIVE SITE, SO THAT MIGHT EXPLAIN THE COMPLETE LOSS OF GAMMA SECRETASE ACTIVITY IN CULTURE. AND ALSO INTERESTINGLY, THIS MUTATION BASICALLY IN HUMAN PATIENTS HAS A LOT OF A BETA 42, WHICH -- MUCH LESS OF A BETA 40, UNLIKE THE TYPICAL AD PLAQUE. SO THE LAST PART, AND FORTUNATELY THIS PART IS PUBLISHED, SO I CAN SHOW YOU ALL OF THE DATA, SO WE ALSO FOUND THAT -- WELL, NOT US. THAT WAS REPORTED BY OTHER GROUPS, IS THAT PS4 MUTATIONS HAVE BEEN IDENTIFIED IN FRONTOTEMPORAL DEMENTIA AND FRONTOTEMPORAL DEMENTIA DOES NOT HAVE -- PATHOLOGY BUT SHARES THE NO DEGENERATION AND DEMENTIA FEATURE WITH AD. AND SO THIS HUMAN DATA ALSO SUGGESTS A LOST OF PRESENILIN FUNCTION TO ADDRESS THIS, AND WE BASICALLY AGAIN USING MOUSE MODELING APPROACH BY GENERATING -- MOUSE, SO TWO MUTATIONS, FOR EXAMPLE, THIS AND THAT, THEY ARE IN THE BOUNDARY AND WE MADE -- MOUSE WITH THIS G183V BECAUSE THIS FAMILY HAS THE NEUROPATHOLOGY AVAILABLE, AND ACTUALLY IF YOU'RE LOOKING TO THE LITERATURE, THERE ARE QUITE A FEW MUTATIONS IN PS1 THAT'S FOUND IN BOTH AD AND FTD, AND I LIST SOME OF THESE MUTATIONS HERE SO THERE IS DEFINITELY SOME KIND OF SHARED MECHANISM BETWEEN AD AND F AFTER D AT LEAST IN THESE -- CARRYING MUTATIONS. UNLIKE THE FAD MUTATION CARRIED THE PS1 L435F MUTATION, THIS SPECIFICALLY THIS NUCLEOTIDE CHAINS DOES NOT HAVE ANY AMYLOID PLAQUE. BUT THEY DO HAVE NO DEGENERATION FEATURES AND ALSO CLINICAL DEMENTIA FEATURE. SO WE MADE A -- MOUSE AND INTERESTINGLY, THIS ONE BASICALLY CAUSED REDUCTION OF PS1NRMA, ABANDONS THIS REDUCTION OF NMRA IS DUE TO SKIPPING OF XON6 AND 7 AS -- THE MUTATION IS IN THE INTRON EX-TRON POWN DRI, SO IT'S NOT SURPRISING AND YOU WOULD CAUSE ABERRANT SPLICE FORMS, NOT PARTICULARLY SURPRISING BECAUSE OUT OF FRAME STOP CODES ARE USED AND TYPICALLY THESE MUTATIONS LEAD TO DEGRADATION OF THESE MRNA, AND WE CONFIRMED THAT BY USING SACCROHEXAMIDE -- WE WOULD RECOVER THIS NORMALLY DEGRADED ABERRANT SPLICE FORMS. ANOTHER INTERESTING FEATURE I WANT TO POINT OUT THAT THE ABERRANT SPLICE FORM PRODUCTION AND ALSO THE DEGRADATION OF THIS ABERRANT SPLICE PRODUCT IS SPECIFIC TO THE BRAIN, AND THIS IS NOT PRESENT IN OTHER TISSUES IN THESE -- MICE, SO INDICATING THAT THIS MUTATION BASICALLY LEADS TO REDUCTION OF MRNA, SPECIFICALLY WHAT'S IN THE BRAIN, NOT THE TISSUES. THE CONSEQUENT AMINO ACID CHANGE DUE TO -- DOES NOT AFFECT GHANA SECRETASE ACTIVITY BECAUSE IF WE INTRODUCE THIS NUCLEOTIDE CHANGE IN CDNA AND THIS MUTANT PROTEIN ACTUALLY EXHIBITS NORMAL GHANA SECRETASE ACTIVITY. WE'RE ALMOST THERE, AND I THOUGHT I COULD EXPLAIN MORE SINCE I CUT OUTLOTS OF SLIDES BUT I GUESS WE'RE RUNNING OUT OF TIME AGAIN. SO THIS MIK -- WHEN WE USE THIS VERY SENSITIVE ASSAY TO MEASURE DE NOVO A BETA DEGENERATION, WE FOUND DRAMATIC REDUCTION OF GENERATION. WHEN WE LOOK AT A STEADY STATE LEVEL OF A BETA, WE SAW MUCH SMALLER REDUCTION IN A BETA 40 AND WE DIDN'T SEE A SIGNIFICANT REDUCTION IN A BETA 42, AND SO ALTHOUGH IN TERMS OF BEHAVIORAL ANALYSIS, WE DID NOT SEE ANY DEFECT IN THE KNOCKING NICE ALONE, BUT BECAUSE IT ONLY CAUSED REDUCTION OF LIKE 40% OF THE MRNA EXPRESSION, BUT IF WE CROSS TO PS2 NO BACKGROUND, MEANING IF WE REDUCE PRESENILIN DOSAGE FURTHER, WE DIDN' DID SEE MILD BUT SIGNIFICANT REDUCTION OF QUADRANT OCCUPANCY IN THE POST TRAINING PATH -- PROBE TRIAL, SO THIS IS QUITE SIMILAR TO TS1 SINGLE CONDITIONALLAL PHENOTYPE, INDICATING INDEED THIS KNOCK IN MICE -- GAMMA SECRETASE ACTIVITY AND ALSO IMPAIRMENT OF MEMORY. SO TO SUMMARIZE THIS TOGETHER, WHAT HAVE WE FOUND, THAT WHEN WE LOOK AT FAD MUTATION OR FTD MUTATION, AND BASICALLY THE MECHANISM IS DIFFERENT AND THE ONE IS CAUSED REDUCE -- EXPRESSION FOR MORAL ACTIVITY OF MUTANT WHEREAS FAD MUTATION AND I DIDN'T SHOW YOU THE DATA, BASICALLY THE EXPRESSION IS NORMAL BUT WE SAW ELIMINATION OF PRESENILIN ACTIVITY, AND TOGETHER THEY BOTH COMPROMISED PRESENILIN DEPENDENT NEURONAL SURVIVAL DURING AGING THAT COULD LEAD TO DEMENTIA. SO HERE I'M PROPOSING A MODEL FOR FAD, AND IT'S THAT PRESENILIN MUTATIONS ARE LOSS OF FUNCTION MUTATION, WHETHER IT'S PARTIAL LOSS OF FUNCTION MUTATIONS OR COMPLETE LOSS OF FUNCTION MUTATION, CAN CAUSE NEURODEGENERATION AND THE MANIFESTATION OF NEURODEGENERATION INCLUDES DEMENTIA, AND TAU HYPERPHOSPHORATION, BUT IN THE HETEROZYGOUS SETTING, THE PATIENT WITH ONLY HAVE ONE ALLYL THAT IS AFFECTED, THEY ALSO PROMOTE AMYLOID DEPOSITION BECAUSE THEY INCREASE THE PRODUCTION OF A BETA 42, AND TOGETHER THEY PROMOTE RESULTING ALZHEIMER'S DISEASE, AND ALSO HOW DO WE EXPLAIN APP MUTATIONS IN THE CONTEXT OF THE PRESENILIN HIGHYPOTHESIS, AND WE THINK -- WHICH LEAD TO INCREASED A BETA MUTATION MUCH, MUCH MORE POTENTLY THAN PRESENILIN MUTATION, HOWEVER ONE THING WE ALWAYS FELT LIKE WE COULDN'T QUITE EXPLAIN YOU'D EUSING THE AMYLOID HYPOTHESIS IS WHY PRESENILIN MUTATIONS THAT ONLY INCREASE A BETA GENERATION MUCH MORE SUTLY COMPARED TO APP MUTATION BUT IT HAS A MORE SEVERE DISEASE PROGRESSION. SO THIS PART IS COMPLETE SPECULATION, WE DON'T HAVE ANY DATA YET. WE THINK THAT THE LONGER A BETA, AS CAN YOU IMAGINE, THE LONGER A BETA ITSELF ARE SUBSTRATES OF GAMMA SECRETASE, THE LONGER A BETA MAY BE A POOR SUBSTRATE OF GAMMA SECRETASE, THEY OCCUPIED ACTIVE SITE TOO LONG BECAUSE THEY CANNOT BE EFFICIENTLY CLEAVED, SO THEY BLOCKED THE EXCESS OF MORE IMPORTANT SUBSTRATE THAT IS INVOLVED IN MEDIATION NEURONAL SURVIVAL. SO ANYWAY, IN TERMS OF OUR PRESYNAPTIC MODEL AND THE HAD AND SURPRISINGLY THAT'S ACTUALLY SHARED WITH PD AS WELL, BECAUSE WHEN WE KNOCK OUT PD QUEE GENES, FOR EXAMPLE, PINK 1, ALL OF THEM LEAD TO -- DEFECT AND -- AS WELL AS THE LOCK 2, KNOCKING ALSO SHOWS IMPAIRMENT OF DOBINERGIC RELEASE AND THE WORK WAS DONE BY OTHER GROUPS, SO I ONLY WANT TO POINT OUT THAT SORT OF LIKE THERE IS INDEED A SYNERGY BETWEEN AD AND THE PD STUDIES IN THE LAB AND THOSE WERE DONE BILL BY COMPLETELY DIFFERENT PEOPLE AND DIFFERENT COLLABORATORS, HOWEVER, WE WERE ABLE TO FIND THIS SORT OF INTERESTING CORRELATION BETWEEN THE TWO DISEASES. SO LASTLY, I WANT TO THANK THE PEOPLE AND THE FORMER POST DOCS IN THE LAB WHO DID THE WORK AND ALSO THE CURRENT MEMBERS OF THE LAB, THAT'S THE ALZHEIMER'S GROUP, THAT'S THE PARKINSON'S GROUP, AS WELL AS MY COLLABORATORS, AND ESPECIALLY RAY KELLEHER, AND ALL THE PEOPLE WHO CONTRIBUTED GAVE US THE AVAILABLE MOUSE LINES AND MAKE OUR STUDY A LOT EASIER. THANK YOU, EVERYBODY. QUESTIONS? >> [INAUDIBLE] >> YEAH, SO WE BASICALLY -- SO HERE, THE HYPOPHOSPHORRATION TAU IS ONLY DETECTED AFTER DEGENERATION HAS OCCURRED. SO FOR EXAMPLE WE SAW IT VERY CLEARLY AT NINE MONTHS OF AGE, AND THE KINASE INVOLVED, FOR EXAMPLE, WE LOOKED AT CDK5, SO WE DIDN'T SEE INCREASED AP25, WHICH IS NORMALLY NOT DETECTABLE, BUT AT FIVE MONTHS OF AGE, WE ALREADY SAW P25 PRESENCE. >> [INAUDIBLE] >> WE DID NOT LOOK AT -- MEASURE THE KINASE ACTIVITY, PER SE, WE KIND OF JUST LOOKED AT THE ACTIVE FORM OF CDK COFACTOR. >> -- IS IT CLEAR THAT THE MUTATION IS ACTING AT THE IS NAPSYNAPSE OR IN THE -- >> WE CANNOT DIFFERENTIATE THAT, AND WE DIDN'T LOOK AT IF PRESENILIN IS PRESENT IN THE PRESYNAPTIC TERMINALS. GIVEN THE FACT THAT ER IS PRESENT IN THE PRESYNAPTIC TERMINALS AND ALSO SORT OF LIKE THE DEFECT WE ARE SEEING, SO I CANNOT DIFFERENTIATE WHETHER THE PRESYNAPTIC DEFECTS IS DUE TO THE ER CALCIUM DEFECTS IN THE -- OR IN THE PRESYNAPSE TERMINAL, BUT I WOULD IMAGINE BASED ON THE PHENOTYPES DETECT AND ALSO THE PRESENCE OF ER AND IN THE PRESYNAPSE TERMINAL, I WOULD IMAGINE AT LEAST THE PRESYNAPTIC ER WOULD BE MORE LIKELY TO BE CONTRIBUTING TO THESE PHENOTYPES. >> I HAVE A COUPLE OF QUESTIONS. LIKE THE FIRST THING, YOU WERE SAYING THAT THE PS1 IS ONE OF THE MAJOR -- I MEAN, LIKE CAUSING FOR THE SYNAPTIC DYSFUNCTION. AND PRESYNAPTIC PROTEINS. MY QUESTION IS -- TWO QUESTIONS. 1S IT THAT THE SYNAPTIC DYSFUNCTION LEADS TO THE FORMATION OF THE FLOCK FOLLOWED BY -- IN THE HUMAN AD, AND MY SECOND QUESTION IS HAVE YOU LOOKED AT ANY OF THE PRESYNAPTIC MODELS, SO THAT CAN THESE BE USED FOR THE EARLY DIAGNOSIS OF THE HUMAN AD, BECAUSE SO FAR, ONE OF THE MAJOR BIOMARKERS WHICH HAS BEEN USED IS MAINLY TAU, BUT IT APPEARS THAT THE PRESYNAPTIC DYSFUNCTION STARTS MUCH, MUCH EARLIER, THEN LATER IT STARTS FORMATION OF THE PLAQUE, THEN FOLLOWED BY THE -- SO CAN ANY OF THIS -- HAVE YOU LOOKED AT ANY OF THE SYNAPTIC PROTEINS WHICH ARE DEREGULATED, CAN THOSE BE USED AS THE BIOMARKERS FOR THE HUMAN AD? >> VERY GOOD QUESTION. SO WE DIDN'T LOOK AT A LOT OF SYNAPTIC MARKERS, AND -- BECAUSE U KNOW, SORT OF WE DID THIS WORK IN COLLABORATION WITH TOM'S LAB SO WE HAVE ALL OF THE ANTIBODIES. WE COULD NOT CONVINCE OURSELVES WE SAW ANY CHANGES IN THE PRESYNAPTIC OR POST SYNAPTIC PROTEIN PER SE, ALTHOUGH WE PREVIOUSLY DID A PUBLISH THAT NMD RECEPTOR LEVEL IS PRODUCED, EVEN AT TWO MONTHS OF AGE, AND -- BUT I WOULD -- WE CONSISTENTLY SEE THAT BUT IT'S LIKE A 10% REDUCTION. I WOULD PROBABLY LIKE NOT TRUST THAT AND GIVE AT MUCH WEIGHT AS WE LATER SAW, NOT LATER SAW, AT THE SAME TIME, WE SAW 50% OF FUNCTIONAL REDUCTION. SO TO ANSWER YOUR QUESTION DIRECTLY, CAN WE USE, YOU KNOW, NMD RECEPTOR FUNCTION AS A BIOMARKER, MAYBE, BECAUSE YOU KIND OF ASKED LIKE THREE, FOUR QUESTIONS TOGETHER. AND OBVIOUSLY THIS IS WHAT I HAVE BEEN THINKING A LOT ALSO, CAN WE USE NMD RECEPTOR AS A MARKER? MAYBE, BECAUSE WE DID SEE A VERY EARLY CHANGE. THE OTHER THING IS THAT WHAT ABOUT -- THE OTHER THING WE SAW MOLECULARLY IS THAT WE SAW -- DEPENDENT GENE EXPRESSION IS AFFECTED VERY EARLY. SO THE FIRST PHENOTYPE WE SAW ARE THE PRESYNAPSE DEFECTS VERY EARLY. WE ALSO SAW -- EXPRESSION IS AFFECTED AT FIVE WEEKS OF AGE ALSO. SO FOR EXAMPLE, CAN WE USE THAT, LIKE -- AND SORT OF LIKE BIOMARKER? MAYBE. AND -- BECAUSE WE DIDN'T SEE THAT EITHER AT FIVE WEEKS OF AGE OR TWO MONTHS OF AGE OR SIX MONTHS OF AGE. SO THAT HAS A FURTHER TWIST BECAUSE PS1 ITSELF IS A -- TARGET GENE ITSELF. SO IN TERMS PEOPLE WOULD ASK, WHAT'S THE RELATIONSHIP TO WHATEVER YOU FIND TO SPORADIC AD? AND -- YEAH. SO THAT COULD ACTUALLY, LIKE, TIE TOGETHER BECAUSE TO KEEP MY BRAIN STRAIGHT, THERE ARE SO MANY ISSUES WE'RE TALKING ALL AT ONCE, IS THAT BASICALLY -- SO FOR EXAMPLE, LIKE THERE IS AGE-DEPENDENT FACT OF BASICALLY NEURONAL ACTIVITY, SO I'M JUMPING, IN SPORADIC AD CASES, THERE IS AGE-DEPENDENT EFFECT OF NEURONAL ACTIVITY AND REDUCED NEURONAL ACTIVITY PROBABLY WILL AFFECT -- EXPRESSION AND THE REDUCED CRA DEPENDENT -- EXPRESSION MAY REDUCE -- SO THERE IS EVIDENCE IN THE AT LEAST PAPERS IN LITERATURE AND THE SHOWING THAT PS1 EXPRESSION IS REDUCED IN THE PART OF THE BRAIN THAT'S MORE AFFECTED IN AD, AND -- BUT I THINK THE BETTER EVIDENCE PERHAPS IS THAT THERE IS A POLYMORPHISM IN PS1 PROMOTER WHICH REDUCED PS1 EXPRESSION THAT PRO MORPHISM WHICH DOES REPRESENT A RISK FACTOR FOR AD. SO BASICALLY THAT'S WHY I TRY TO EXPLAIN AGING NEURONAL ACTIVITY AND ALSO PRESENILIN EXPRESSION AND ALSO BIOMARKERS. >> EXAC EXACTLY. JUST ONE MORE QUESTION. REGARDING THE NEURONAL LOSS WHICH YOU HAVE SHOWN WITH RESPECT TO PS1, IS IT THAT THE MAJOR NEURONAL LOSS IN THE AD CONTRIBUTED WITH THE PS1, FOR EXAMPLE, IN THE DOUBLE -- MODELS LIKE APP/PS1, THE ONES WHICH YOU HAVE SHOWN WITH THE PS1? >> SO IN A SENSE KIND OF VERY SURPRISING IS THAT PRESENILINS THAT POTENT IN TERMS OF PROMOTING NEURONAL SURVIVAL, AND IN TERMS OF CELL TYPE, IT IS THE RIGHT CELL TYPE, BECAUSE THE -- PARAMETER NEURONS ARE MOST VULNERABLE IN AD AND ALSO NORMALLY EXPRESS THE HIGHEST LEVEL OF PRESENILINS, AND HERE WE SPECIFICALLY TARGET THESE CELLS SO THEY HAVE A CELL -- EFFECT IN TERMS OF SUPPORTING NEURONAL SURVIVAL. SO WITHOUT THAT, THEY WILL HAVE THIS, LIKE -- THAT'S WHY I ELABORATE HOW BASICALLY THE NEURODEGENERATION CAUSED BY LOSS OF PRESENILIN IS KIND OF VERY INTERESTING BECAUSE IT'S NOT IMMEDIATE. IT HAS A DELAY. AND ALSO THIS DELAY, AND -- WOULD BE SUGGESTIVE THAT BASICALLY LOSS OF PRESENILIN PROBABLY LIKE DISRUPTS SOME KIND OF HOMEOSTASIS IN THESE CELLS, ONLY A SMALL PERCENTAGE OF CELLS, AND THE DISRUPTION OF THE HOMEOSTASIS WOULD REACH TO CERTAIN LEVEL THAT CAUSE THE CELLS TO DIE. >> THANK YOU. >> LAST QUESTION. >> OKAY. VERY NICE TALK. I THINK MOST -- YOU FOCUS YOUR WORK ON -- [INAUDIBLE] >> WE DID NOT ADDRESS THAT, AND IT'S POSSIBLE. THE REASON IS BECAUSE, LIKE, I DID NOT FIND A GOOD GLIO SPECIFIC CLE LINE BECAUSE WHEN WE GET TO THE GSAP CRE, THE GFAP CRE EXPRESSED CRE IN -- SO IT'S NOT CRE. SO I DON'T LIKE TO STUDY THAT BECAUSE ANY OF THE GLOI PHENOTYPE -- THAT'S THE REASON WE ARE NOT. BUT AS YOU CAN SEE FROM -- IN THE ADULT BRAIN, AT LEAST PS1 IS EXPRESSED THE HIGHEST IN THE PARAMETER NEURONS, WHERE THE PS2 IS MORE GLOBAL. SO THAT KIND OF MAKES US FEEL LIKE, YOU KNOW, WE WANT TO FOCUS THOSE CELL TYPES. OF COURSE PRESENILIN ALSO -- I DIDN'T GET INTO IT. PRESENILIN ALSO PLAYS A ROLE IN INHI TRINHIBITORY NEWER ORANGES WHICH WNEURONS, WHICH WEONLY DID IN CULTURE. OUR PRESENILIN -- WE ACT THE SERRATED IT IN ALL CELL TYPES, IN VIVO, IT'S ONLY -- NEURONS, AND IN VIVO, WE DO SEE STILL 50% OF THE PRESENILIN PROTEIN REMAINING IN THE ADULT VEEB VEEB RAL CORTEX, THOSE EXPRESSIONS ARE DUE TO EXPRESSIONS INTERNEURONS, SO I THINK, YOU KNOW, OF COURSE IF I HAVE FUNDING, I WILL DO INTERNEURON AND ALL OF IT, AND ALSO I WILL DO GENETICS BASICALLY AS I'M ALMOST EMBARRASSED LIKE IF YOU ASK ME, WHICH MOLECULE IS THE TARGET OF PRESENILIN IN NEURONAL SURVIVAL, BECAUSE I THINK THAT WOULD BE A REALLY GOOD DRUG TARGET BECAUSE THE PHARMACEUTICAL COMPANIES ARE ALSO ASKING, YOUR THEORY IS INTERESTING, GIVE ME A DRUG TARGET. I DON'T HAVE IT BECAUSE I CAN'T APPLY FOR A GRANT TO DO A FISHING EXPEDITION. SO I'M KIND OF EMBARRASSED, I DON'T KNOW, BUT ALTHOUGH I THINK IT WOULD BE REALLY -- LIKE I EMPHASIZE THE FACT THAT IT'S ONLY 20% OF NEURONS THAT ARE DYING, HOW CAN I DO BIOCHEMISTRY? I CAN ONLY DO GENETICS. I CAN'T AFFORD TO DO GENETICS IN MICE, SO I NEED TO DO IT IN FLY, BUT NO ONE HAS LOOKED AT PRESENILIN FLY FUNCTION IN THE ADULT. YOU CAN DO FLIES, FISH, WORMS, BUT YOU HAVE TO FIRST RECAPITULATE WHAT WE FOUND IN MICE, THEN DO GENETIC SCREENING. LOTS OF MONEY. >> PLEASE THANK ME IN -- [APPLAUSE]