>> I WANT TO WELCOME EVERYONE TO THE SECOND DAY OF THE NEUROSCIENCE MEETING AND WE APPRECIATE THE SPEAKERS MAKING TIME TO SPEAK AT THIS IMPORTANT SYMPOSIUM. I WANT TO THANK ALL THE COLLEAGUES HELPING US TO PUT THE MEETING TOGETHER. YESTERDAY WE HAD MANY EXCELLENT LECTURES ON THE TOOLS PRODUCED BY THIS COMMON FUND INVESTIGATORS AND SHOWED HOW IT CAN HELP US TO UNDERSTAND IMPORTANT IMMUNOSCIENCE AREAS AND HELP IN THE OUTCOME OF ALZHEIMER'S DISEASE AND ALL OUR LECTURES FROM THE MEETING WILL BE ARCHIVED ON OUR NIH VIDEO WEBSITE SO YOU CAN VISIT ALL THE LECTURES LATER. THIS MORNING WE HAVE FOUR EXCELLENT SPEAKERS. DR. KAREN DUFF AND DR. HYMAN AND DR. FINKBEINER AND DR. HOLTZMAN AND I WANT TO THANK THEM FOR SPEAKING. IF YOU WANT TO FIND OUT MORE ON OUR SPEAKERS WE PROVIDED A LINK ON THE MEETING WEBSITE. WITHOUT FURTHER DELAY OUR FIRST SPEAKER IS DR. KAREN DUFF. SHE IS A DIRECTOR OF DEMENTIA RESEARCH INSTITUTE AT UNIVERSITY OF COLLEGE OF LONDON IN THE U.K. AND ON AND THE TITLE OF HER LECTURE IS PATHOGENIC MECHANISMS DRIVING TAUOPATHY. >> THANK YOU VERY MUCH. THIS HAS BEEN VERY INTERESTING SO FAR AND I'M SURE IT WILL CONTINUE TO BE. I'M GOING TO BE TALKING ABOUT ALZHEIMER'S DISEASE AND TAUOPATHIES AND IT'S MAINLY DEFINED AS HAVING TWO DIFFERENT PATHOLOGIES, AMYLOID PLAQUES AND TAU WITH TANGLES. THESE DEVELOP IN SLIGHTLY DIFFERENT AREAS OF THE BRAIN AND IMPACT DIFFERENT AREAS OF THE BRAIN AS THE DISEASE PROGRESSES. WITH AMYLOID PLAQUE BEING MORE DISTRIBUTED THROUGH THE BRAIN AND TANGLES FOLLOWING A VERY STEREOTYPICAL DISTRIBUTION SPATIAL TEMPORAL DISTRIBUTION WE'LL GO INTO IN A MINUTE. HERE IS SOME NEWER PATHOLOGY TALKING ABOUT TAU TANGLES AND I'LL TALK ABOUT TAU AND I'M NOT COVERING THE AMYLOID PART THOUGH I CONSIDER IT IMPORTANT IT'S NOT THE FOCUS OF MY RESEARCH. THE TAU PROTEIN BINDS WHICH BINDS IN ALZHEIMER'S DISEASE OR ONE OF THE PRIMARY TAUOPATHY AND THE MICROTUBULES AND THE DISEASES THAT CAUSE TAUOPATHY IS DIFFERENT. THIS IS A FASCINATING ASPECT TO THE DIVERSITY OF DISEASES THAT ARE ASSOCIATED WITH TAU PATHOLOGY AND YOU SEE ALZHEIMER'S DISEASE AND PSP AND THE AREAS SHADED INDICATE WHERE THE TAU PATHOLOGY STARTS AND SPREAD AS THE DISEASE WORSENS. THE DIFFERENT CELL TYPES AFFECTING THE DISEASES. NEURONAL IN MOST BUT ALSO GLIAL PATHOLOGY DEVELOPING WITH TAU IN SOME OF THE OTHER DISEASES. THE DISEASES ARE CATEGORIZED BY DIFFERENT ISO FORMS OF TAU AND DIFFERENT THREE AND FOUR REPEAT CONTAINING TAU MANIFESTING THE DIFFERENT DISEASE. THE REPEAT DOMAINS ARE CONTAINED IN MICROTUBULE FINDINGS. IT CAN TAKE DIFFERENT PATHOLOGIES AND ONE PROTEIN CAN TAKE DIFFERENT CONFIRMATIONS. ONE OF THE ASPECTS WE'RE INTERESTED IN THE TAU PATHOLOGY WE CREATED A MOUSE MODEL IN THE CORTEX AND LOOKED AT THE DISTRIBUTION AS MICE AGED. IN THE EARLY MICE WE SHOWED THE DISTRIBUTION MAINLY LOCATED IN THE CORTEX AND THE PATHWAY BUT THE DISTRIBUTION CHANGED WITH MICE AGE SPREADING TO AREAS NOT PREVIOUSLY SEEMING TO HAVE ANY TAU PATHOLOGY AND THE DISTRIBUTION SUGGESTS TAU WOULD CROSS FROM ONE TO ANOTHER WITH AREAS SUCH AS THE GRANULE CELL AND THE GYRUS BECOMING POSITIVE FOR TAU AS THE DISEASE WORSENED. NOW THE MECHANISM WHICH TAU CAN CROSS FROM ONE TO ANOTHER IS OF GREAT INTEREST TO US. THE QUESTION HAS BEEN HOW DOES IT GO FROM CELLS IN THE CORTEX THESE CELLS AND TAU RELEASED FROM A DEGENERATING NEURON COULD BE TRANSFUSED AND TAKEN UP BY IT CAN GO BY CYTOSIS AND THIS IS WHERE THE GLYCANS MAY COME INTO AFFECT OR CAN GO BY VESICLES AND ONCE IT GETS IN THE RECIPIENT NEURON IT HAS TO CREATE MORE COPIES OF THE AGGREGATED FORM AND CAN LEAD TO THE PROPAGATION FROM NEURON TO NEURON AS THE AGGREGATED TAU Cs SPREAD FROM NEURON TO NEURON. WE FOCUSSED ON THE VESICLE MEDIATED TRANSFER OF TAU BETWEEN NEURONS AND THERE'S A LITTLE BIT OF THE BIOLOGY OF THE EXTRA CELLULAR VESICLES. THERE'S A NUMBER OF DIFFERENT TYPES. GET MICROVESICLES SLIGHTLY LARGER BUDDING OFF OR SHEDDING OF THE PLASMA MEMBRANE. YOU GET EXOSOMES SLIGHTLY SMALLER AND APOPTOTIC BODIES AND THIS IS THE METHOD WE USED AND IF YOU FRACTIONATE YOU CAN IDENTIFY FRACTIONS THAT CONTAIN DIFFERENT VESICLES AND SOME OF THE CRYOEM STRUCTURES ARE SHOWN BELOW. WE TOOK THE EXTRA VESICLES AND PERFORMED SEVERAL EXAMINATIONS OF WHAT TAU IS CONTAINED IN THEM. WE SEE TAU IN THREE OF THE FRACKS OF THE SEVEN OR EIGHT -- FRACTIONS OF THE SEVEN OR EIGHT AND WHEN YOU DO EM AND LOOK AT THE TAU CONTAINED WITHIN THE VESICLE THEY LOOK LIKE FILL AMOUNT S -- FILAMENTS FROM THE HUMAN BRAIN AND THIS IS IN THE FORM IT COULD GO OFF AND CAUSE AGGREGATES OF TAU TO FORM THROUGH TEMPLATING. WE TOOK THOSE EXTRA CELLULAR VESICLES AND HAVE INJECTED THEM INTO A MOUSE WHICH HAS SOME ONGOING TAUOPATHY AND THIS IS A FAIRLY STANDARD TECHNIQUE IN OUR FIELD WHERE YOU TAKE TAU AND PUT IT IN THE BRAIN AND YOU SEE WHETHER IT ACCELERATES THE TAUOPATHY AND CHANGES THE DISTRIBUTION AS THE TAU IN THE VESICLES IS SOMEHOW TEMPLATED ONTO THE MOUSE TAU. WHEN WE DO THIS WE SEE A SITE INJECTED AND WE SEE EXACERBATION OF THE TAUOPATHY SUGGESTING THIS TAU IS COMPETENT TO FORM SEEDS AND THE SEEDS CAN IMPACT AND QUIN EFFECT CELLS IN THE BRAIN AND SPREAD THE PATHOLOGY. NOW, WE'VE BEEN DOING SOME PROTEOMICS AND THIS IS THE VESICLE PREPS FROM THE NUMBERS B, C AND D THE TAU CONTAINING EVs AND WHEN WE DO PROTEOMICS OF THESE EVs AND WE LOOK FOR PROTO GLYCAN RELATED PROTEINS WE CAME UP WITH A LIST OF ABOUT 11 RELATED PROTEINS AND THOSE ARE SHOWN HERE. NOW, ANOTHER STUDY WAS PERFORMED AND HAS BEEN PUBLISHED -- WE HAVEN'T PUBLISHED YET AND FOUR OF THEIRS OVERLAPPED WITH OURS SUGGESTING WE ARE PICKING UP EV RELATED GAGS. NOW, WE DIDN'T SEE MUCH OF A DIFFERENCE WHEN WE DID THIS BETWEEN AB AND CONTROLS WE SAW A REDUCTION IN APOE AND WE'RE NOT SURE WHAT THAT MEANS. WE'RE GOING TO EXPLORE IT FURTHER. OF NOTE, MOST OF THE PROTEO GLYCANS WE THINK MIGHT DEFLECT THE FACT THEY'RE THE MOST ABUNDANT IN THE EXTRA CELLULAR MATRIX AND THEREFORE MOST ABUNDANT IN THESE EVs. WE'RE CURIOUS ABOUT THE CELLULAR SOURCE OF THE PROTEOGLYCANS. THERE'S MORE REACTION IN THE BRAINS OF THE MICE AND MAYBE A LOT OF THESE ARE COMING FROM ASTROCYTES BUT THIS SUGGESTS TO US THEY CONTAIN A PROTEOGLYCANS AND THE CSPGs. ANOTHER ANOTHER STUDY SHOWED WHEN YOU TAKE THEIR EVs AND INJECTED THEM IN THE MOUSE MODEL THIS A WILD TYPE MOUSE AND THEIR EV TAUOPATHY WENT TO NEURONS AND THE TYPES IMPACTED MOSTLY BY TAU ON THE BUT THEY SHOWED UPTAKE IN INHIBITORY NEURONS. THESE EV RELATED TAUOPATHY WAS VERY ROW BUST AND MORE ROBUST THAN IF YOU JUST INJECTED FILAMENTS OF TAU SO IT'S VERY MUCH THE TAU SEEDS CONTAINED IN THE EVs ARE VERY COMPETENT. WE'RE CURIOUS WHAT RECEPTORS ARE PICK UP THE EVs AND A NUMBER HAVE BEEN IDENTIFIED AS TAKING UP EVs. AND WE'RE WONDERING WHETHER THERE'S DIFFERENCES IN THE EVs BETWEEN INHIBITORY AND EXCITATORY NEURONS. NOW TAU HAS BEEN SHOWN TO INTERACT WITH THE GAGS A NUMBER OF TIMES STARTING WITH THE WORK IN THE MID '90s AND WITH MARK DIAMOND'S WORK WHERE HE LOOKED AT HSPGs SHOWING TAU CAN BE INTERNALIZED BY CELLS AND SHOWING SPECIFIC MODIFICATIONS FACILITATING THE TAU INTERNALIZATION AND THE IDEA IS TAU, ESPECIALLY TAU C CAN BIND TO THE SPGs AND GO ACROSS THE MEMBRANE OR INTERNALIZE IN SOME WAY THROUGH THEIR BINDING TO PROTE IIE IIO -- PROTEOGLYCANS AND TAU GENERATED EXPERIMENTALLY TO MAKE MONOMERS OR AGGREGATES WHERE WE'VE LOOKED AT EXTRA CELLULAR VESICLE TAU SO THERE MAY BE A DIFFERENCE IN TERMS OF UPTAKE. THE IMPLICATIONS HAVE BEEN SHOWN IN A COUPLE STUDIES AND THIS IS A STUDY BY MARK DIAMOND'S LAB SHOWING HE CAN CREATE WHAT CAN PREVENT THE UP TAKE OF TAU INTO CELLS AND THERE'S BEEN WORK USING ANTIBODIES TO BLOCK THE BINDING SITE. THIS HAS IMPLICATIONS. OUR ONGOING WORK ON THIS IS MULTIFOLD AND I THINK WE'LL DO MORE WORK NOW ON THIS AND EXCITED TO TAKE THIS ON. WE'RE DOING PROTEOMICS TO LOOK AT THE DIFFERENT TYPES OF EVs IN THE DIFFERENT FRACTIONS. WE'RE LOOKING AT PROTEOMICS FROM EV COMPONENTS TO SEPARATE THE MEMBRANES FROM THE LUMEN AND DOING PROXIMITY PROTEOMICS TO LOOK AT WHAT IS INTERACTING WITH PERHAPS THE MEMBRANE PROTEINS OF HSPGs PLUS TAU. WE'RE DOING A LOT OF EM AND LOOKING AT THE COMPOSITION OF THE TAU WHETHER IN FRAGMENTS OR ISO FORMS OF TAU AND MODIFIED WITH MODIFICATIONS AND THE CONFIRMATION OF IT. WE'RE DOING MASS SPEC TO DO THIS WITH CRYOEM AND CRYO-EMET AND WE'RE LOOKING AT SURFACE DENSITIESES THE GLYCANS ON THE MEMBRANES DOING THIS BY CRYO-EM AND CRYO-ET AND I'M SHOWING WORK DONE ON VIRUSES SHOWING GLYCANS ASSOCIATED WITH THE SURFACE MEMBRANE OF VIRUSES AND I THINK THIS IS AMAZING. YOU CAN REALLY START TO IDENTIFY WHAT TYPE OF GLYCAN YOU HAVE BY DOING CRYO EM AND CRYOET AND WE'RE LOOK AT CELLULAR COMPARTMENTS AND DID THEY COME TO EXOSOMES OR INTO MICROVESICLES AND WE'RE LOOKING AT WHAT TYPE OF CELLS ARE SECRETING THE TAU CONTAINING EVs. WE THINK THE NEURONS IS WHERE THE TANGLES FORM BUT COULD BE GLIIA -- GLIA AS WELL AND WE'RE LOOKING AT THE NEURONS AND GLIA AND WHAT TYPES OF NEURONS THEY'RE GOING INTO. HOW DID THE TAU GET INTO SPECIFIC NEURONS. SO WE'RE USING CRYO-ET AND LOOKING AT FUNCTIONAL IMPACT AND THIS IS WORK WE'RE DOING WITH DR. FINKBEINER AND IN VIVO. I WANTED TO TALK ABOUT SELECTED VULNERABILITY AND HOW THAT'S PERHAPS IMPACT CELL SURFACE PROTEO GLYCANS AND THE TAU PATHOLOGY OF PARTICULAR NEURONS IS FASCINATING AND YOU CAN SEE HERE ON THE LEFT THREE AFFECTED NEURONS WITH TAUOPATHY AND MULTIP MULTIPLE NEURONS SPARED AND THIS IDEA DIFFERENT NEURONS ARE IMPACTED AN DIFFERENT REGIONS HAVE TEMPORAL VULNERABILITY HAS BEEN LOOKED AT IN MANY WAYS AND HERE'S A TABLE SHOWING YOU WHAT'S KNOWN ABOUT THE IMPACT OF IMPACTED TYPES OF AREAS EARLY IN THE DISEASE, LATER IN THE DISEASE AND UNAFFECTED. THE EARLIEST AFFECTED ARE THE LARGE NEURONS IN THE ENTORHINAL CORTEX AND INHIBITORY NEURON SPARED AND ALSO IN DISEASES. WE HAVE A BIOINFORMATICS APPROACH AND THIS IS A SINGLE DATA SET GENERATED BY CARVING UP THE NORMAL WILD TYPE MOUSE BRAIN INTO AREAS AND PERFORMING SINGLE NUCLEUS RNA SEQ TO IDENTIFIED CLUSTERS OF GENES DISTRIBUTED IN DIFFERENT CELL TYPES AND LAYERS OF THE MOUSE BRAIN. WE WANTED TO LOOK AT WHETHER EARLY REGIONS LIKE THE ENTORHINAL CORTEX SHOWED VULNERABILITY TO EXPLAIN WHY THEY WERE EARLIER AFFECTED THAN LATER AFFECTED IN THE HIPPOCAMPUS. WE TOOK THE DATA SETS AND YOU CAN SEE HERE CIRCLES -- AND THIS LED TO THE CA 1, CA3 AND INHIBITORY NEURONS IMPACTED EARLY TO LATE AND DID TRANSCRIPTOMICS TO IDENTIFY GENE CLUSTERS IN THE DIFFERENTIALLY EXPRESSED BETWEEN MAJOR AFFECTED AREAS AND INHIBITORY NEURONS AND ONE IMPLICATED TRANSFERASE AND WHEN WE LOOKED IN A BIT MORE DETAIL AT THESE WE WERE ABLE TO SHOW SULFATE TRANSFERASE GENES AND THE PROTEO GLYCAN METABOLIZING GENES WERE EARLIER EXPRESSED FROM THE EARLIER AFFECTED NEURONS TO THE LATE AND WENT IN THE OPPOSITE DIRECTION. WE'RE NOT SURE WHAT THIS MEANS BUT IT'S SOMETHING TO FOLLOW-UP. WE DID A QUICK TEST THAT LOOKED LIKE THE GENES WERE EXPRESSED DIFFERENTIALLY CRASS THE AREAS. HERE'S THE -- ACROSS THE AREAS AND HERE'S THE SET FOR ONE OF THE GENES AND NDST AND IT WAS EXPRESSED DIFFERENTIALLY BETWEEN ENTORHINAL CORTEX AND THE INHIBITORY NEURONS. WE TOOK ANOTHER DATA SET AND THIS WAS INTERESTING. THIS HAD ISOLATED NEURONS FROM TANGLE NON-BEARING NEURONS IN THE SAME PIECE OF HUMAN AD BRAIN AND SEPARATED THEM AND PUT THEM INTO CELL TYPES. AND WE WERE INTERESTED IN LOOKING AT ONE OF THE PARTICULAR CELL TYPES. THEY'RE ALL CELL TYPES IN THE AREA AND THESE ARE VULNERABLE TO THE TANGLES AND WHEN YOU LOOK AT DATA SETS BETWEEN THE POSITIVE TANGLE NEURONS VERSUS THE NEGATIVE TANGLE BEARING NEURONS YOU SEE DIFFERENTIAL EXPRESSION ESPECIALLY HERE AT THE TRANSFERASE GENES SUGGESTING THERE'S AN INTRINSIC VULNERABILITY BETWEEN CELLS IMPACTED WITH TANGLES AND THOSE THAT ARE NOT. WHEN WE COMPARED THE DATA SETS WE WERE INTERESTED TO SEE ACTUALLY BOTH THE BOTH REGIONAL DISTRIBUTION AND THE SELECTED VULNERABILITY OF TIME BEARING NEURONS MATCHED AND WE WERE GETTING THE DATA SETS FROM THOSE WHO EXPERIMENTS. WE HAVE ALSO BEEN LOOKING AT -- SORRY, MY BAR'S ON THE TOP. INTERESTINGLY, WHEN WE LOOKED TO OTHER GAGS AT THE TIME BEARING NEURONS WE WERE INTERESTED TO SEE A LOT OF THE PROTEO GLYCAN GENES CAME IN AS DIFFERENTIALLY EXPRESSED MATCHING THE PROTEOMICS WITH THE SYNTHETIC GENES PARTICULARLY IMPACTED AND RESPONSIBLE IN TANGLE-BEARING NEURONS. I WANTED TO FINISH OFF TALKING ABOUT THE RESILIENCE OF INHIBITORY NEURONS TO TAUOPATHY. THIS COPS BACK TO THE IDEA OF THE NEURONAL NETS WE STARTED TO TALK ABOUT YESTERDAY AND THIS IDEA THAT INHIBITORY NEURON PARTICULARLY RESISTANT TO TANGLES IS ENCOMPASSING THE IDEA THAT THE NETS ARE INVOLVED AS THEY MAINLY IMPACTED THE INHIBITORY NEURONS AND DID A SMALL STUDY TO START WITH ANTIBODIES WITH THE MARKERS SHOWING AND TAU ANTIBODY AND THE INHIBITORY NEURONS WERE UNVULNERABLE SHOWN IN ALZHEIMER'S DISEASE AND MOUSE MOLD AND OTHER TAUOPATHIES. THIS INVULNERABILITY IS CONSISTENT. NOW, WHY ARE THE NEURONAL NETS INVOLVED IN THIS AND WE WERE WONDERING WHETHER PERHAPS THEY SHIELD THE INHIBITORY NEURONS FROM TAU SEEDS BEING TAKEN UP BY THE EVs OR FREE TAU OR TAU SOMEHOW TRANSFERRING INTO THEM AND SOMETHING I'D LIKE TO THINK ABOUT FURTHER ABOUT WHAT'S THE INVOLVEMENT PERHAPS IN THE TAU SPREAD. JUST TO FINISH OFF, A COUPLE THINGS WE'RE LOOKING AT NOW FOR THE BIOINFORMATICS WORK WE'RE LOOKING AT THESE USING SPATIAL TRANSCRIPTOMICS AND NEEDS MORE RNA AND DATA SETS FROM THE AFFECTED AND UNAFFECTED NEURONS AND WE'RE LOOKING AT TRANSCRIPTOMICS APPROACHES AND IN SITU PROTEOMICS AND WE HAVE WORK OF MY LAB DEVELOPING WAYS OF GOING FROM GENE SETS TO ONTOLOGY PATHWAYS WHICH IS A LITTLE BIT OF A WEAKNESS IN THE FIELD AND NEEDS WORK AND WORKING OUT COMPUTATIONAL METHODS FOR DOING THAT AND DEVELOPING RELEVANT CELL AND MOUSE MODELS SO WE CAN ACTUALLY HAVE TOOLS TO LOOK AT THESE DIFFERENCES AND THESE INCLUDE NEURONS DIVIDED BY PATIENTS WITH TAUOPATHIES. NOT THE PROGRAMMING NEURONS BUT A DIRECT VERSION WHICH MAINTAIN SOME OF THEIR DISEASE PHENOTYPE AND AGING CHARACTER OF TF ISTICS AND NEW MOUSE MODELS TO MAINTAIN THE PATHOLOGY WITHOUT OVER EXPRESSION, ETCETERA. I JUST WANT TO THANK THE PEOPLE WHO CONTRIBUTED TO THIS TALK. AND BEN FALCON AT CAMBRIDGE UNIVERSITY WHO IS THE EXPERT ON ALL THINGS EM AND WE'RE WORKING ON THE ED UPTAKE AND WHAT IT DOES IN NEURONS THROUGH USING ROBOTIC MICROSCOPY. WE HAVE A NUMBER OF COLLABORATORS FOR PROTEOMICS AND DATA SET ANALYSIS AND I WANT TO THANK MARK DIAMOND. THANK YOU VERY MUCH. CRYO >> THANK YOU, KAREN. WE HAVE ONE QUESTION DOES THE TAU HAVE MODIFICATION SUCH AS THE ACID DO THE EV CONTAINING TAUS HAVE PTMs. >> THE PTM ANALYSIS REQUIRES MORE SPECIALIZED PROTEOMICS WE DON'T HAVE THE EXPERTISE IN. THEY DO PATHOGEN -- PATHOGENIC TAU HAS POSTTRANSLATIONAL MODIFICATIONS. I DON'T KNOW IF IT WILL BE TALKED ABOUT. IT IS GLYCOSYLATED. I THINK IT'S BEEN SHOWN TO BE MODIFIED MANY DIFFERENT SITES BOTH WITH GLYCOSYLATION AND ACETYLATION AND PHOSPHORYLATION. DO THE EVs CONTAIN PTMs? WE'RE LOOKING AT THAT. WE EXPECT WILL DO AND CAN DO SOME OF THAT WORK WITH THE PROTEOMICS AND SOME OF THE EM WORK. AT THE MOMENT I DON'T HAVE DATA ON THAT. >> ANY QUESTIONS FROM OUR PANELISTS? >> IN THE TAU CONTAINING EV THERE'S RTPRZ1 WHICH WE FOUND MAKE THE TARGETS THAT WOULD TAKE IT TO MICROGLIA. THE QUESTION IS DO MICROGLIA PLAY ANY ROLE IN MOVING TAU? >> VERY INTERESTING PART OF THE FIELD IS WHETHER THE GLIA ARE CONDUITS WHERE THE EVs PASS FROM NEURON TO NEURON. EVIDENCE ASTROCYTES WILL ACCUMULATE TAU AND THE DENDRO DENDROCYTES BUT THERE'S EFFORTS THEY'LL TAKE UP TAU AND THAT'S PERHAPS HOW IT'S GETTING PASSED ON. IT WAS A GLIAL TRANSFERRING ONE. >> THERE'S WORK BEING DONE. >> I THINK WE SAW THAT ONE BOTH IN THE TANGLE BEARING DATA SET AND ALSO IN OUR PROTEICS FROM THE EVs. I'LL GO BACK AND CHECK THAT ONE. >> GREAT, THANK YOU. >> SO WE HAVE ONE MORE QUESTION FROM THE AUDIENCE. HOW DO YOU THINK THE EV ARE INTERNALIZED? YOU MENTIONED THE RECEPTOR WHICH MAKES IT SOUND LIKE THEY ARE PICKING UP IMPACT. IF THIS IS CORRECT, HOW DO YOU THINK THE TAU ESCAPED FROM THE EV TO THE CELLULAR TAU. >> THIS IS ONE OF THE QUESTIONS HOW IT GETS IN BUT HOW DOES IT GET OUT OF WHATEVER IT'S IN. I THINK THE EVs WILL FUSE WITH THE MEMBRANE AND YOU COULD DISCOURAGE THE CONTENTS OF THE LUMEN INTO THE RECIPIENT CELL. COULD BE MOVED THROUGH THE CELL INTACT AND THE TAU CAN GET ACROSS THE MEMBRANE AND MARK'S WORKED ON THIS A LOT THAT TAU IS A MEMBRANE ASSOCIATED PROPENE THAT CAN BE TRANS LOCATED ACROSS THE MEMBRANE. WE DON'T KNOW EXACTLY HOW THIS HAPPENS. WE KNOW THE TAU GETS PUT INTO LYSOSOMES BUT IT'S A BIT OF A MYSTERY STILL, AS FAR AS I'M AWARE HOW A VESICLEAR TAU CAN MOVE AND HOW IT HAPPENS IS STILL IN QUESTION. >> THE FINAL QUESTION. >> THIS IS LINDA'S QUESTION. >> CAN YOU SEE THE QUESTION? >> YOU WORKED ON THE PAPER, RIGHT? THIS IS INTERESTING THE UP AND DOWN OF THE PROTEO GLYCANS. WE HAVEN'T DONE ANYTHING MORE ON IT BUT I CAN'T REALLY COMMENT ON THE DATA SETS WE HAVEN'T HAD A CHANCE TO LOOK AT THEM IN DETAIL AND I'D BE VERY HAPPY TO SHARE THEM WITH YOU AND GET YOUR INPUT ON THEM. I CAN'T ANSWER YOUR QUESTION AT THIS MOMENT. WE'VE JUST STARTED LOOKING AT THIS. >> GREAT. I LOVE TO LOOK AT THEM. >> THEY BECOME LESS POPULATE AND YOU GET MORE INFORMATION. OBVIOUSLY IT'S NOT THE ONLY DATA SET THAT'S SIGNIFICANTLY CHANGED. THERE'S LOTS OF DIFFERENT MODULES. THE PROTEIN GLYCANS IS JUST ONE OF THEM BUT HAPPY TO SHARE THEM. >> AND POSSIBLY SOME OF THE ANTIBODIES OR THE HPLC ANALYSES AND THE STANDARDS WE'VE GENERATED MIGHT BE USEFUL FOR DECIPHERING SOME OF THIS. >> IT'S GREAT. THANKS. >> THANK YOU VERY MUCH. >> I'M GOING TO MOVE ON TO THE NEXT LECTURE FROM DR. BRAD HYMAN. >> CAN YOU GUYS SEE MY SLIDE. >> DR. BRAD HYMAN THE PROFESSOR OF NEUROLOGY AT HARVARD MEDICAL SCHOOL. DR. BRAD HYMAN HAS MANY INTERESTS IN THE AREA OF ALZHEIMER'S DISEASE AND HE WAS ALSO THE COUNCIL MEMBER AND DR. HYMAN HAS DEVELOPED MANY OF THE IMAGING TOOLS TO HELP US UNDERSTAND MANY IMPORTANT BIOLOGY. WITHOUT ANY FURTHER DELAY, BRAD. >> OKAY, THANK YOU, AUSTIN. AND THANK YOU FOR THE INVITATION TO PARTICIPATE IN THIS REALLY INTERESTING SYMPOSIUM. CAN YOU CONFIRM MY SLIDES ARE UP AND YOU CAN HEAR ME? >> I CAN HEAR YOU. >> GOOD, THANKS. I'D LIKE TO TALK ABOUT SOME OF THE SAME ISSUES THAT KAREN ADDRESSED AND THEN HIGHLIGHT SLIGHTLY DIFFERENT ASPECTS OF IT. I DON'T KNOW IF THESE SORTS OF DISCLOSURES ARE NECESSARY FOR THIS KIND OF MEETING BUT IT SEEMS GOOD TO SAY THAT INTERACTIONS WITH A VARIETY OF GROUPS. I'D MOSTLY LIKE TO THANK NIH AND OTHER FUNDING AND WOULD LIKE TO POINT SOME OF THE WORK HAS BEEN DONE WITH MERCK AND HIGHLIGHT COLLABORATIONS WITH THE STEEN LAB AND THE STRICKLAND LAB AND THE OTHER LABS WE'VE COLLABORATED WITH. AS KAREN POINTED OUT THE PROCESS OF ALZHEIMER'S DISEASE INVOLVES A SPREAD OF THE PATHOLOGIC CHANGES OUT TO THE NEOCORTEX IN A PROCESS THAT TAKES UP TO DECADES AND THIS IS VERY STEREOTYPED TO THE POINT THEY WERE ABLE TO CREATE A STAGING SCHEME FROM ONE TO SIX AND ABOUT 95% OF ALL ALHEIMER CASES FALL ON THIS STAGE IN SCHEME. IT SEEMS IT'S A VERY STEP YO TYPICAL HIERARCHICAL PATTERN OF VULNERABILITY OF THE TANGLES. AND THE GENETICS OF ALZHEIMER'S DISEASE STRONGLY IMPLIES THE AMYLOID DEPOSITION INITIATES THE DISEASE AND THE PART OF THE BRAIN THAT LEADS TO A CLINICALLY DEVASTATING DEMENTIA AND THE TANGLES CORRELATES BEST WITH CLINICAL SYMPTOMS. FROM THE POINT OF VIEW OF DESTRUCTION OF NEURAL SYSTEMS TANGLES SEEM TO BE QUITE INTEGRAL. AND THESE ARE CONNECTED BY ANATOMICAL PATHWAYS. THERE'S TWO POSSIBILITIES FOR WHY THAT PATTERN CAN EMERGE. THE FIRST POSSIBILITY IS NEURON ARE CONNECTED TO EACH OTHER SHARE MULTIPLE ATTRIBUTES THEY SEND TO BE EXCITATORY AND LARGE AND RELATIVELY LARGE AXONS. THEY SHARE A LOT OF CHARACTERISTICS AND MAYBE AMONG THOSE CHARACTERISTICS IS THE ABILITY TO DEVELOP A TANGLE UNDER CERTAIN CIRCUMSTANCES. OF COURSE THE OTHER POSSIBILITY THAT AMONGST THE REASONS WHY CONNECTED NEURONS ARE VULNERABLE IS THAT THE CONNECTION ITSELF CONNOTES SOME LEVEL OF THAT VULNERABILITY AND IN MY OWN OPINION BOTH OF THOSE THINGS ARE [AUDIO DIGITIZING] HOWEVER, THE CONNECTIVITY IS IMPORTANT DERIVES HAVE A SERIES OF EXPERIMENTS SOME OF WHICH YOU INJECT TAU ONE FORM OR ANOTHER INTO THE BRAIN AND CAN BE TAKEN UP BY VULNERABLE NEURONS AND AGGREGATE TO SOMETHING THAT LOOKS LIKE A TANGLE. THERE'S ALSO A SERIES OF MOUSE EXPERIMENTS THAT KAREN'S LAB AND MY LAB [AUDIO DIGITIZING] AND TAU IN ONE PART OF THE BRAIN AND THE ENTORHINAL CORTEX AND THE HUMAN TAU PROTEIN CAN BE EXPRESSED AND PROPAGATE TO THE NEXT NEURON IN THE ZONE SO THAT'S ANOTHER LEVEL OF EVIDENCE. AND A THIRD LEVEL OF EVIDENCE IS THE WORK I'M SHOWING HERE WHICH IS ISOLATING SYNAPSES FROM DIFFERENT AREAS OF THE BRAIN IN INDIVIDUALS WHO HAD DIFFERENT LEVELS OF THE DISEASE PROCESS. AN EARLY INDIVIDUAL WITH DISEASE IN THE ENTORHINAL CORTEX IF YOU LOOK AT THEIR TERMINAL ZONE OF THE SYNAPSES -- COMPARED TO THE AFFECTED PERSON THE BRAAK6 PERSON WHERE VISUAL CORTEX IS AFFECTED. TURNS OUT YOU CAN FIND TAU BIO ACTIVITY AS SEEDING ACTIVITY IN SYNAPTIC PREPS THAT MATCH THE PATHWAY TO CROSS THE SYNAPSE AND BE TAKEN UP BY THE POSTSYNAPTIC NEURON. IF ONE ACCEPTS AT LEAST IN PART THE SPREAD OF TAU PATHOLOGY ACROSS THE BRAIN IS DUE TO THIS PROPAGATION PHENOMENA, THEN IT STANDS TO REASON THAT MODIFICATIONS OF THAT PROPAGATION PHENOMENA COULD IMPACT THE RATE IN WHICH PROPAGATION IN WHICH THE SPREAD OCCURS ACROSS THE ENTIRE BRAIN AND THAT WOULD CORRELATE WITH IN PRINCIPLE AT LEAST THE RATE OF PROGRESSION OF DISEASE. IT TURNS OUT AS A CLINICIAN, THIS IS OBVIOUS BUT I THINK IT'S OBVIOUS TO ANYONE WHO KNOWS INDIVIDUALS WHO HAS ALZHEIMER'S DISEASE. SOME PEOPLE GET ALZHEIMER'S DISEASE AND JUST HAVE AN AWFUL COURSE. IN THE COURSE OF TWO OR THREE YEARS PROGRESS RAPIDLY TO SEVERE DEMENTIA AND OTHERS HAVE MILD IMPAIRMENT AND CLEARLY GET A LITTLE WORSE FROM YEAR TO YEAR, REALLY NOT FROM A RAW CLINICAL PERSPECTIVE. THIS IS TRUE NOT ONLY IN SPORADIC ALZHEIMER'S DISEASE BUT IN DEFINED POPULATIONS, GENETICALLY DEFINED POPULATION WHERE AS THE AN AUTOSOMAL DRIVER AND AGE IS YOUNG YOU ARE BUT THERE'S A SPREAD WITH SOME HAVING FAST PROGRESSIONS AND OTHER INDIVIDUALS HAVING SLOW PROGRESSION. WE BEGAN TO EXPLORE WHAT THE UNDERLYING BIOLOGY OF THIS PROPAGATION PHENOMENA COULD BE TO SEE IF WE COULD UNCOVER ANYTHING THAT WOULD CORRELATE WITH THE RATE OF [AUDIO DIGITIZING] THE SAME BIOLOGY DONE BY MARK DIAMOND IN HEX CELLS. WE MEASURE THE BIOACTIVITY IN TAU PRESENT IN 32 INDIVIDUALS WE FOLLOWED IN OUR CLINICAL WHO ULTIMATELY CAME TO AUTOPSY AND WE MEASURED THE AMOUNT OF TAU BIOACTIVITY AND SINCE WE FOLLOWED THE INDIVIDUALS IN CLINIC WE SAW WHICH HAD A FAST RATE AND WHICH HAD A SLOW RATE OF PROGRESSION. [AUDIO DIGITIZING] WE SHOWED THOSE WITH SEEDING THE HIGHEST HAD THE HIGHEST RATE OF PROGRESSION AND THE RANK ORDER OF SEEDING AND PROGRESSION. THIS SUGGESTS SOMETHING ABOUT THE ISOLATED TAU WE HAD COLLECTED FROM THESE BRAINS WAS IN FACT DIFFERENT FROM INDIVIDUAL TO INDIVIDUAL. IN COLLABORATION WITH JUDITH STEEN WE WERE ABLE TO SHOW THE POSTTRANSLATIONAL MODIFICATIONS OF THESE INDIVIDUALS UP TO OVER 40 DIFFERENT PHOSPHORYLATION, ACETYLATION AND IN GENERAL THE NUMBER OF POSTGENERATIONAL MODIFICATIONS WAS HIGHER IN THOSE INDIVIDUALS WITH A FASTER RATE OF PROGRESSION AND THAT IN FACT THERE WERE CERTAIN PATTERNS THAT EMERGED SUGGESTING PARTICULARLY UNIQUE PHOSPHORYLATION SITES WHICH ARE HIGHLIGHTED ON THIS SLIDE. SO WITH HIGHER RATES OF SEEDING, CERTAIN CLUSTERS OF PHOSPHORYLATION, ACETYLATION AND SITES SEEM TO OCCUR. THERE'S SOMETHING ABOUT THE POST-TRANSLATIONAL MODIFICATIONS THAT OCCUR AND THOUGH IT'S NOT SHOWN ON THIS SLIDE IN WHICH WE MAPPED OUT EVEN MORE POST-TRANSLATIONAL MODIFICATIONS WITH GLYCOSYLATION WERE DEEPLY UNDER STUDIED BECAUSE THE MASS SPEC TECHNIQUE USED TO IDENTIFY THESE POST-TRANSLATIONAL MODIFICATIONS IS NOT ESPECIALLY EFFICIENT TO STUDY GLYCATION AND GLYCOSYLATION EVENTS. THE SAME STORY CAN BE TOLD WITH THE CASES [AUDIO DIGITIZING] WE AGAIN DEMONSTRATED DIFFERENT TAU PATTERNS OF POSTTRANSLATIONAL MODIFICATION CORRELATED WITH DIFFERENT RATES OF PROGRESSION IN THESE INDIVIDUALS. AND IN FACT THERE'S A THIRD DATA SET NOW FROM YET ANOTHER POPULATION OF INDIVIDUALS THAT DEMONSTRATES EXACTLY THE SAME THING. SO IT RAISES THE QUESTION WHAT ALTERS THE RATE OF PROPAGATION? ONE IS THE OBSERVATION THAT TAU CAN BE TAKEN UP BY AS A NUMBER OF INTERESTINGLY NOT BY ALL CELLS IN CULTURE TO ASTROCYTES AND MICRO GLIA EFFICIENT AND SOME CANNOT ONLY TAKE UP TAU WHEN PRESENTED WITH A TRANSDUCTION AGENT. WE CONFIRMED IN PRIMARY NEURONS AND IPS CELLS AND RECOMBINANT TAU AND [AUDIO DIGITIZING] OF A PATIENT WITH ALZHEIMERS DISEASE BE PRESENT ON THE CELL SURFACE AND HAVE BEEN ABLE TO DEMONSTRATE AND AGAIN IN COLLABORATION WITH THE STRICKLAND LAB THE UPTAKE OF FREE TAU APPEARS TO BE DEPENDENT LARGELY ON THE CELL SURFACE RECEPTOR, LRP1. SO ON THE NEXT PART OF THE TALK I'D LIKE TO TALK ABOUT LRP1 MEDIATED UPTAKE THEN HOW IT MOVES FROM BEING TAKEN UP TO ESCAPING INTO THE CYTOPLASM AND THEN TALK ABOUT THE PROCESS OF SEEDED AGGREGATION BECAUSE ALL THESE HAVE TO OCCUR FOR THE TANGLE PROPAGATION OCCUR FROM CELL TO CELL. NOW THREE DIFFERENT LABORATORIES HAVE DEMONSTRATED THAT LRP1 IS A NECESSARY AND PRIMARY UPTAKE MECHANISM FOR FREE TAU. LRP1 DEPENDS ON A LOW AFFINITY LIGAND ON THE CELL SURFACE WHICH TRANSFERS TO LRP1 FOR AN ENDOSOMAL UPTAKE PROCESS. SO THERE'S A CLEAR IMPLICATION IN THIS PROCESS AND THIS IS DISTINCT FROM THE EVs A DIFFERENT PATHWAY KAREN HIGHLIGHTED AND I THINK BOTH OF THESE PATHWAYS EXIST IN THE BRAIN. THOUGH IT'S A LITTLE BIT HARD TO KNOW EXACTLY WHAT PERCENTAGE OF TAU IS TAKEN UP BY ONE MECHANISM OR ANOTHER AT LEAST IN VIVO WORK IT APPEARS AS IF THE RELEASE OF TAU FROM ONE NEURON CROSSING THE SYNAPSE AND BEING TAKEN UP BY THE NEXT SYNAPSE CAN AT LEAST OCCUR WITH LRP1 AS THE PRIMARY MEDIATED SYSTEM. IF YOU ASK ABOUT OTHER LRP1 LIGANDS AND IT'S A MULTIFACETED THERE'S CERTAIN COMMONALITIES AMONGST THE DOZENS OF LIGANDS FOR LRP1 AND DR. STRICKLAND WHO CLONED IT AND HAS DONE WORK NOTICES THERE'S A SERIES OF LYSINES AND THEY HAVE A CHARACTERISTIC SPACING BETWEEN THEM AND WHEN WE LOOKED AT THE CRYO EM STRUCTURE OF PHOSPHO TAU WE SAW A HANDFUL OF SEVEN DIFFERENT LYSINES SEPARATED THAT QUALIFY IT AS AN LRP1 LIGAND AND DEMONSTRATED THE LYSINES ARE CRITICAL FOR THE UPTAKE OF TAU AND THE UPTAKE OF TAU IN CELLS AND CULTURE AND IF YOU MODIFY THIS SPACING OF THE LYSINES OR PUT PHOSPHORYLATION SITES NEAR THEM OR ACETYLATE THEM YOU CAN MEDIATE THE UPTAKE. SO WHICH OF THE UPTAKE MECHANISMS IS USED BY A NEURON IS PROBABLY DEPENDENT ON WHICH POST-TRANSLATION MODIFICATIONS ARE PRESENT ON TAU. ONCE IT'S TAKEN UP IT GOES TO THE ENDOSOME AND THE VAST MAJORITY OF THE LIANE -- LIGANDS ARE DEGRADED FROM THE ENDOSOME INTO THE CYTOPLASM AND WE STARTED TO STUDY AUTOPHAGY AND RE-SECRETION AND THE RELEASE INTO THE CYTOPLASM WHICH WE THINK IS THE MOST CRITICAL PART THERE ARE THE TAU PROPAGATION PHENOME PHENOMENA. AS I MENTIONED IN CELLS AND CULTURE, WHEN YOU TAKE UP RECOMBINANT TAU AND SAME FOR HUMAN BRAIN DERIVED TAU, THE VAST MAJORITY IS INTERNALIZED. THEN MOST IS DEGRADED WITHIN ABOUT AN HOUR. THAT'S REALLY STRIKING THAT MOST THE TAU THAT CAN PRESENT TO A CELL IN THIS CASE A CELL IS TRUE IN NEURONS BUT THE TIME FRAME'S A LITTLE BIT DIFFERENT. MOST OF IT GETS DEGRADED. IT TURNS OUT NOT ALL OF IT GETS DEGRADED. AND ESPECIALLY IF YOU PRESENT ALL THE WAY TAU IS POSTTRANSLATIONALLY MODIFIED AND APPEARS TO BE THE FORM OF TAU THAT IS MOST LIKELY TO BE TAKEN UP BY NEURONS ATAU COMPONENT. IT END UP ESCAPE FROM THE ENDOSOMAL COMPARTMENT AND YOU CAN SEE, I HOPE, WHERE THE WHITE ARROWS ARE, WHEN YOU USE THIS TAU OPPOSED TO PHYSIO LOGIC LOW MOLECULAR WEIGHT TAU DIFFERENTIATED BY THE HIGH MOLECULAR TAU YOU SEE RUPTURED ENDOSOMES IN THE STAINING. VERY RAPIDLY WITHIN AN HOUR OF EXPOSURE TO THE CELLS, THE TAU GETS DEGRADED OR SOME SMALL PART OF IT RUPTURES IN THE ENDOSOMES AND GETS DISTRIBUTED AND IN THIS PART OF THE SLIDE I'M SHOWING THE FIRST IMAGES THAT I TOOK ON A NEW SUPER RESOLUTION MICRO SCOPE IN WHICH WE'RE ABLE TO STAIN TAU AND THIS IS WITHIN AN HOUR OF EXPOSURE OF NEURONS TO HIGH MOLECULAR TAU AND IF WE REMOVED THE ENDOGENOUS MOUSE TAU WE CAN SEE THE TAU IS PRESENT NOT ONLY IN THE AXONS AND DENDRITES OF THE NEURON BUT RELEASED IN THE CYTOPLASM AND INTRIGUINGLY ENOUGH THIS TAU UPTAKE MECHANISM IS ABLE TO DROP IT IN AREAS OF THE CELL WHICH ARE POTENTIALLY BIOLOGICALLY ACTIVE. SO IN THE LAST FEW MINUTES I'D LIKE TO TALK ABOUT OTHER ASPECTS OF THE PROPAGATION PHENOMENA WE'VE BEEN STUDYING WHICH I THINK MAY GIVE US THE TOOLS WE MIGHT BE ABLE TO USE TO ASK HOW GLYCOSCIENCE IMPACTS THE SORT OF TAU PROPAGATION PHENOMENA THAT FASCINATES ME SO MUCH. SO IN ORDER FOR TAU PROPAGATION FROM ONE AREA TO THE NEXT YOU CAN DO A VARIETY OF THINGS LIKE INJECTING TAU DERIVED FROM HUMAN BRAIN AND SEE WHETHER OR NOT IT'S TAKEN UP. ORDINARILY IT'S IN MICE THAT OVER EXPRESS THE FORM OF HUMAN TAU AND LOOKING FOR ACCELERATION OF THE PROCESS OF AGGREGATION BUT TO DO IS IN A WAY THAT MORE FLEXIBILITY IN THE MOUSE MODELS WE DEVELOPED THE TAU MOLECULE WHICH CAN BE MODIFIED IN A VARIETY OF WAYS. A SMALL 2A CONSTRUCT WHICH IS ANALOGOUS TO AN IRIS. IT'S A SELF-CLEAVAGE MOLECULE AND THEN A FLUORESCENT PROTEIN. AND THE IDEA IS THE CELLS IN WHICH THE AV IS TAKEN UP EXPRESSED BOTH TAU THEN IF YOU STAIN THE TAU RED THE PROTEIN IS GFP THE CELLS WHERE THE VIRUS WERE TAKEN UP ARE BOTH RED AND GREEN. IF YOU LOOK FOR CELLS IN WHICH THE TAU HAS BEEN PROPAGATED TO SO RECIPIENT NEURONS WILL ONLY HAVE HUMAN TAU IN THEM AND WON'T HAVE ANY GFP IN THEM SO THEY'LL BE ONLY RED. SO IF YOU MAKE THE INJECTION AND WAIT THAT PERIOD OF TIME AND THEN LOOK TO THREE -- 3% TO 4% OF NEURONS WHERE IT PROJECTED TO HAVE ONLY HUMAN TAU AND A WAY TO MEASURE WHERE PROPAGATION OCCURS TO LOOK AT THE MOUSE'S LIFE DURING THE EXPERIMENT AND ALLOWS YOU TO MANIPULATE THE TYPE OF MOUSE YOU'RE USING QUITE EASILY. IT'S OBVIOUS ALZHEIMERS DISEASE IS A RAGE RELATED PHENOMENON. EVEN INDIVIDUALS WHO HAVE AN INHERITED ALZHEIMER'S DISEASE DON'T DEVELOP IT UNTIL THEIR EARLY 40s. THERE'S FOUR DECADES OF LIFE IN WHICH THERE'S SOMETHING GOING ON BUT ALZHEIMER'S DISEASE HASN'T HAPPENED. SO WE WERE CURIOUS WOULD THIS PROPAGATION PHENOMENON WOULD OCCUR MORE LIKELY IN AN ELDERLY ANIMAL THAN YOUNGER ANIMAL AND TURNS OUT IT'S TRUE THERE'S AN TWOFOLD INCREASE AND THERE'S A CONFIRMATIONAL CHANGE IN THE TAU IN THE OLD ANIMALS AS WELL. AND NOW IN A SERIES OF EXPERIMENTS WE'RE CARRYING OUT IN COLLABORATION WITH JACKSON LABORATORY AND THE INITIAL EXPERIMENTS WERE DONE IN COLLABORATION WITH INVESTIGATORS FROM LILY WE LOOKED AT MOUSE STRAIN AND IF THAT MAKES A DIFFERENCE AND TURNS OUT IT DOES AND SOME MOUSE STRAINS HAVE A LOT OF PROPAGATION AND OTHER MOUSE STRAINS HAVE VERY LITTLE PROPAGATION AND I'M POINTING OUT THE C56 ARE RELATIVELY LOW PROPAGATORS THAT IS THERE'S SEEMINGLY RESILIENT PROPAGATING TAU FROM ONE BRAIN AREA TO THE REST. IT'S CERTAINLY PRESENT AND DETECTABLE AND MEASURABLE BUT FAR LESS ROBUSTLY THAN OTHERS. SO WE'RE LOOKING TO SEE WHETHER OR NOT OTHER LINES MAY BE IMPORTANT AND ONE CAN IMAGINE THROUGH THE SAME KIND OF EXPERIMENT WHERE GLYCOSCIENCE RELATED TRANSGENIC LINES ARE INVOLVED OR GLYCOSCIENCE RELATED PHARMACEUTICALS ARE USED EVEN IN DIABETIC MICE. I'LL CLOSE WITH THE EXPRESSION OF TAU CHANGES OTHER CELL TYPES TO GLIA, MICROGLIA AND IN THIS INSTANCE ENDOTHELIAL CELLS WHICH SEEM TO LEAD TO THIS UNUSUAL CHANGE IN THE VASCULATURE SIMILAR TO THE RETINA IN THE DIABETIC RETINOPATHY WHERE THERE'S A CASCADE OF FUNNY LOOKING SMALL VESICLES AND IS DUE TO AN UPTAKE OF SENESCENT GENES. SO IN SUMMARY THERE'S HETEROGENEITY AMONG PATIENTS WITH ALZHEIMER'S DISEASE. THE REASONS FOR THAT WILL BE PROVEN TO BE COMPLEX BUT IT SEEMS AS IF ONE CONTRIBUTOR TO IT PROPERTIES OF TAU THAT INDIVIDUALS GENERATE AND THAT COULD BE DUE TO A VARIETY OF ENVIRONMENTAL AND GENETIC INFLUENCES AND SO FORTH. IS THERE ROLE FOR GLYCATION OR OTHER NOT YET IDENTIFIED PTMs IN THE PROCESSES? I THINK LIKELY TO BE THE CASE BUT NOT YET STUDIED. AND BIOLOGY TO CANCER. WILL THERE BE OTHER THINGS THAT ARE ENVIRONMENTAL OR GENETIC THAT IMPACT THIS UNDERLYING PROCESS THAT CHANGE ITS RATE OF PROGRESSION AND POTENTIALLY THE WAY WE TREAT IT. AND I'LL JUST CLOSE THERE BY SAYING THANK YOU VERY MUCH. IT'S BEEN INTERESTING TO HEAR THE DISCUSSIONS. >> THANK YOU, BRAD. LET'S SEE IF WE HAVE ANY QUESTIONS POSTED IN THE CHAT BOX. >> CAN YOU START MAKING THE TAU IN DIFFERENT GENETIC BACKGROUNDS? >> I THINK RIGHT NOW THAT'S A VERY PLAUSIBLE THING. THE OUTBREAK STRAIN I THINK IS A GOOD STARTING POINT BUT IN ADDICTION LIKE DIABETES, FOR EXAMPLE, THERE'S OBVIOUSLY STRAIN DIFFERENCES IN HOW THE ANIMAL RESPONDS TO IT AND I BELIEVE THAT'S GOING TO PROVE TO BE THE CASE FOR TAU BIOLOGY AS WELL. AS WE FINISH UP THE STUDIES WITH CATHERINE WE'LL MAKE THE DATA AVAILABLE AS SOON AS WE CAN. ONE KEY WILL BE THE PRACTICAL CONSEQUENCE TAU EXPRESSERS BUT THE OTHER IS THIS INSIGHT INTO NEW GENES THAT MIGHT IMPACT >> THE NEXT GENERATION OF TAU CAN YOU COMMENT ON THIS? >> AUSTIN, SORRY, CAN YOU REPEAT THE QUESTION? >> WE'RE TALKING ABOUT THE STRAIN VARIATION. YOU WERE IN THE PROCESS OF MAKE THE NEXT GENERATION TAU ANIMAL WOULD THAT BE CONSIDERED AT SOME POINT. >> WE'RE MAKING THEM BUT I ALSO HAVE A PROJECT TO DO THIS BXD STRAIN CROSS WHERE YOU HAVE AT LEAST 28 DIFFERENT STRAINS OF MICE TO LOOK AT LOTS OF MEASURES. IT'S AN EXTENSION OF BRAD'S STUDY AND WHETHER THAT WAS FIVE OR SIX DIFFERENT STRAINS. WE'RE DEFINITELY GOING TO HAVE THE TOOLS TO DISSECT OUT SOME OF THOSE GENETIC DIFFERENCES THAT MAY GIVE RISE TO SOME OF THESE THINGS. THAT WOULD BE ONE TO LOOK AT AND THAT STUDY IS JUST STARTING. AND THAT'S GOING TO BE WITH A COUPLE DIFFERENT TYPES OF TAU MOUSE. >> I HAVE A QUESTION. >> WE NEED TO MOVE ON TO THE NEXT SPEAKER. >> OKAY. >> WE'LL HAVE ANOTHER ROUNDTABLE MEETING AT THE END OF THE DAY SO WE CAN TALK ABOUT THIS FURTHER. SO OUR NEXT SPEAKER IS DR. STEVEN FINKBEINER. HE IS THE SENIOR INVESTIGATOR AT THE INSTITUTE OF NEUROLOGICAL DISEASE AND PROFESSOR OF THE DEPARTMENT OF NEUROLOGY AT UCSF. HE HAS BEEN TRYING TO USE MODELSES IN TRYING TO UNDERSTAND THE MANY DIFFERENT ASPECTS OF THE TAUOPATHY TOGETHER WITH HIGH THROUGHPUT IMAGING ANALYSIS. >> THANK YOU, AUSTIN, AND THANK YOU FOR THE INVITATION TO THE MEETING. IT'S BEEN INTERESTING AND I LEARNED A LOT AND I THINK I CHANGED MY TALK AND ONE WAS TO BRING THE GROUPS TOGETHER TO LOOK FOR INTERSECTIONS AND THOUGHT I'D BEGIN BY STARTING THE TALK TELLING THE GLYCOSCIENCE COMMUNITY ABOUT NOVEL TOOLS IF YOU'RE INTERESTED IN COLLABORATION AND USE THE REST OF THE TALK TO TALK ABOUT SOME OF THE ALZHEIMER'S DISEASE AND THE ROLES GLYCANS WOULD PLAY AND I KNEW THEY'D DO A GOOD JOB TALKING ABOUT TAU BUT I'LL TALK ABOUT RELATED DEMENTIA CALLED FTD. SO PART OF OUR TECHNOLOGY DEVELOPMENT REALLY BEGAN WITH AED QUESTION IN NEURO DEGENERATION. YOU HEARD FROM KAREN AND BRAD ABOUT TAU AGGREGATION AND IT'S A FEATURE OF ALZHEIMER'S DISEASE AND A VARIETY OF OTHER DEGENERATIVE DISEASES AND WE COULDN'T FIGURE OUT A WAY TO ADDRESS THE QUESTION UNLESS WE WERE ABLE TO FOLLOW THE BIOLOGY AS IT UNFOLDED OVER TIME AND THEN TO BE ABLE TO RELATE WHAT WE SAW TO THE EVENTUAL FATE OF THE CELL. SO WHAT WE HAD TO DO WAS DEVELOP NEW PLATFORM TO DO THAT. WE CALL THESE ROBOTIC MICROSKOPS AND HAVE ROBOTIC INCUBATORS AND WE HAVE A ROBOTIC ARM AND THERE'S A FOURTH GENERATION SYSTEM IN THE FOREGROUND AND THIRD IN THE BACKGROUND. AND THE KEY CAPABILITY WE BUILT IN THE INSTRUMENTS IS TO PUT THE PLATE BACK AND FORTH TO OBSERVE BIOLOGY AS IT UNFOLDS AND DETERMINE WHAT THE ULTIMATE FATE OF THE CELL IS AND RELATE THE TWO AND YOU GET A SENSE HOW FINELY ALIGNED THE SYSTEMS ARE BY THE MOVIE WHERE YOU SEE IMAGES COLLECTED ONCE PER DAY AS THE CELLS GO BACK AND FORTH AND SEE HOW NEURONS EXTEND AND GROW OVER TIME. WE HAVE ANALYSIS PIPELINES BECAUSE THESE GENERATE 2 TO 3 TERABYTE OF DATA PER DAY AND YOU CAN FOLLOW THE BIOLOGY AS REPEATED MEASURES IN INDIVIDUAL CELLS AND WE CAN INTRODUCE DIFFERENT BIO SENSORS AND SEE HOW THEY RELATE TO EACH OTHER AND TAKE ADVANTAGE OF THE FACT WE CREATE PHYSICAL MAPS AND SPACE AND KAREN AND BRAD MENTIONED TAU PROPAGATION FROM CELL TO CELL. AND YOU CAN FIX THE CELLS AT THE END IF YOU WANT TO LABEL THEM AND LOOK AT THE BIO SENSORS AND WHAT WE FOUND IS WITH THIS APPROACH WE GET 100,000 FOLD SENSITIVITY COMPARED TO CONVENTIONAL SNAPSHOT APPROACHES. WE APPLIED THIS APPROACH FOR A LONG TIME NOW PARTICULARLY LOOKING AT THE TITLE OF MY TALK WHICH WAS STRESS RESILIENCE AND PROTEO STASIS AND HAVE BEEN ABLE TO UNFOLD A DYNAMIC RESPONSE THAT SEEMS TO BE STEREOTYPED ACROSS NEURONS WITH A VARIETY OF PROTEINS AND TAU AND OTHERS IN WHICH THESE TOXIC CONFORMERS MAY BE SEQUESTERED INTO THESE INCLUSION BODIES. THIS IS ONE EXAMPLE OF HOW WE USED THIS TO UNRAVEL CONTEXT MECHANISMS AND THINK THERE MAY BE OPPORTUNITIES TO LOOK AT OTHER BIOLOGIES LIKE PROTEIN GLYCOSYLATION. IN PARTICULAR ONE THING THAT OCCURRED TO ME WITH A PROGRAM CALLED THE NEIGHBORHOOD WHERE WE TRIED TO UNDERSTAND DOES IT MAKE A DIFFERENCE WHAT NEIGHBORHOOD THE CELL IS IN AND WE INTRODUCED TDP43 AND A TRANS VECTION MARKER IN A DIFFERENT POPULATION OF CELLS AND WE CAN SEE THEM INDEPENDENTLY AND MONITOR THEIR BIOLOGIES LONGITUDINALLY. THERE SEALS -- SEEMS TO BE AN ACTION BETWEEN THE CELL TYPES AND HOW CLOSE YOUR TO A TDP NEURON THE CHANCE OF SURVIVAL WILL BE CHANGED DRAMATICALLY AND THESE ARE RISK OF DEATH CURVES AND THE HIGHER THE CURVE THE HIGHER CHANCE THE CELL HAS TO DIE AND WE HAVE THE PROGRAM IDENTIFIED THE CELLS AND THEN LOOK SPECIFICALLY AND QUANTIFY THEIR RELATIONSHIP TO NEIGHBORS. WE CAN DO THIS WITH WHATEVER CELL TYPES WE HAVE LABELLED. ANOTHER THING WE'VE DONE IS TO DEVELOP BIO SENSORS TO LOOK AT DIFFERENT BIOLOGIES FOCUSSED ON NEUROSCIENCE AND WE HAVE FEW THAT LOOK AT GLYCANS AND WE WOULD BE VERY INTERESTED IN THINKING ABOUT POTENTIAL NEW TOOLS IN THAT SPACE TO BE ABLE TO STUDY THE BIOLOGY OF SOME OF THESE PROTEINS IN SITU. ONE WAS MADE BY GROTZKE ET AL LOOKING AT PROTEIN GLYCOSYLATION AS IT RELATE TO ERAB. AND ONE THING OCCURRED TO ME LOOKING AT GLYCANS AND DIFFERENT CELLS WE'VE BEEN DEALING WITH THAT ISSUE ON ADHERENCE CELLS SO NEURONS DON'T LIKE TO BE FACT SORTED SO WE HAVE COME UP WITH NEW TECHNOLOGIES TO DO BAR CODING ON ADHERENT CELLS LIKE NEURONS AND WHAT WE'VE TRIED DO IS RESOLVE THE FACT THE PROTEINS HAVE AN ABSORPTION MAXIMUM AND THESE ARE A TRIES OF TREATABLE FILTERS TO ENABLE THE ROBOTIC MICROSCOPES TO GATHER SPECTRA AND USE MIXING TO RESOLVE THE DIFFERENT FLUOROPHORS AND LOOK AT SMALL SECTIONS OF THE -- FLUOROFORS AND TAKE THE IMAGES OF DEEP LEARNING AND RESOLVE THE COMPONENTS PIXEL BY PIXEL AFTERWARDS. WITH THIS WE CAN DO THIS IN LIVE CELLS AND OUR ULTIMATE GOAL IS TO TRY TO SEE IF WE CAN USE THIS IN A VARIETY OF WAYS TO BAR CODE CELLS AND HELP WITH IDENTIFICATION AND ALSO TO BE ABLE TO LOOK AT MORE BIOLOGY AT ONCE AND AT MORE SYSTEMS. I COULDN'T HELP BUT THINKING ON YESTERDAY'S TALK CAN WE LABEL GLYCANS ON AN ADHERENT BRAIN TO LOOK AT RELATED DEMENTIAS. ANOTHER TECHNOLOGY WE'VE DEVELOPED BASED ON THE LARGE AMOUNT OF DATA WE'VE STARTED TO GENERATE IS NEW A.I. TOOLS TO EXTRACT INSIGHTS WE COULDN'T OTHER WISE. THIS IS A NEW BIOSENSOR GOT PUBLISHED LAST WEEK AND WE HAVE AND HUMANS HAVEN'T BEEN ABLE TO MAKE THE CALL FROM MORPHOLOGY AND WE DEVELOPED THE BIOSENSOR BUT WE HAVE TAKEN ADVANTAGE OF THE FACT OUR ROBOTICS GENERATE THESE LARGE DATA SETS. WE HAVE TAKEN EXAMPLES OF THE BIOSENSOR FROM CELLS COMMITTED TO DIVERSE US THOSE HEALTHY TO SEE IF WE CAN TRAIN A DEEP LEARNING NETWORK TO TRAIN HUMANS TO MAKE THE CALL FROM MORPHOLOGY AND SO WE'VE TRAINED DEEP TRAINING NEURAL NETWORKS AND WITH THE DATA COLLECTION A COUPLE HOURS OF DEEP LEARNING WE COULD ACHIEVE ACCURACIES HUMANS COULDN'T DO. AND ONE OF THE COOL THINGS AND A CHALLENGE WITH DEEP LEARNING IS A LITTLE BIT OF A BLACK BOX MORE RECENTLY NEW TOOLS HAVE BEEN DEVELOPED AND WITH THE NEW TOOLS THE ONE HERE YOU CAN FIGURE OUT WHAT THE NETWORK IS SEEING TO MAKE SUPER HUMAN CLASSIFICATIONS. THIS IS ARCHIVE IF PEOPLE ARE INTERESTED WE APPLIED THIS AND I WAS THINKING ABOUT THE GLYCOSCIENCES AND WOULD THIS BE USEFUL AND MAKE THE PERTURBATION NETWORKS TO MAKE DISTINCTIONS AND WHAT ARE THE SIMPLE CHANGES UNDERGOING RESPONSE. ANOTHER TECHNOLOGY AUSTIN MENTIONED EARLY ON WE'VE BEEN PRETTY COMMITTEED TO IPS CELL DEVELOPMENT AND ONE OF THE FOUNDING TEAMS TO DEVELOP THESE FOR DISEASE AND SO BY NOW WE HAVE OVER 2,000 IPS LINES EASILY ACCESSIBLE. MOST ARE PATIENT DERIVED AND SOME ARE CRISPR ENGINEERED AND HAVE CONTROLS AND HERE YOU SEE A SAMPLE OF THE ALZHEIMER'S DISEASE LINES WE HAVE FTD LINES AND LEWY BODY DEMENTIA WORK. WE HAVE BEEN DEVELOPING DIFFERENTIATION PROTOCOLS AND HAVE THEM FOR A VARIETY OF CELL TYPES SOME RELEVANT TO ALZHEIMER'S DISEASE INCLUDING BRAINS AND MICROGLIA, ETCETERA. WE'VE ALSO BEEN ABLE TO TAKE THESE APPROACHES AND DEVELOP THE CULTURE METHODS AND ORGANOIDS AND STUDY THE CONTEXT AND THERE'S AN IMPORTANT ROLE FOR RAGING IN ALZHEIMER'S DISEASE AND TO COMPLIMENT THE IPS LINES WE DEVELOPED METHODS TO TRANS DIFFERENTIATE NEURONS FROM FIBROBLASTS AND A LOT OF THE LINES WE HAVE WHOLE GENOME ANALYSIS. WE HEARD ABOUT THE CONNECTION BETWEEN GENETICS AND BIOSYNTHESIS AND GLYCANS. IF FOLKS ARE INTERESTED WE CAN LOOK AT OUR REPOSITORY TO SEE IF WE HAVE LINES WITH PATIENTS WITH VARIANTS. LET ME USE THE TIME THAT REMAINS TO TALK A LITTLE BIT ABOUT SOME OF OUR WORK ON ALZHEIMER'S DISEASE RELATED DEMENTIA FROM TEMPORAL DEMENTIA WHERE WE THINK THERE'S SOME CONNECTION TO PROTEIN GLYCOSYLATION. WE WOULD CERTAINLY WELCOME THE HELP OF THE COMMUNITY. WE HAVE IN GENERAL NOT REALLY WORKED IN THAT AREA MUCH BECAUSE WE DIDN'T HAVE TOOLS FOR NEXT STEPS TO TAKE. I'LL TALK ABOUT TWO GENE MUTATION MUTATIO MUTATIONS FRONTOTEMPORAL DEMENTIA IN PATIENTS 60 YEARS OLD OR LESS AND HAVE A DIFFERENT PROFILE THAN ALZHEIMER'S DISEASE AND INTERESTINGLY HAS A LOT OF OVERLAP WITH ALS. THE GENES I'LL TALK ABOUT C9ORF72 IS THE MOST COMMON AND THE NATURE OF THE MUTATION IS A HEXO NUCLEOTIDE AND THE FUNCTION IS STILL BEING WORKED OUT BUT IS BEING WORKED OUT IN INFLAMMATION AS WELL AS AU -- AUTOPATHY AND VAS VARIANTS SHOWED UP WITH CONNECTION TO ALZHEIMER'S DISEASE AND MORE CLEARLY IMPLICATED IN INFLAMMATION AUTOPATHY. AND HERE'S AN INTEREST IN INTRACELLULAR MATRIX AND HERE'S WHERE THE STORY ENDED BECAUSE WE DIDN'T HAVE TE TOOLS TO TAKE IT FURTHER AND THIS IS THE LINCS PROGRAM AND WE WERE FOCUSSED ON NEUROLOGICAL DISEASE IN OUR GROUP IN THE PROGRAM AND THE IDEA WAS TO TAKE IPS LINES WITH AN ALS FOCUS WITH C9 OR 72 MUTATIONS DIFFERENTIATE THEM INTO MOTOR NEURONS AND DEEPLY PHENOTYPE THEM AND DO WHOLE GENOME ANALYSIS AND TRANSCRIPTOMICS AND IMAGING AND AND LOOK AT PATIENT CELLS FROM HEALTHY CONTROLS AND IT'S ALL PUBLICLY AVAILABLE. WE CAN LOOK AT IT ON NEURAL LINCS DOT-ORG AND WE'RE MAKING IPS LINES FROM ALL OF THEM AND DOING THE SAME SORT OF EFFORT TO SEE IF WE CAN ON A LARGER DATA SET WHICH INCLUDES A LOT OF IDIOPATHIC OR SPORADIC DISEASE FIND SIGNATURES WE CAN CONNECT. AND WE LOOKED AT DATA CELLS INCLUDING PROTEOMICS AND ATTACK SEQ AND LOOKED AT PRIOR DATA PUBLICLY AVAILABLE PARTLY FROM THE TWO HYBRID SYSTEMS AND USE BIOINFORMATIC APPROACHES TO BE ABLE TO INTEGRATE THEM. IT'S SOMETHING CALLED A PRIZE COLLECTOR WITH THE MODEL THAT'S A MACHINE LEARNING APPROACH TO BE ABLE TO TRY TO TAKE THESE DATA SETS AND LOOK FOR WAYS THAT CONNECT THEM BOTH WITH THE SIGNALS AND THE DATA THEMSELVES AND LEVERAGING THE INTERACTIONS TO GENERATE NETWORKS. AND WITH THE IPS LINES IT'S NOT ENTIRELY CLEAR WHETHER THEY'RE RELEVANT BUT WE DID GO ON TO TAKE CERVICAL SPINAL CORD TISSUE FROM PATIENTS FROM ALS AND WERE ABLE TO CONFIRM A NUMBER OF THESE CHANGES AND DID PERTURBATION TO SEE IF THE MODELS WERE BETTER AND WORSE AND THE EXTRA CELLULAR MODELS SEEMED TO MAKE IT WORSE BUT THERE WERE CHANGES IN COLLAGEN AND FIBRILLIN AND OTHER PROTEINS THAT MAY BE RELEVANT TO THE DISCUSSIONS WE HAD YESTERDAY. FRANKLY, THAT'S WHERE THE STORY ENDED. AND LOOKING FORWARD TO POSSIBLE THERAPEUTIC STRATEGIES AND DUE TO A DIFFERENT GENE MUTATION THIS IS THE MUTATION OF THE GENE CALLED PROGRANULIN I WANTED TO MENTION. THIS IS CHARACTERIZED BY PATHOLOGY AS NEURON LOSS IN THE FRONTAL TEMPORAL LOBE AND A STRONG MICROGLIOSIS AND A PROTEIN CALLED TDP43 AND THE MICROGLIAL ACTIVATION IN THE DISEASE WAS SO PROMINENT EARLY ON SOME SPECULATED IT MAY BEGIN IN MICROGLIA AND MY OWN OPINION NOW IS IT PROBABLY BEGINS IN NEURONS BUT IT MAY BE A PROGRANULIN LOSS AND MAY LEAD TO NEURODEGENERATION. WE'VE BEEN VERY INTERESTED IN AND THE PROGRANULIN MAY LEAD TO PFTD AND SOME ARE LINKED TO PARKINSON'S DISEASE AND MAY BE A LINK TO ALZHEIMER'S AND MAY BE A LINK TO CONNECT ALZHEIMER'S DISEASE AND RELATED DEMENTIAS AND ONE OF THE THINGS WE DISCOVERED WHEN WE DID A GENOME WIDE SCREEN AT PROGRANULIN LEVELS IS IT PLAYS A CRITICAL ROLE IN THIS DISORDER BOTH IN TERMS OF REGULATED PROGRANULIN LEVELS AND ALSO THE PROGRANULIN MAY BE ABLE TO REGULATE LYSOSOMAL FUNCTION. IN THE PROCESS OF STUDYING PROGRANULIN WE HAD SYSTEMS AND A KNOCK DOWN OF PROGRANULIN AND LOOKING AT LEVELS AT TRANSGENIC MICE OR PATIENTS AND WE'VE SEEN CONSISTENT PHENOTYPES WHEN WE KNOCKED DOWN PROGRANULIN OR WHEN WE GENETICALLY ALTER IT TO MIMIC A PATIENT IN THAT WE SEE SIGNIFICANT LOSS OF NEURO ITS. WE ALSO SEE AN INCREASE IN THE RISK OF DEATH AND WHEN WE LOOK AT MICROGLIA WE SEE A LOSS OF LEVELS AND IF WE TAKE ME MEDIA INTEREST THE CULTURES AND APPLY IT TO THE NEURON WE SEE A DRAMATIC DECREASE IN THE RISK OF DEATH. THERE'S SOMETHING ABOUT MICROGLIAL ACTIVATION LEADING TO THE PROFICIENCY AND WHAT THE MICROGLIA SEAL TO BE HARMFUL FOT NEURONS AND WE CREATED CO-CULTURES HUMAN MICROGLIA AND NEURONS DERIVED FROM PATIENT-DERIVED IPS CELLS AND WE HAVE THE ABILITY TO MAKE THESE AND THERE'S A NUMBER OF PROTOCOLS AND SO WE CAN DEVELOP DIFFERENT PROTOCOLS. WE HAVE TO MAKE THE CELL SEPARATELY AND CAN COMBINE MIX AND MAX TO LOOK AT WHAT AFFECTS THEY HAVE AND WHAT WAS STRIKING TO US WAS THERE SEEMS TO BE A REALLY STRIKING SYNERGY BETWEEN AND IF YOU LOOK AT THEM SEPARATELY SO WHEN WE LOOK AT THE CELLS SEPARATELY WHETHER IT'S NEURONS OR MICROGLIA WE SEE MODEST CHANGES IN A VARIETY OF CYTOKINES BUT WHEN THEY'RE CO-CULTURED TOGETHER PARTICULARLY FTD AND PLUS THE MICROGLIA WE SEE DRAMATIC INCREASES IN A NUMBER OF CYTOKINES. SO WE'RE VERY INTERESTED ON THE CELL INTERACTIONS IN THIS PROCESS SO THIS WOULD BE ANOTHER PLACE WHERE WE WOULD BE VERY INTERESTED IF THERE'S TOOLS AVAILABLE TO BE ABLE TO PERTURB THE SYSTEM AND LOOK AT THE GENETICS OF ALZHEIMER'S DISEASE STRONGLY IMPLICATES MICROGLIA AND THE VARIANTS LINKED TO ALZHEIMER'S DISEASE CAN BE FOUND IN THE PROTEINS WE TALKED ABOUT YESTERDAY AND TODAY INCLUDING CD33, AND THERE'S A RECEPTOR FOR PROGRANULIN AND IT'S INTERESTING TO ME SOME OF THE PATHWAYS CULMINATE IN THE AUTOPHAGY AND ONE QUESTION I HAD FOR THE GLYCOSCIENCE COMMUNITY IS TO LEARNING THE MAJOR PATHWAYS IN WHICH THE GLYCANS GET TURNED OVER AND WHAT ROLE AUTOPHAGY PLAYS AND THERE'S A PRIMARY ROLE OF PROGRANULIN SECRETION AND UPTAKE BY OTHER RECEPTORS THAT CULMINATES AND TARGETING TO THE LYSOSOME WHERE IT REGULATED LYSOSOME FUNCTION AND WE'VE BEEN THINKING ABOUT IT PRIMARILY IN THE IMPAIRMENT THAT MAY PRODUCE TO THE PROTEOSTASIS NETWORK AND SEEMS TO BE A RECURRING FEATURE OF DISEASE AND LYSOSOMAL DYSFUNCTION. I'LL FINISH UP THERE. TO REMIND FOLKS I THINK WE'VE TRIED TO DEVELOP A NUMBER OF TECHNOLOGIES WITH ALZHEIMER'S DISEASE AND RELATED DEMENTIA QUESTIONS IN MIND INCLUDING THIS AUTOMATED LONGITUDINAL IMAGING SYSTEMS AND BIO SENSORS AND PATIENT MODELS AND MAY THEY BE USEFUL TO STUDY THE ROLE OF GLYCANS AT A SINGLE CELL LEVEL AND IN SITU IN WAYS THAT MAY BE MORE DIFFICULT RIGHT NOW AND THE OTHER POINT IS WE DO SEE A LOT OF BIOLOGY IN ALZHEIMER'S DISEASE AND RELATED DEMENTIAS LIKE FRONTOTEMPORAL DEMENTIA THAT SEEM TO INDICATE GLYCANS AND WOULD BE INTERESTING TO LOOK AT THE TOOLS DEVELOPED IN THE COMMON FUND PROJECT AND HOW THEY MAY BE USED TO UNDERSTAND BETTER WHAT ROLE WILL THEY PLAY AND THINK ABOUT THERAPEUTIC STRATEGIES AND WAS PLEASED TO HEAR THE POSSIBILITY OF ADMINISTRATIVE SUPPLEMENTS TO COLLABORATE AND WE'D LOVE TO COLLABORATE AND HOPE THERE'S OPPORTUNITIES. I'LL FINISH BY HIGHLIGHTING SOME OF THE FOLKS WHO HAVE DONE THE WORK. SPECIAL THANKS TO JEREMY LINDSAY AND JULIA KAY WHO HAVE DONE WORK THEN MICROSCOPY AND I'LL END NHL. >> THANK YOU. WHILE WE'RE WAITING FOR QUESTIONS COMING IN FROM THE IN BOX I'M WONDERING WHETHER THERE WERE QUESTIONS FROM OUR PANELISTS AT THIS POINT. >> GREAT TALK. THANK YOU SO MUCH FOR THAT. REALLY POWERFUL TECHNOLOGY. I'D LIKE TOE ASK A SELF SERVING QUESTION. YOU DISCUSSED AT THE END THE IMPORTANCE OF ENDOSOMAL PATHWAYS AND MICROGLIA AND EARLIER ON AND YOU TALKED ABOUT MICRO GLIAL IPS C. WE'VE BEEN STRUGGLING WITH THE POWER OF THESE TO LOOK AT THE PATHWAYS. WHAT ARE YOUR THOUGHTS ON THAT? >> TO THIS POINT MOST OF THE WORK WITH I MICROGLIA IS TO LOOK AT THEIR BEHAVIOR COMPARED TO HEALTHY CONTROLS AND IT'S BEEN MAINLY FOCUSSED AT THE LEVEL OF FAIRLY SIMPLE PHENOTYPES LIKE CYTOKINE PRODUCTION AND PHAGOCYTOSIS. IS THE DIFFICULTY YOU'VE EXPERIENCED SO FAR -- WHAT SPECIFICALLY HAVE YOU -- >> THROUGH THE RECEPTORS. >> OH, INTERESTING. >> IF YOU HAVE LINES THAT APPEAR THAT ARE FAIRLY STABLE AND SHAREABLE WE CAN TEST THEM FOR THE PRESENCE AND SEE IF ANY OF THEM DO RETAIN THOSE. ESSENTIALLY ANYTHING WE'VE DONE FOR ANY PERIOD OF TIME AND WOULD LOVE TO HEAR FROM OTHER PEOPLE BECAUSE IT'S NOT OUR EXPERTISE HAS RESULTED IN THE LOSS OF THESE RECEPTORS FROM THE CELL SURFACE. >> I SEE. THAT WOULD BE VERY INTERESTING TO LOOK AT. I DON'T KNOW WHICH PROTOCOLS YOU'VE USED AND SOME ARE RELATED AND WE'VE BEEN MORE FOCUSSED ON THE DISEASE CAUSING PROTEINS RATHER RECEPTORS BUT WE HAVE NOTICED SOME DIFFERENCES. PERHAPS THAT MAY BE IMPORTANT AS WELL. >> GREAT, THANKS. >> WE HAVE ONE QUESTION IN THE CHAT BOX. STEVE, CAN YOU SEE IT OR DO YOU WANT ME TO READ IT BACK TO YOU? >> WE HAVE NOT LOOKED SPECIFICALLY FOR SIGNAL RECEPTORS BUT WE HAVE LOOKED AT AND LIKE I SAID WE HAVEN'T REALLY EXPLORED THE LIKE GLYCANS IN THESE SYSTEMS. AS WE'VE STUDIED THESE WE HAVE DONE A LOT OF WORK AS I MENTIONED CHARACTERIZING THE PROTEOME AND I CAN LOOK AT THE DATA THAT WE HAVE ON THE PROTEOMICS TO SEE SPECIFICALLY. I DIDN'T LOOK AT THAT BEFORE BUT I CAN GET BACK TO YOU IF YOU WANT TO SHOOT ME AN E-MAIL TO REMIND ME. THE PROTEOMICS WE'VE DONE SO FAR WE HAVEN'T MADE A HUGE EFFORT WITH POST TRANSLATIONAL MODIFICATIONS BECAUSE AS KAREN MENTIONED IT'S A DIFFERENT KIND OF MORE SPECIALIZED PROTEOMICS AND A LOT OF THE WORK WE'VE BEEN SO FAR IS TRYING TO GET LARGE SCALE PROFILING AND IT'S MOSTLY MORE FOR NETWORK ANALYSIS AND DISCOVERY RATHER THAN LOOKING AT SPECIFIC BIOLOGIES BUT WE WE HAVE LOOKED AT SPECIFIC SIGNAL TRANSACTION PATHWAYS AND CAN REPLICATE SOME SIGNALLING FOR TRANSLATIONAL MODIFICATIONS BUT I THINK THAT WOULD TAKE A MORE SPECIALIZED LOOK. BUT IF YOU WANT TO SHOOT ME A MESSAGE I CAN ASK THE FOLKS WHO HAVE THOSE DATA SETS TO SEE WHAT THE EXPRESSION LEVELS LOOK LIKE. >> THE FINAL QUESTION, IN THE COMMON FUND WE HAVE A LOT OF INVESTS MAKING A LOT OF CHEMICAL PROBES FOR THE PROTEIN GLYCOSYLATION SO THE QUESTION FOR CATHERINE I'M WONDERING THE COMMON FUND IS MAKING AN IMAGING PROBE BECAUSE AS YOU CAN SEE FROM OUR LECTURE THE IMAGING APPROACH IS AN IMPORTANT PART OF THE ANALYSIS TO STUDY BECAUSE YOU CAN GET A HIGHER RESOLUTION FROM THESE TYPE OF SINGLE CELL ANALYSIS. I'M WONDERING WHETHER WE HAVE ANY TOOLS AVAILABLE FOR OUR INVESTIGATORS? >> THE TOOLS THAT COME TO MIND ARE THE ONE WHERE WE CAN METABOLICALLY ENGINEER THE GLYCANS AND IMAGE AFTER BUT THOSE ARE THE TOOLS THAT COME TO MIND IN THINKING OF DOING THE HIGH RESOLUTION IMAGING EXPERIMENTS ON THE GLYCAN SURFACE DOING SOME KIND OF -- YOU HAVE TO INCORPORATE SOME SORT OF PROBES YOU CAN THEN CAPTURE. THE OTHER PROBES TO MIND ARE THING OF HIGH AFFINITY LECTINS OR ANTIBODIES AN CAN BEGIN TO DO IMAGING ON THE CARBOHYDRATE LEVEL. DOES THAT ANSWER YOUR QUESTION? >> WE'RE MAKING A LOT OF TYPE OF IPS LINES NOW FROM VARIOUS DEGENERATIVE DISEASES BUT LOOKING AT THE CELLS EXPRESSING THE GLYCANS AND CARBOHYDRATES IN GENERAL AND THINK IT WILL HAVE A PROFOUND IMPACT ON THESE TYPES OF CELLS. >> I THINK IT'S HARD BECAUSE THE CHANGES COULD BE VERY DYNAMIC AND THE TOOLS I JUST DESCRIBED YOU HAVE AN IDEA WHAT THE GLYCAN IS YOU'RE LOOKING FOR. >> SO STEVE, MAYBE YOU CAN INTERACT WITH CATHERINE'S GROUP AND OTHER COMMON FUND INVESTIGATORS TO SEE IF ANY TOOLS ARE AVAILABLE. KAREN. >> I'M JUST WONDERING, WHAT'S THE CAPACITY FOR EM TO GET TO IS THE SPECIFICS OF WHAT THE GLYCAN AND THE SITE CHAINS? I'VE MENTIONED AND I'M WONDERING HOW POWERFUL OF A TOOL THAT IS AND WHAT ARE THE LIMITATIONS? AND DOING EM, CRYO ET. >> I'M NOT SURE YOU CAN DISTINGUISH BETWEEN WHAT CARBOHYDRATE THAT IS USING EM. I DON'T THINK WE CAN DO THAT YET. >> YOU SHOULD BE ABLE TO DO CRYO EM. >> I THINK PART OF THE PROBLEM IS THE CHAINS AREN'T THAT FLEXIBLE. BUT JUST WONDERING IF ANYBODY HAS EXPERTISE. THE EV ARE MICE ISOLATED STRUCTURES LIKE VIRUSES SO NOT LIKE YOU'RE TRYING TO DO THIS IN TISSUE. >> ALL RIGHT. SO LET'S MOVE ON TO THE LAST LECTURE OF THIS MORNING FROM DR. DAVE HOLTZMAN. HE IS A PROFESSOR FROM THE WASHINGTON UNIVERSITY SCHOOL OF MEDICINE. HE HAS BEEN A CHAIR OF NEUROLOGY THE LAST 17 YEARS AND DECIDED TO FOCUS ON HIS RESEARCH INTERESTS. THE PAST FEW YEARS HE'S BEEN INTERESTED IN UNDERSTANDING THE ROLE OF APOE AND IN ALZHEIMER'S DISEASE. AS WE KNOW THE APOE IS ONE OF THE MOST IMPORTANT RISK FACTORS IN ALZHEIMER'S DISEASE. WE STILL DON'T KNOW TOO MUCH ABOUT IT. SO TODAY DAVE'S GOING GIVE US A LECTURE ON ROLE OF APOE, ASTROCYTES AND MICROGLIA IN ABETA AND TAU-MED IATED PATHOGENESIS. >> I'M SORRY I COULDN'T BE AT THE OTHER PARTS OF THE CONFERENCE SO FAR BUT WILL TRY TO RELATE IN SOME WAY THE GLYCOSCIENCES THAT GO ALONG THOUGH THE DATA PER SE DOESN'T DIRECTLY ADDRESS THAT BUT THERE'S A NUMBER OF AREAS AND I'LL TALK ABOUT THE ROLE OF APOE ASTROCYTES AND MICROGLIA AND TAU-MEDIATED PATHOGENESIS. IT'S IMPORTANT IN THINKING ABOUT WHAT APOE IS DOING IN ALZHEIMER PATHOLOGY AND LOOK AT TAU MEDIATED DISEASE. IN ALZHEIMER'S DISEASE THERE'S AGGREGATION OF THE AMYLOID PEPTIDE AND THE TAU AND TANGLES AND INSIDE NEURAL PROCESSES. THERE'S ALSO PROMINENT INFLAMMATORY RESPONSE THAT OCCURS. IF YOU LOOK AT THE CLINICAL TIME COURSE OF THE DISEASE WE KNOW IN DOMINANTLY INHERITED ALZHEIMER'S DISEASE AS WELL AS LATE ONSET ALZHEIMER'S DISEASE AMYLOID DOSES STARTS MANY YEARS BEFORE SYMPTOMS BEGIN, 20 YEARS AND STARTS INCREASING AND BY THE TIME YOU'RE SYMPTOMATIC THERE'S ALSO A MASSIVE AMOUNT OF AMYLOID IN THE NEOCORTICOID REGIONS AND THERE'S A STRONG IN INFLAMMATORY RESPONSE IN RESPONSE TO THE AMYLOID AND THE ABETA PEPTIDE IS A KEY ELEMENT BUT PROBABLY NECESSARY BUT DEFINITELY NOT SUFFICIENT TO GET THE DISEASE COMPLETELY AND WE KNOW ABETA SOMEHOW IS REGULATING HOW MUCH TAU IS SEEDING AND SPREADING IN THE BRAIN AND WE NOW ARE LEARNING MORE ABOUT THE ROLE OF INFLAMMATION AND THE PROCESS OF THE DISEASE AND TALK ABOUT THESE IN APOE IS THE STRONGEST GENETIC RISK FACTOR FOR THE LATE ONSET FORM OF ALZHEIMER'S DISEASE. IT'S SOMEWHAT UNIQUE IN THAT IT'S A RELATIVELY FREQUENT -- THE PRESENCE OF APOE 4 THE RISK FACTOR IS RELATIVELY FREQUENT AND IN 25% OF THE CAUCASIAN PEOPLE PRESENT IN ONE IN 5 ALLELE AND IF YOU HAVE ONE COPY OF APOE VERSUS TWO COPIES OF APOE 3 THE INCREASE IS INCREASED THREE AND A HALF FOLD TO GET DISEASE AND IF YOU HAVE TWO COPIES OF APOE 4 IS TWO FOLD AND IF YOU HAVE ONE COPY HAVE YOU A DECREASED RISK BY 40%. ALL THESE CHANGES APPEAR TO BE DUE TO A SINGLE AMINO ACID DIFFERENCE OF THE ACID PROTEIN. SO APOE IS A GLYCOPROTEIN. IT'S PRODUCED AT HIGH LEVELS IN THE LIVER AND ALSO AT VERY HIGH LEVELS IN THE BRAIN WHERE IT'S MADE PREDOMINANTLY BY ASTROCYTES AND THEY BECOME ACTIVE AND PRODUCE CELLS AROUND THE BLOOD BRAIN BARRIER. APOE WHEN IT'S PRODUCED BY ASTROCYTES IS SYILATED AND IT ALSO BINDS STRONGLY TO ASPG. IF YOU STAIN A BRAIN WITH AN APOE ANTIBODY THAT'S GOOD IT STAINS IN ASTROCYTES AND MICROGLIA BUT STAINS STRONGLY PROBABLY BECAUSE IT'S BOUND AROUND THE BLOOD LEVELS AND IT'S PRESENT IN PROTEIN PARTICLES AND WE KNOW THAT ANY TIME A PROTEIN THAT FORMS AN AMYLOID FORMS IN THE BOLLY, APOE CO-AGGREGATES WITH IT. IF YOU LOOK AT THE TIME COURSE OF THE PATHOLOGY OF ALZHEIMER'S DISEASE IF ONE OF THE MAIN AND IT AFFECTED AMYLOID DEPOSITION YOU EXPECT APOE 4 DRIVES IT LATER AND THAT'S WHAT YOU SEE IN HUMAN BEINGS AND AT OUR RESEARCH CENTER WE STUDIED PEOPLE COGNITIVELY NORMAL AGE 50 TO 70 AND SIMPLY ASKED THE QUESTION WHAT'S THE PERCENTAGE OF PEOPLE WITH AMYLOID IN THE BRAIN AND THAT ARE SEEMINGLY NORMAL AND YOU SEE THE LEVELS OF THE BETA AND IF YOU'RE E44 WITH AN AVERAGE AGE OF 60, ALREADY MORE THAN 60% OF THESE PEOPLE HAD AMYLOID IN THE BRAIN WHEREAS IF YOU HAD NO COPIES OF E4 IT WAS 20% AND IF YOU HAVE AN E2 ALLELE ALMOST NO ONE HAD AMYLOID DEPOSITION AT THAT POINT AND THIS KIND OF FINDING HAS BEEN SEEN BY OTHERS. IF YOU MODEL THIS IN ANIMALS YOU SEE MICE THAT TAKE AMYLOID DEPOSITION AND YOU SEE THIS ISO FORM DEPENDENT AFFECT AND IT LOOKS LIKE APOE ISO FORM CAN INFLUENCE THE CLEARANCE OF SOLUBLE FORMS OF MONOMERIC A BETA AND ALSO THE APOE CAN SOMEHOW AFFECT THE SEEDING PROPENSITY OF THE ABETA MOLECULE TO HIGHER ORDER STRUCTURES. AND WE'VE LOOKED AT THIS AND IF YOU GENETICALLY DECREASE THE COPY OF APOE 3 AND 4 IN MICE IT STRONGLY ATTENUATES AMYLOID DEPOSITION. YOU CAN LOWER THE LEVEL IN THIS CASE LOWER THE LEVEL OF APOE BY 50% AND WHEN WE DO THAT AND START GIVING THE ASOs AFTER BIRTH OF THE ANIMALS IT DECREASES AMYLOID DEPOSITION BY ABOUT 50%. IF YOU DO THIS LATER, IF YOU LOWER APOE LEVELS AFTER THERE'S AMYLOID DEPOSITION WHEN YOU LOWER IT BY 50% IT DOESN'T SEEM TO FURTHER AFFECT THE AMOUNT OF AMYLOID DEPOSITION BUT IN RECENT EXPERIMENTS WHEN WE LOWER IT AFTER AMYLOID IS FORMED BUT TO GREATER LEVELS YOU CAN APPEAR TO AFFECT FURTHER AMYLOID DEPOSITION. I DON'T HAVE THE DATA HERE TODAY. SO WHAT IS AFFECTING THE DIFFERENT TYPE AND ONE REASON WE'RE INTERESTED IS WHEN APOE IS MADE AT LEAST IN CULTURE BY MICROGLIA OR ASTROCYTES WHAT WE FIND IN THE MEDIA WHERE APOE IS RELEASED BY THE CELLS IS ASTROCYTE IS IN LARGE PARTICLES 13 TO 20 NANOMETERS AND MICROGLIA RELEASE A PARTICLE LESS LIPIDATED. BECAUSE OF THE AMOUNT OF LIPID IN A PROTEIN PARTICLE IS LIKELY TO INFLUENCE ITS FUNCTION WE THINK MAYBE CELL TYPE SPECIFIC EXPRESSION OF APOE MAY HAVE DIFFERENT AFFECTS AND WE'VE LOOKED AT THIS IN MICE THAT EXPRESS THEIR AMYLOID DEPOSING MICE EXPRESSING E4 OR E3 AND HAVE ALLELE AND WE CAN KNOCK IT OUT IN WHATEVER CELL TYPE WE'RE INTERESTED IN. IN THIS CASE WE USE AN ALDH1 CREE DRIVER IN A TOMOXIFEN AND LOOKED TO SEE IF IT AFFECT THE PATHOLOGY AND THE LEVELS OF PROTEIN GOES DOWN ABOUT 70% TO 80% WHICH IS CONSISTENT WITH THE FACT THAT MOST THE APOE UNDER NORMAL CONDITIONS IS PRODUCED BY ASTROCYTES AND THERE'S STILL REACTIVE MICROGLIA PROBABLY ACCOUNTING FOR THE REMAINING AMOUNT IN THE BRAIN AND WHEN WE KNOCK OUT THE ASTROCYTE ONLY WE SEE A STRONG AFFECT ON EXPRESSION SIMILAR TO WHAT YOU SEE IF YOU COMPLETELY REMOVE APOE FROM THE ANIMALS. IT LOOKS LIKE UNDER AT LEAST CONDITIONS LIKE THIS THAT ASTROCYTE APOE IS THE PRIMARY CONTRIBUTOR TO PATHOLOGY AND WHETHER MICROGLIAL APOE WE'RE NOT SURE. ONE OF THE OTHER THINGS THAT HASN'T BEEN ADDRESSED UNTIL MORE RECENTLY IN THE LITERATURE IS, IS IT POSSIBLE WHILE WE KNOW APOE AFFECTS DOSES WOULD IT AFFECT THE PATHOLOGY OR SOMETHING LINKED TO THE PATHOLOGY AND THE CHANGES IN TAU THAT OCCUR AND ANY OF THE DAMAGE THAT IS LINKED WITH TAU PATHOLOGY. IN ORDER TO ADDRESS THE EXPERIMENTALLY, WE BEGAN TO USE THE TAU MUTANT MICE. ONE OF THE SEVERAL DIFFERENT ANIMALS THAT HAVE BEEN CREATED THAT DEVELOPED TAU PATHOLOGY AND OVER EXPRESSED. THESE WERE MADE BY VIRGINIA LEE'S LAB YEARS AGO AND OVEREXPRESSED A MUTANT FORM OF TAU THAT CAUSES DEMENTIA DUE TO TAUOPATHY. WHEN YOU OVEREXPRESS THIS MUTATION OF TAU IN THE BRAIN YOU SEE ABOUT FIVE, SIX MONTHS OF AGE TAU BEGINS TO AGGREGATE AND AS THERE'S PROGRESSIVE AGGREGATION YOU GET SIGNIFICANT NEURONAL LOSS AND VOLUME LOSS IN THE BRAIN WHICH IS SIMILAR WHAT YOU SEE IN TAU RELATED FTD AND ALZHEIMER'S DISEASE WHERE TAU IS BEGINNING TO ACCUMULATING DAMAGE. WE CROSSED TAU TRANSGENIC MICE WITH THESE AND THE MICE BY MONTHS OF AGE IF THEY EXPRESSED E4 HAVE DEATH AND EXPRESS ATROPHY AND THAT'S ALSO IN THE PRESENCE OF HUMAN APOE 3 AND 4 BUT NOT AS MUCH AND WHAT'S REALLY STRIKING TO ME IS THAT WHEN YOU REMOVE APOE FROM THE ANIMAL COMPLETELY THEY ESSENTIALLY GET ALMOST NO NEURAL DEGENERATION. WE REPEATED THIS MANY TIMES WITH A CONSISTENT RESULT. SO WHEN WE SAW THIS WE BEGAN TO LOOK AT WHAT HAPPENS TO THE GENES RELATED TO INFLAMMATION. FIRST WE LOOKED AT GENES PREDOMINANTLY EXPRESSED BY MICROGLIA AND THERE WAS A BIG INCREASE IN THE TAU MICE EXPRESSING APOE 4 REFERRING TO THE DISEASE RELATED GENE WITH A HOST OF OTHER GENES. THIS SIGNATURE IS MOSTLY ABROGATED YOU CAN SEE WITH STAINING WITH CD68 AND MICROGLIA IT'S MOSTLY GONE WHERE HOMO STATIC MICROGLIAL GENES ARE LOWERED IN THE ABSENCE OF APOE 3 AND 4. WE SAW SIMILAR RESULTS WITH REACTIVE ASTRO TYPE GENES AND THIS IS MOSTLY IN THE ABSENCE OF APOE. YOU CAN SEE ASTROCYTES MAKE NORMAL APOE IN THE BRAIN BUT IN INJURY YOU SEE A BIG INCREASE IN APOE AND MICROGLIA. WE WANTED TO LOOK ARE NOT JUST AT THE AMYLOID MODEL BUT IN THE TAU MODEL I SHOWED YOU THE CELL SPECIFIC ROLE IN APOE AND THE TAU RELATED CHANGES I JUST SHOWED YOU. IN THIS STUDY WE JUST PUBLISHED WE LOOKED AT THE TAU MICE AND REMOVED APOE ASTROCYTES RIGHT BEFORE THEY START TO DEVELOP NEURODEGENERATION AND FOLLOWED THEM OUT TO WHERE THEY HAVE A LOT OF BRAIN INJURY. WHAT WE COULD SEE WHEN WE REMOVE APOE FROM ASTROCYTES WE SEE A REDUCTION IN LEVELS IN THE BRAIN BY 50%, 60%, IF YOU DO SINGLE RNA SEQ ANALYSIS OF THE CELL TYPES IN THE BRAIN YOU SEE ASTROCYTE APOE IS HIGH NORMALLY AND REMOVED WHEN WE GIVE THE ANIMALS TAMOXIFEN WHICH IS UNDER CONTROL OF THE ASTROCYTE SPECIFIC PROMOTER AND THE MICROGLIAL APOE STAYS QUITE HIGH. WHEN WE LOOK AT THE OUTCOME AND WHAT HAPPENS TO THE BRAIN IN THE TAU MODEL, WHAT YOU CAN SEE AGAIN IS NORMALLY THE MICE HAVE A LOT OF HYPE -- HIPPOCAMPAL AFFECT AND IN THE CASE OF APOE THERE'S NOT AS MUCH INJURY TO THE BRAIN AND WASN'T A SIGNIFICANT FURTHER PROTECTION WHEN WE REMOVED ASTROCYTE E3 AND SOME PARAMETERS WHICH I DON'T HAVE TIME TO SHOW DO SHOW SOME NEURO PROTECTION OCCURRING. WE ALSO LOOKED AT A SIMPLE BEHAVIORAL TEST OR FESTING BEHAVIOR WHERE THE ANIMALS SCORE A 5 ON THE TEST IF THEY'RE NORMAL. IN THE TAU TESTS THEY'VE PERFORMED POORLY ON THE TEST AND THEIR FUNCTION IS IMPROVED WHEN IT RETURNS TO THE LEVEL IN THE E3 MICE. INTERESTINGLY, WHEN WE REMOVE ASTROCYTE E4 THERE'S NOT ANY MAJOR AFFECT ON INSOLUBLE TAU. THERE'S A LITTLE AFFECT BUT WHEN WE LOOK AT PHOSPHORYLATED FORMS THERE'S A DECREASE PROBABLY DUE TO THE FACT IT'S AFFECTING SIGNALLING IN SOME WAY TO INFLUENCE TAU PHOSPHORYL STATE. THAT'S JUST A SPECULATION. AND DOING ANALYSIS IN DIFFERENT CELL TYPES AND SHOW YOU THE MICROGLIAL RESULTS WHEN WE SEE TWO MAJOR POPULATIONS OF MICROGLIA AND THE TAU MICE AND THE FORM LABELLED 4 TO 0 OR ONE LABELLED 1 YOU SEE THE TAU GENE SIGNATURE THIS IS NOT ABROGATED WHEN IT'S REMOVED FROM THE ASTROCYTES BUT IF YOU LOOK AT SPECIFIC DISEASES ASSOCIATED MICROGLIAL GENE EXPRESSION IT'S REDEVED WHEN WE LOWER APOE LEVELS FROM ASTROCYTES AND IF WE LOOK AT MICROGLIAL STAINING THERE'S A MARKER OF DISEASE-ASSOCIATED MICROGLIA AND WHEN REMOVE IT IT DECREASES STRONGLY CONVERSELY IF YOU LOOK AT A HOMO STATIC MICROGLIAL MARKER AND WHEN YOU REMOVE THE E4 IT STRONGLY INCREASES. WHEN WE LOOK AT SYNAPSES IN THE MODEL THERE'S A DECREASE IN PSD95 WHEN THE DAMAGE OCCURS AND THIS SYNAPTIC DECREASE THIS IS PROTECTED WHEN WE REMOVE ASTROCYTE E4 AND IF YOU LOOK AT THE MICROGLIA YOU SEE THE PSD95 AND WHEN WE REMOVE ASTROCYTE E4 THERE'S A SYNAPTIC PHAGOCYTOSIS AND TO SUM UP IN THE SHORT TALK IT LOOKS LIKE APOE 4 DRIVES TAU MEDIATED NEURO DEGENERATION AND WE HAVE OTHER EXPERIMENTS SUGGESTING MICROGLIA ARE REQUIRED FOR THE PROCESS. IT LOOKS LIKE APOE PRODUCED BY ASTROCYTES PLAYS A ROLE IN THE TAU MEDIATED NEURO DEGENERATION AND WE'RE IN THE MIDST OF COLLABORATIVE EXPERIMENTS TO TRYING TO ADDRESS THAT. IN ADDITION TO ASSESSING THE ROLE OF APOE IN MICROGLIA LOOKING AT INTRODUCTIONS WITH OTHER MOLECULES SUCH AS MICROGLIA CONTRIBUTING TO NEURO DEGENERATION AND WILL BE IMPORTANT TO SORT OUT AND ONE POSSIBILITY AS NEURONS AND SYNAPSES BEGIN TO DEGENERATE APOE MAY SERVE IN REGIONS LIKE THIS FOR RECEPTORS ON MICROGLI JA THAT MIGHT THEN ENABLE PHAGOCYTOSIS AND THERE'S THE ABILITY OF APOE TO REGULATE LIPID METABOLISM IN MICROGLIA MAY PLAY A ROLE AND WE'RE ASSESSING THAT WITH OTHER TECHNIQUE. ONE OF THE OTHER THINGS WE'VE ADDRESSED RECENTLY IS IF WE OVEREXPRESS THE LDL RECEPTOR STRONGLY LOWERS APOE LEVELS IN THE BRAIN AND IS VERY NEURO PROTECTIVE AGAINST TAU MEDIATED NEURODEGENERATION. WE'RE TRYING TO BETTER UNDERSTAND THAT PHENOMENON AS WELL. THESE ARE MANY OF THE PEOPLE WHO CONTRIBUTED TO THE STUD AND FOR THE SAKE OF TIME I'LL STOP SHARING AND TAKE ANY QUESTIONS. >> WHILE WE'RE WAITING FOR QUESTIONS FROM THE AUDIENCE, ARE THERE ANY QUESTIONS FROM THE PANELISTS? >> I'M GLAD SOMEBODY SENT A NOTE ABOUT APOE BEING GLYCOSYLATED AT SEVEN SITES. WE'RE IN THE PROCESS OF LOOKING AT THAT OURSELVES WHETHER THE GLYCOSYLATION DIFFERS DEPENDING ON WHICH CELLS PRODUCE THE APOE. THAT'S IMPORTANT TO SORT OUT. >> BASED ON YOUR LECTURE IT LOOKS LIKE LRP MIGHT HAVE TWO DIFFERENT ROLES. ONE POTENTIALLY IS PROTECTED OR CAN HELP PROPAGATE THE TAU PATHOLOGY. >> WE HAVEN'T LOOKED -- IN THE EXPERIMENTS MY LAB HAS BEEN DOING RECENTLY WE HAVE LOOKED AT THE LDL RECEPTOR NOT LRP. I SUSPECT THE TWO MOLECULES HAVE DIFFERENT ROLES IN RELATION TO NOT JUST APOE METABOLISM BUT ALSO TAU MEDIATED SEEDING AND SPREADING AS I'M SURE BRAD DISCUSSED. THERE'S MANY LIGANDS AND IT MAY BE THAT THAT'S WHY IT HAS DIFFERENT AFFECTS THAN THE LDL RECEPTOR. >> WE HAVE ANOTHER QUESTION FROM THE AUDIENCE. I DON'T KNOW IF YOU CAN SEE THE CHAT BOX. HOW DO YOU THINK THE SITE OF APOE PROTEIN -- >> LETS SEE IF I CAN SEE THIS. HOW DO YOU THINK THE SIZE OF THE PROTEINS CARRYING CHOLESTEROL PLAYS A ROLE IN THE DISEASE. I THINK ONE POSSIBILITY IS THAT IN THE BRAIN, RECENT DATA THAT'S BEEN PUBLISHED FROM DENALI THERAPEUTICS IN A NEURON PAPER SHOWED FOR MANY YEARS WHEN PEOPLE BEGAN TO LOOK AT LIPID AND CHOLESTEROL METABOLISM AND WHETHER APOE PLAYED A ROLE THE RESULTS WERE NEGATIVE JUST MEASURING PHOSPHO LIPIDS IN THE ABSENCE OF APOE THERE WASN'T MUCH SEEN BUT TORNADOES OUT IF YOU LOOK MORE CAREFULLY AND THEY SHOWED THIS IN THEIR PAPER THAT APOE WHEN YOU KNOCK OUT APOE IN THE BRAIN IT AFFECTS THE LEVEL OF DIFFERENT PHOSPHOLIPIDS AND TO SOME DEGREE ASTROCYTES SO I WONDER WHETHER THE SIZE OF GLYCOPROTEINS IN THE BRAIN MAY PLAY A ROLE IN REVERSE CHOLESTEROL TRANSPORT OR ENABLING DIFFERENT LIPIDS TO BE REMOVED OR SECRETED FROM CELLS. SO THE TYPE OF GLYCOPROTEIN MAY BE RELATED TO THE CELL THAT'S GETTING RID OF PHOSPHOLIPID AND PROTEINS IN THE CELL FOR DIFFERENT REASONS. >> ANOTHER QUESTION IS CAN YOU COMMENT ON THE ACCOUNTS OF APOE AND THERAPEUTICS. >> I THINK AT LEAST IN THE PRESENCE OF AT LEAST APOE 4 FOR SURE MAYBE 3, IF YOU LOWER APOEs IN THE BRAIN BY AS MUCH AS 50%, 60%, I'M NOT AWARE THERE'S NEGATIVE CONSEQUENCES OF THAT IN TERMS OF AFFECTING A NORMAL FUNCTION IN SOME WAY. SO I THINK THAT WOULD PROBABLY BE ASSUMING YOU COULD DO THIS AND IT'S PRACTICAL IT SHOULD BE A REASONABLE TARGET TO TRY TO DECREASE NEURODID GENERATION. -- NEURODEGENERATION. IN THE PERIPHERY YOU DON'T WANT TO LOWER APOE MORE THAN 85% BECAUSE YOU START GETTING ELEVATED LARGE GLYCOPROTEINS BUT YOU CAN LOWER IT WITHOUT A PROBLEM YOU JUST DON'T WANT TO LOWER IT TOO MUCH. I THINK IT DOES HAVE AA LOT OF POTENTIAL OTHER THAN THE METHODS OF ADMINISTERING IT IN THE CSF SPACE. THAT'S NOT OPTIMAL BUT IT'S DOABLE. >> AS THE APOE BIOLOGISTS WHAT DO YOU THINK IS THE TOOL YOU'D LIKE TO SEE THE COMMON FUND INVESTORS CAN MAKE TO HELP US UNDERSTAND THE AREA RELATED TO GLYCOSYLATION, FOR EXAMPLE. >> I THINK FROM A QUESTION STANDPOINT, ONE THING THAT'S POORLY UNDERSTOOD AND MAYBE THERE'S A TOOL THAT CAN BE MADE TO ADDRESS THIS, IT WOULD BE HELPFUL TO FOLLOW -- ONCE APOE IS PRODUCED BY A CELL SAY IN THE BRAIN, IT WOULD BE NICE TO BE ABLE TO TRACK IT. WHERE DOES IT ACTUALLY GO AND WHAT DOES IT THEN BIND TO WITHIN THE BRAIN. IN ONE OF THE -- IT'S VERY LIKELY TO BE BINDING TO HSPG. IF THERE'S TOOLS THAT CAN BE MADE TO BETTER UNDERSTAND THAT PATHWAY OF APOE EFFORTS RELEASED AND WHAT DOES IT BIND TO AND WHAT'S THE AFFECT OF HSPG. THERE'S WAYS -- I KNOW THERE'S ANIMALS WHERE YOU CAN MODIFY HSPG PRODUCTION BY CELLS, AND I THINK TRACKING -- BEING ABLE TO TRACK APOE WHEN IT'S MADE ENDOGENOUSLY WOULD BE IMPORTANT TO TRACK. I'VE SEEN A TOOL WHERE YOU CAN BASICALLY PRODUCE APOE 1 THAT ARE FLUOROESCENT SO MAYBE YOU CAN TRACK WHERE IT GOES AFTER IT'S PRODUCED. >> CATHERINE DO YOU HAVE A COMMENT WHERE THE TOOL COULD BE MADE THE LARGER PROTEIN MOLECULE. >> I'M HERE. I THINK RON COULD COMMENT BETTER THAN I COULD ABOUT THE LIBO TOOLS AND THINGS HE THINKS ABOUT BUT I'M NOT SURE ABOUT THAT ONE. >> OKAY. WELL, AGAIN THANK YOU EVERYBODY FOR THIS MORNING'S SECTION. I'LL THANK OUR SPEAKERS AGAIN. IT'S LUNCH TIME BUT AGAIN WE'LL HAVE A ROUNDTABLE SECTION AT 3:30 P.M. IF ANY OF YOU CAN COME BACK AND JOIN US TO SEE WHAT WE CAN DO OR ANY OF THE TOOLS FOR THE BIOLOGISTS COME BACK AND JOIN US. THANKS AGAIN, EVERYONE. >> WE'LL RESUME AT 1:00 WITH OUR NEXT SECTION. >> THANKS, AUSTIN. >> THANK YOU. >> WELCOME MY NAME IS KEVIN VEMURI FROM THE NATIONAL INSTITUTE ON DRUG ABUSE AND I'LL BE YOUR CHAIR FOR THIS SESSION FEATURING TALKS FROM FIVE PROMINENT SCIENTISTS WHO HAVE PROFOUND RESEARCH WHICH IS ON OUR INTERFACE OF THE GLYCOSCIENCES AND EACH SPEAKER WILL GET 30 MINUTES AND POSSIBLY ONE OR TWO QUESTIONS. THERE'LL BE A TIMER SO YOU CAN WATCH OUT FOR THAT. IF WE CAN'T GET TO THE QUESTIONS WE'LL GET TO IT THROUGH THE PANEL SESSION LATER AT 3:30. WITHOUT FURTHER ADO LET ME INTRODUCE DR. ALBERT WONG. . HE IS PROFESSOR OF PSYCHIATRY AND PHARMACOLOGY AT THE UNIVERSITY OF TORONTO. HIS TALK TODAY WILL BE ON THE MOUSE MODELS OF PSYCHIATRIC DISORDER. >> THANK YOU VERY MUCH. I HAVE PICTURES IN MY TALK FROM MY TRAVELS HOPING TO REMINISCE ABOUT THE DAYS WHEN WE COULD JUST GET ON A PLANE. THIS IS FROM ICELAND. THE TOPIC WHICH IS GLYCOSCIENCE WHICH IS NOT REALLY MY AREA BUT I HOPE SOME OF THE THINGS I TALK ABOUT WILL BE HELPFUL AND USEFUL. I WANT TO HAVE A BROAD DISCUSSION HOW ANIMALS CAN BE USED IN PSYCHIATRIC DISORDERS. WE KNOW OF MANY RISKS FOR PSYCHIATRIC DISORDERS BUT WHAT REMAINS OBSCURE IS THE PHENOTYPES LINKING THESE AND MUCH OF OUR DIAGNOSTIC CRITERIA IN PSYCHIATRY RELY ON HISTORY AND MICE CAN'T GIVE THAT. THE SOLUTIONS ESSENTIALLY ARE ALL WORKAROUNDS. IT'S SEEN MORE IN PSYCHIATRY THAT OTHER AREAS OF MEDICINE. FOR MOST OTHER AREAS THERE'S SUBJECTIVE TEST, IMAGING, BIOCHEMICAL TESTS TO BE USED TO MAKE THE DIAGNOSIS OR CHARACTERIZE THE PHENOTYPE AND IN PSYCHIATRY WE DON'T HAVE ANY OF THOSE. THE APPROACHES INCLUDE USING DIFFERENT MOUSE MODELS KNOWING NO MODEL OF ANY RODENT OR EVEN A PRIMATE CAN REALLY RECAPITULATE ALL THE FEATURES OF A PSYCHIATRIC DISEASE IN HUMANS AND THEY HAVE TO MODEL MORE THAN ONE ASPECT AND SOME AREAS AMENABLE TO THIS APPROACH WHICH ARE AREAS SUCH AS LOOKING AT BRAIN STRUCTURE AND DEVELOPMENT. ANOTHER AUTO APPROACH IS REFINING TESTING AND THERE'S THE TOUCH SCREEN APPARATUS ESSENTIALLY IS LIKE AN iPAD ON THE SIDE OF THE MOUSE CAGE HAVING IT RESPONSE WITH THE NOSE TOUCHING THE SCREEN WHICH IS VERY MUCH LIKE A HUMAN USING THEIR FINGER ON THE TOUCH SCREEN. THESE HAVE THE ADVANTAGE OF BEING TRANSLATABLE BETWEEN SPECIES. IN SOME CASES THE HUMAN CAN USE THE SAME TESTING PARADIGM AS THE MOUSE DOES. ANOTHER CAVEAT IS TO ATTACK TRACKABLE PROBLEMS AND THERE'S ASPECTS OF PSYCHIATRIC DISORDER WHICH ARE NOT AMENABLE TO THIS MODEL BASED APPROACH. THE CHOICE OF WHAT PROBLEM OR PARADIGM IS CRUCIAL. AND ONE OF THESE APPROACHES IS TO LOOK FOR ENDO PHENOTYPES OR MEASURES OF BEHAVIOR LIKE THE COMMON CELLS IDEA NOT LOOKING AT THE CLUSTER OF SYNDROME WITH THE NAME OR DIAGNOSIS BUT LOOKING AT SPECIFIC BEHAVIORAL MODULES WITH A CLEARLY IDENTIFIABLE PHENOTYPE THAT CAN BE TRANSLATED BETWEEN MOUSE AND MAN. FINALLY, LOOKING AT ENVIRONMENT INTERACTIONS CAN BE FRUITFUL BECAUSE REALLY NONE OF THE COMMON PSYCHIATRIC DISORDER ARE SINGLE GENE DISORDERS, OF COURSE, TO UNDERSTAND HOW THE PATHOPHYSIOLOGY WORKS WE NEED TO MOVE BEYOND JUST GENETIC MUTANTS AND A BROAD WAY OF CREATING A MODEL. THIS IS A LIST OF THE MODELS I'VE USED IN MY LAB. THEY'RE KIND OF ALL OVER THE PLACE. THIS IS MY SHORT ATTENTION SPAN GOING FROM ONE TOPIC TO THE OTHER AND ONE INTERSECTS WITH THIS MGAT MODEL. I WANT TO TALK ABOUT AND ONE PARTICULAR IS THE ONE I HAVE MOST EXPERIENCE THE EXAMPLE OF A CONVENTIONAL MUTANT WE LEARN SOMETHING ABOUT THE PATH OWE PHYSIOLOGY ABOUT SCHIZOPHRENIA AND I WANT TO TALK ABOUT TWO OTHER APPROACH USING ANIMAL MODELS IN RELATION TO PSYCHIATRIC DISORDER. ONE IS FOR PTSD. IN THIS CASE WE'RE NOT USING A GENETIC MUTANT BUT A BEHAVIORAL PARADIGM WHICH I THINK PROBABLY IS CLOSER TO THE HUMAN SITUATION THAN MOST OTHER MOUSE PARADIGMS FOR PSYCHIATRIC DISORDERS. IN THIS CASE THE BEHAVIORAL PARADIGM IS SOMETHING WE USE TO INDUCE A DISEASE LIKE STATE AND AN INDEX AND ANOTHER WAY OF USING ANIMAL MODELS TO UNDERSTAND MORE ABOUT PSYCHIATRIC DISORDERS AND USING A WAY OF MEASURING SENSORY DATING AND REGULATORS OF THIS PHENOTYPE. ALSO, IF THERE ARE QUESTIONS, FEEL FREE TO ASK THEM IN THE MIDDLE. I'M NOT WAITING IN THE END. OF COURSE, THERE'S NO CURRICULUM HERE SO IF WE GET DISTRACTED BY QUESTIONS EITHER IN THE CHAT OR JUST INTERRUPT, I'M VERY HAPPY TO ADDRESS THEM AS WE GO RATHER THAN WAITING UNTIL THE END. SO THE STORY BEGINS IN SCOTLAND WHERE THERE WAS A FAMILY DISCOVERED CLINICALLY THAT HAD A HIGH LOADING FOR PSYCHIATRIC DISORDERS. ALL THE COLORED SHAPES REPRESENT PEOPLE IN THE FAMILY AFFECTED BY SOME KIND OF PSYCHIATRIC DISORDER AND SCHIZOPHRENIA IS COMMON AND A MOOD DISORDER AND OTHER DIAGNOSIS AND THE FAMILY CARRIES THIS DRAFT IS MUTATION THAT SEVERS A GENOME CHROMOSOME 1 AND SHOULD BE DISRUPTED IN MANY PSYCHIATRIC DISORDER AND SCHIZOPHRENIA AND THE TRANS LOCATION THIS FAMILY HAS CUTS THE GENE ALMOST EXACTLY IN HALF. IT TURNS OUT NOW THAT THIS IS THE ONLY KNOWN FAMILY WITH THIS PARTICULAR MUTATION. THIS IS MAYBE A PRIVATE MUTATION, YOU WOULD SAY. THE WHOLE AREA IS SOMEWHAT CONTROVERSIAL WHERE IT'S BEEN ABOUT THE FUNCTION OF THE PARTICULAR PROTEIN BUT IT'S NOT CLINICALLY APPLICABLE TO ANYONE ELSE NOT A MEMBER OF THIS FAMILY. NOW, THERE ARE GENETIC ASSOCIATIONS AT A POPULATION LEVEL WITH VARIANTS IN THE GENE AND SCHIZOPHRENIA AS WELL AS OTHER PSYCHIATRIC ILLNESSES AS WITH OTHER TYPES OF THE GENETIC ASSOCIATIONS THEY'RE FAIRLY WEAK AND THE RELATIVE RISK INFERRED IS QUITE SMALL AND IT'S THE STORY OF MOST COMMON COMPLEX DISEASE GENETICS FROM DIABETES TO CANCERS. SO DISC 1 IS A SCAFFOLDING PROTEIN AND INTERACTS WITH OTHER PARTNERS KNOWN TO BE INVOLVED IN EITHER PSYCHIATRIC DISORDER OR BRAIN DEVELOPMENT OR DRUG TARGETS WE USE TO TREAT PSYCHIATRIC DISORDER SUCH AS GSK3 BETA IS A WELL KNOWN STAR GET FOR A MODULATED DOWN STREAM BY LITHIUM, FOR EXAMPLE, TO TREAT PSYCHIATRIC DISORDER. WHAT WE WERE INTERESTED IS THE ONE RELATED TO NEURON MIGRATION. IN PARTICULAR IN THE CYTOSKELETON THAT PROVIDE THE LOCOMOTIVE FORCE FOR NEURONS TO MIGRATE DURING EARLY BRAIN DEVELOPMENT. THERE'S BEEN A LONG HISTORY OF STUDY HISTOLOGY AND SCHIZOPHRENIA AND ALZHEIMER'S AND THERE WAS NEVER ANY PATHOLOGICAL PATHWAYS DISCOVERED AND THERE'S BEEN MANY REPORTS OF FAIRLY SUBTLE CHANGES IN THE ORGANIZATION OF THE CORTEX AS AN ARCHITECTURE AND THESE USUALLY INVOLVE FEWER NEURONS OR SOME CHANGE IN THE MORPHOLOGY AND SIZE AND THE HYPOTHESIS IS BECAUSE DISC 1 IS IMPORTANT IN THE MIGRATION OF NEURONS DURING DEVELOPMENT THAT MUTATIONS COULD OR VARIANTS COULD IMPAIR THIS MIGRATION AND IF IT WAS A SUBTLE AFFECT YOU MIGHT EXPECT TO SEE WHAT WE SEE IN THIS PICTURE MORE NEURONS IN THE DEEPER LAYERS THAT'S BECAUSE THE CORTEX LAM LAMINATES AND THE NEURON AND AND LATER BORN NEURONS APPEAR IN THESE LAYERS. THIS IS NOT THE WAY ONE WOULD FORWARD ENGINEER AN ASSEMBLY OF STRUCTURE BECAUSE IT MEANS THE NEURONS BORN LATEST HAVE THE SHORTEST TIME TO MIGRATE AND HAVE TO SQUEEZE THROUGH THE EARLIER BORN NEURONS. THE NEURONS BORN EARLIER ON ONLY HAVE TO MOVE A SHORT DISTANCE AND HAVE A LOT OF TIME THOUGH THEY USUALLY DO IN THEIR TIME WINDOW. THE HYPOTHESIS WOULD BE IF THERE'S A SUBTLE CHANGE IN NEURON MIGRATION ONE COULD OBSERVE THE NEURONS GOING TO THE SUPERFICIAL LAYERS DIDN'T HAVE ENOUGH TO TIME TO GET TO THE TOP LAYER. WHILE THIS IS HAPPENING IN THE CORTEX THE RADIAL MIGRATION AND THERE'S THE TANGENTIAL MIGRATION FROM THE STRUCTURES IN THE EMBRYO AND THIS BUMP HERE IN THE EMBRYO GIVES RISE TO INTERIN THE TRAJECTORY AND ENTER TANGENTIALLY TO THE PERIMETER NEURON. THE WHOLE PROCESS REQUIRES SOME COORDINATION AND DISRUPTIONS CAN CHANGE THE TIMING OF ARRIVAL OF THE TYPES OF CELLS INTO THE CORTEX WHERE THEY'RE SUPPOSED TO FORM CONNECTIONS. IF NOT THERE THEY CAN BE DISRUPTED. I WANT TO SHARE PICTURES FROM A TALENT SCIENTIST AND STUDIED AN OLD TECHNOLOGY OF MUTO GENESIS AND THE MUTATIONS THE MICE CARRY ARE ESSENTIALLY RANDOM AND LAND WITHIN THE DISCIPLINE GENE AND THIS IS WHAT JOHN ROTOR AT MT. SINAI AND HIS GROUP FIRST OBTAIN THE MICE AND CHARACTERIZED THEM AND HAVE A SERIES OF BEHAVIORAL AND CELLULAR ABNORMALITIES AND DISORDERS AND SCHIZOPHRENIA AND THE L MUTANT. THE P MUTANT HAS IMPAIRED COGNITION, PRE PULSE INHIBITION, SENSORY DATING AND HYPO LOLOCO HYPOLOCOMOTION AND NOT A GOOD STARTING POINT FOR A MODEL THAT ALLOWED US TO LOOK MORE AT BRAIN STRUCTURE AND DEVELOPMENT AND THE FIRST THING FRANKIE FOUND WAS OVERALL NEURO DENSITY WAS LOWER IN THE CORTEX, IN THE DRAMATICALLY LOWER BUT SLIGHTLY LOWER AND THE SAME MAGNITUDE IN POST-MORTEM STUDIES WITH SCHIZOPHRENIA. AS WE HYPOTHESIZED AND THIS LAYER IS THICKER WHICH LED US TO INFER PERHAPS NEURONS DIDN'T HAVE ENOUGH TIME OR ENERGY TO GET TO THAT LAYER SO IT'S NOT AS COMPACT AS IT SHOULD BE. THE DENDRITE SHORTER. THESE ARE GOLGI STAIN IMAGES AND SPINE DENSITY IS ALSO REDUCED. ALL THESE THINGS ARE CONSISTENT WITH WHAT WE SEE IN POST MORTEM HUMAN STUDIES LIKE ALL STUDIES HAVE VARIANCE AND INCONSISTENCY. WE USED BRDU WHICH WAS INJECTED DURING DEVELOPMENT AT DIFFERENT TIMES AS YOU CAN SEE HERE, 12, 15, 18 WHICH BRACKETS THE PERIOD OF TIME DURING GESTATION WHEN THE CORTEX IS LAMLAMINATED AND THE WILD TYPE GIVES A CLEAR PICTURE OF NEURONS QUITE NICELY DEFINED AND THESE ARE NEURONS BORN LATE IN THE DEVELOPMENT. THESE NEURONS IMAGINE THE NEURONS IN THE SUPER OFFICIAL LAYERS AND THAT'S WHAT HAPPENS IN THE WILD TYPE AND WE SEE NEURONS ON THIS DAY END UP SCATTERED THE CORTEX AND IT'S MORE OF A SNOWSTORM HERE. SO THAT WAS ENCOURAGING TO US SUGGESTING OUR HYPOTHESIS WAS CORRECT AND THERE WAS SOME DEFICIT IN MIGRATION DUE TO THE MIGRATION. WE ALSO SAW A DECREASED NEUROGENESIS WHICH CAN EXPLAIN THE LOWER OVERALL NUMBERS. I'LL PAUSE TO SEE IF THERE'S ANY QUESTIONS AT THIS POINT? IF NOT I WANT TO BRIEFLY TELL YOU ABOUT TWO ADDITIONAL EXPERIMENT EXPERIMENTS AND THEY DID A GENE ENVIRONMENT INTERACTION STUDY WHERE THEY EXPOSED THE MUTANT MICE TO EITHER SOCIAL DEFEAT THIS WOULD BE AS ADULTS SO WOULD SIMULATE THE KIND OF PSYCHOLOGICAL AND SOCIAL STRESSORS WE THINK ARE IMPORTANT IN THE GENESIS OF DEPRESSION, MAJOR DEPRESSIVE DISORDER VERSUS MATERNAL PERIOD AND INFLUENZA AND CORONAVIRUS ARE NOT GOOD FOR SIGNALLING AND THROUGH IL6 IT SEEMS TO HAVE A BAD AFFECT ON BRAIN DEVELOPMENT AND INCREASE RISK FOR SCHIZOPHRENIA AND AUTISM AND SO ON. THERE WAS AN INTERACTION BETWEEN THESE ENVIRONMENTAL STRESSORS AND MUTATIONS ESPECIALLY WHEN WE LOOKED AT THE HETEROZYGOTES AND WHEN COMBINED WITH THE ENVIRONMENTAL STRESSORS THERE WAS AN INTERACTION REINFORCING THE IDEA AS THE A CORRECT MODEL FOR HOW CONFLICTS LIKE PSYCHIATRIC DISORDER ARISE. AND I WANT TO TALK ABOUT THE MGAT 5 MODEL. THE MICE WERE GIVEN TO ME AND WE WERE INTERESTED IN HOW THIS MUTATION WOULD AFFECT PSYCHIATRIC PHENOTYPES ESPECIALLY RELATED TO DEPRESSION. SO MGAT IS A MENTAL TRANSFERASE. IT REMODEL GLYCOPROTEINS AND WE LOOKED AT THE KNOCKOUT IN THE GENE AND STRUCTURE TO EARLY DEVELOPMENT EITHER FOSTER REARING OR MATERNAL SEPARATION AS AN EARLY LIFE ADVERSITY. WHAT WE FOUND IS THAT THESE MUTANTS HAD BETTER PHENOTYPE MEASURES FOR EXAMPLE THEIR PHYSICAL HEALTH AND HAD BETTER GLUCOSE TOLERANCE, HIGHER BONE DENSITY AND GREAT GLUCOSE CONSUMPTIONS THE LATTER TESTSES ARE RELATED TO DEPRESSION AND WE KNOW ANTIDEPRESSANTS INCREASE THE SWIM TEST AND SUCROSE IS A MEASURE OF DEPRESSION AND WHEN SUBJECTED TO EARLY LIFE STRESS AND MOST MICE SHOW DIFFERENCES AND MGAT SHOW PROTECTION SO IT'S A RESILIENCE MODEL. I WANT TO TALK ABOUT ANOTHER EXPERIMENT AND THIS IS FROM MYANMAR. WE STARTED WITH THIS PHENOTYPE OF ACOUSTIC STARTLE AND THE IDEA IS WHEN THERE ARE MULTIPLE LOUD AUDITORY STIMULUS CAN APPLY TO OTHER MODALITIES OF SENSATION AND MOUSE WILL STARTLE WITH A LOUD BANG IT'S A WHITE NOISE BURST AND THIS STARTLE CAN BE ATTENUATED WITH A LOWER INTENSITY SOUND LIKE A WARNING SOUND BEFORE THE MAIN STARTING STIMULUS AND PATIENTS WITH SCHIZOPHRENIA CAN HAVE A WARNING SOUND NOT ATTENUATING COMPARED TO A HEALTHY CONTROL. AND THIS IMPAIRED PPI CAN BE PARTIALLY CORRECTED IN PATIENTS WITH SCHIZOPHRENIA AND THERE'S HAVE AN IMPAIRMENT IN THEIR PRE PULSE INHIBITION AND CAN BE TRANSLATED BETWEEN SPECIES. THE MAIN PROBLEM IS IT'S UNCLEAR HOW IT'S RELATED TO THE SYMPTOMS PER SE. THERE'S MANY THEORIES. SOME RETROSPECTIVELY TRY TO CONSTRUCT AN IDEA HOW THESE PHENOTYPES ARE RELATED TO THE SYMPTOMS BUT THE TRUTH IS WE DON'T HAVE A CLEAR IDEA AT THIS POINT. NEVERTHELESS BECAUSE PPI CAN BE AN INDEX OF PSYCHOSIS WE APPROACHED IT BY LOOKING AT SIX DIFFERENT STRAINS OF MOUSE. WE ORDERED SIX DIFFERENT STRAINS OF MICE BASED ON THEIR KNOWN PREVIOUSLY LEVEL OF PREPULSE INHIBITION TRYING TO EXPLOIT DIFFERENCES INHERIT TO THAT STRAIN FOR WHATEVER REASON WHETHER INCIDENTAL OR DELIBERATE AND TO MEASURE THE PRE PULSE INHIBITION LEVEL AND THEN LOOK AT GENE EXPRESSION LOOKING AT RNA IN PARTICULAR TO SEE IF WE CAN IDENTIFY TRANSCRIPTS WHOSE LEVEL IN THE BRAINS OF MICE WAS CORRELATED OR ANTI-CORRELATED WITH THE LEVEL OF PREPULSE INHIBITION. WE IDENTIFIED MANY GENES AND FILTERED THEM IN DIFFERENT WAYS AND EVENTUALLY ARRIVED AT A GENE CALLED PDXDC1 IT'S A DECARBOXYLASE BUT NOT A PROTEIN WELL CHARACTERIZED. WE FOUND IT TO BE ASSOCIATED THE LEVEL OF THE TRANSCRIPT WAS ASSOCIATED WITH THE LEVEL OF PPI AND WE CREATED A VIRAL LECTOR WITH HSRNA TO SUPPRESS THE LEVELS OF THE TRANSCRIPT AND SHE WAS ABLE TO CREATE PRE PULSE INHIBITION IN TE INHIBITION . THIS PARTICULAR GENE COULD BE IMPORTANT IN REGULATING PPI AND COULD BE A TARGET FOR NOVEL ANTI-PSYCHOTIC TREATMENT. IT'S 1:23 AND TELL YOU ABOUT ONE MORE STORY AND LEAVE IT AT THAT. THIS IS AN EXAMPLE OF USING ANIMAL MOLDS NOT WITH GENETIC MUTANTS BUT AS A WAY OF UNDERSTANDING AND IDENTIFYING POSTTRAUMATIC STRESS DISORDER. I'LL START WITH THE PUNCH LINE FIRST. OUR MODEL IS A NEURON. THIS IS GR THE CORTICOID RECEPTOR AND NOT VERY USER FRIENDLY NAMES AND THE IDEA IS OUR HYPOTHETICAL MODEL THIS COMPLEX AND THIS IS A RESPONSE AND IT'S PERSISTENCE AND HAS A HIGH LEVEL SO TOO MUCH FOR TOO LONG AND THE APPROACH HAS BEEN SUCCESSFUL IN OTHER PARADIGMS TO IDENTIFY THE PROTEIN COMPLEXES AND THEN TO IDENTIFY WHAT THE BINDING REGIME IS AND THE IDEA IS WHEN IN PTSD IF THE COMPLEX IS TOO PERSISTENTLY ELEVATED IT WILL AFFECT A NUMBER OF THINGS INCLUDING GENE EXPRESSION DOWN STREAM AND THAT USING THE PEPTIDE AND WE FOUND THAT THIS PROTEIN COMPLEX IS ELEVATED. THIS IS THROUGH OUR COLLABORATION WITH EMORY WHO IS NOW IN BOSTON WAS PART OF THE TRAUMA PROJECT WHERE THEY COLLECTED A LARGE POPULATION SAMPLE OF PEOPLE PROSPECTIVELY AND FOLLOWED LONGITUDINALLY PEOPLE AT HIGH RISK FOR EXPERIENCING TRAUMA. WE COMPARED PEOPLE WITH PTSD WITH PEOPLE WITH A SIMILAR TRAUMA HISTORY BUT NO PTSD AND SAMPLED WITH THE NON-PTSD CONTROL GROUP AND COMPARING THEM AGAINST PATIENTS OR PEOPLE WHO DO NOT HAVE PTSD. AND THIS BLOW TEEN COMPLEX WAS ELEVATED -- PROTEIN COMPLEX ELEVATED IN THE MOUSE AND FOUND EXPOSING THE MOUSE TO A SHOCK WITH A CUE THIS WAS A SIMULATED TRAUMATIC EVENT BECAUSE THEY RECEIVE A SHOCK IN THE CAGE AND ARE UNABLE TO ESCAPE THIS. FEAR CONDITIONING WAS SUFFICIENT TO ELEVATE THE PROTEIN COMPLEX TO A MAGNITUDE SIMILAR TO WHAT WE SEE IN PTSD. AND INTERESTINGLY THE OF GIVING THE PEPTIDE TO DISRUPT THE COMPLEX WAS ABLE TO OWE -- OBLITERATE THE FEAR CONDITIONING RESPONSE AND WE GAVE THE PEPTIDE AT DIFFERENT TIMES EITHER DURING THE CONDITIONING PHASE OR RETRIEVAL PHASE AND IN BOTH CASE THE PEPTIDE SEEMS TO BLOCK THE LEARNING OR RECALL OF THE FEAR MEMORY ASSOCIATE. OUR IDEA IT CAN BE USED FOR TRAUMA FOR THOSE WHO EXPERIENCE TRAUMATIC EVENT WHETHER SOLDIERS ON THE BATTLEFIELD FOR CIVILIANS WHO ARE VICTIMS OF ASSAULT OR DISASTERS AND IF SOMEBODY WAS EXPOSED TO A TRAUMATIC EVENT THEY CAN BE GIVEN THE PEPTIDE IMMEDIATELY AFTERWARDS LIKE A PEPTIDE MODEL AND OUR HOPE IS THEY WOULDN'T DEVELOP PTSD LATER. IT'S A DIFFERENT PARADIGM THAN IN PSYCHIATRY WHERE WE USE DRUGS WITH VARIABLE EFFECTIVENESS CHRONICALLY THAT HAVE TO BE TAKEN MANY YEARS OR SOMETIMES A LIFE TIME. THIS IS A DIFFERENT APPROACH WE'RE CURRENTLY TRYING TO DEVELOP FURTHER. AGAIN, DIFFERENT WAY OF USING ANIMAL MODELLING LOOKING AT THE MODEL AS A SIMPLE TRANSLATION BETWEEN A TRAUMATIC EVENT IN A HUMAN'S LIFE VERSUS A COMMON FEAR CONDITIONING PARADIGM. I THINK I'LL STOP THERE. THANK YOU. >> THANK YOU, DR. WONG. DO WE HAVE ANY QUESTIONS FROM THE PANEL, FROM THE AUDIENCE? DR. WANG, I HAVE ONE QUESTION. HAVE YOU LOOKED AT SEX DIFFERENCES IN ANY OF THESE MODELS? ARE THERE SEX DIFFERENCES? >> YES, WE LOOKED AT THE SEX DIFFERENCES IN THE MGAT5 MODEL, FOR EXAMPLE, WE LOOKED AT SEX DIFFERENCES. THERE'S NO CLEAR PATTERN. THAT WOULD BE THE CONCLUSION BUT THERE ARE SEX DIFFERENCES BUT THEY'RE COMPLICATED DEPENDING ON WHAT PHENOTYPE WE LOOKED AT SO THERE'S SEVERAL FACTORS THE SEX, MUTATION AND ENVIRONMENTAL EXPOSURE. AND BEING MORE VULNERABLE OR RESILIENT AND IT'S CONSISTENT WITH WHAT WE SEE CLINICALLY AND THERE'S SOME WITH A CLEAR SEX DIFFERENCE BUT USUALLY THE PATIENTS HAVE BEEN MORE COMPLICATED THAN THAT. >> THE MGAT MODE IS INTERESTING BECAUSE IT'S KIND OF GLOBAL WITH GLYCANS OVER ALL THE DIFFERENT TISSUES. IT'S BEEN TRACKED TO POTENTIAL CHANGES IN GLYCOPROTEIN LIKE RECEPTORS DUE TO GALECTINS. THE QUESTION IS ARE THE GALECTIN MUTATIONS THAT SHOW UP IN SCHIZOPHRENIA? GLANCING AT THE LITERATURE, THERE'S A LITTLE BIT OF THAT COMING THAT MIGHT BE WORTH -- >> THAT'S FOR THAT INSIGHT. THAT'S INTERESTING. AGAIN, THIS IS NOT MY AREA SO THAT'S VERY HELPFUL. THANK YOU. >> THE OTHER THING I'LL BRIEFLY POINT OUT IS FOR A WHILE THERE HASN'T BEEN A LOT GOING OUT AND LATELY THE MORE UNIQUE RARER MODULATION OF GLYCOSYLATION AND POLY SIALIC ACID HAS BEEN LINKED TO SCHIZOPHRENIA HAS WORK BEEN DONE IN MOUSE MODELS. J A HUNDRED YEARS AGO CANCER HAD A MEANING BUT IT'S HUNDREDS IF NOT THOUSANDS OF DISORDERS AND I THINK SCHIZOPHRENIA IS LIKE THAT TOO. AS WE DISSECT THIS WE'LL FIND THERE'S DIFFERENT SYMPTOMS INVOLVED AND DIFFERENT TYPE OF SCHIZOPHRENIA. I'M NOT AWARE OF ANY SPECIFICALLY. THANK YOU. >> THANK YOU, DR. WONG. WE HAVE OUR NEXT SPEAKER DR. ROBIN POLT. HE IS A PROFESSOR OF CHEMISTRY FROM THE UNIVERSITY OF ARIZONA SPEAKING ON CONTROL OF MEMBRANE AFFINITY VIA GLYCOSYLATION AND BRAIN-PENETRANT GPCR AGAINST FORMS OF ENDOGENOUS NEUROTRANSMITTERS AND HORMONES. >> THANK YOU FOR INVITING ME AND HOPE IT WILL BE EXCITING TO THE ARC TICKETS IN THE ROOM. NY TOAD MAKE A DISCLOSURE I HAVE IP AND WE'RE INVOLVED WITH A COMPANY CALLED TELEPORT PHARMACEUTICALS HERE IN TOWN. IN COLLABORATION FOR THIS I THOUGHT I SHOULD GO BACK AND CHECK AGAIN THAT GLYCOPEPTIDE DRUGS AND PEPTIDE DRUGS ARE STILL ON THEIR WAY UP AND INDEED WE THOUGHT IT'S INTERESTING. SOMEBODY HERE SEEMS TO BE AHEAD OF THE FIELD AND THOUGHT I'D SEE WHAT THAT WAS AND IT'S A PAPER WE'RE FAMILIAR WITH. THEY SAID PEPTIDE BASED DRUGS ARE NOT TOO PROMINENT BUT THE SITUATION IS RAPIDLY CHANGED AND IF YOU'D ASKED ME IN 1974 ARE THINGS RAPIDLY CHANGING I WOULD HAVE SAID YES BUT I GUESS IT DEPENDS ON WHAT YOU MEAN BY RAPID. KEY TO THIS IS YOU HAVE TO FOLLOW THE CONFIRMATIONAL AND CHEMICAL MODIFICATIONS OF THE COMPOUNDS TO TO MAKE PROGRESS TOWARDS A PHARMACEUTICAL. IT'S IMBOBBLE -- IMPOSSIBLE TO GO FROM A CLINICAL DESIRE TO AN INTERVENTION. WE'LL COME BACK AND I'LL MENTION OXYTOCIN BUT THE WHOLE STORY ON THAT IS NOT READY TO BE TOLD YET. I THINK MOST PEOPLE ARE FAMILIAR WITH THE CLASSICAL DRUG DEVELOPMENT PIPELINE BY NOW IT'S THE PREDOMINANT PARADIGM FOR DRUG DISCOVERY AND HAVE A PEPTIDE LIGAMENT FOR AFFINITY FOR ITS STRUCTURE TYPICALLY GPCR AND THOUGH IT LOOKS LIKE WE'RE JUST SHORTENING THE PATHWAY HERE, IN FACT IT'S A DIFFERENT PATHWAY. THE EVENTS PRECEDING THE CHOICE OF TARGET INVOLVE A DIFFERENT PREPARATION AS WELL AS THE PATHWAY TOWARDS DEVELOPMENT. HOPEFULLY I THINK I'LL BE ABLE TO CONVINCE YOU WE CAN ACTUALLY HIT THE SAME TARGET THAT YOU WOULD FIND VIA A MORE TRADITIONAL APPROACH. OF COURSE THERE'S A LOT OF INTEREST IN MAKING PEPTIDES MORE STABLE. AND CORE VIABILITY IS AN AREA WHERE THERE'S BEEN A LOT OF ENERGY SPENT BUT IT'S NOT CLEAR TO ME ANYWAY THIS IS THE BEST MODE OF DELIVERY. WHAT'S KEY FOR US IN TERMS OF BRAIN PENETRANTS AND SERUM STABILITY IS TO CONTROL THE PATHOCITY OF THE MOLECULES AFFECTING HOW THEY INTERACT WITH MEMBRANES. IN THE EARLY DAY THE SYNTHETIC APPROACH WAS DIFFICULT TO MAKE ENOUGH MATERIAL SO YOU COULD DO BINDING STUDIES AND OF COURSE WE DID THAT SORT OF THING TO MAKE GRAMS OF A PEPTIDE DRUG WAS CHALLENGING IN THE EARLY DAYS. WE GOT DOWN TO ONE OR TWO STEPS AND REVOLVES THE NOTION AROUND MEMORY COMPETENCY ACIDS AND THE TRADITIONAL APPROACH LOOKS LIKE THIS. WE GENERATE A STRONG PROTON THAT NEEDS TO BE ABSORBED TO GET THIS CHEMISTRY TO WORK. BASICALLY THIS IS NOT A CATALYTIC PROCESS AND PEOPLE GO TO LOWER AND LOWER TEMPERATURES AND MORE AND MORE ACTIVE DONORS AND HERE WE'RE USING AN INTERMEDIATE NOT AN ACTIVATED DONOR AND BY USING A MINIMALLY COMPETENT ACID AND ELIMINATING THE BASE THEN THE STRONGEST ACID WE HAVE TO WORRY ABOUT IS ACETIC ACID. AND WE CAN MAKE MULTIGRAMS TO FRACTIONAL GRAMS OF THE MATERIALS IN ONE STEP AND WE'RE READY TO INCORPORATE THAT IN THE PEPTIDE SYNTHESIS. THE TARGET STRUCTURE IS THE GOLD STANDARD IN THE AGONISM AND BY GLYCOSYLATING IT WE CAN PRODUCE GLYCOPEPTIDE DRUGS THAT HAVE THE SAME BINDING AFFINITY AS THE STARTING MATERIAL. THE KEY TO GETTING THESE TO WORK IS TO FIND THE SWEET SPOT WHERE WE HAVE THE AFFINITY TO BE ABLE TO FIND THE RECEPTOR AND UNDER GO ACROSS MEMBRANE BARRIERS. NOT SO MUCH WE PULL AWAY FROM THE MEMBRANE ENTIRELY. WE STILL NEED TO HAVE A LOT OF MEMBRANE AFFINITY IN ORDER TO GET BRAIN PENETRATION AND NOTICE TOPOLOGICALLY WE'RE ALWAYS ON TOP OF THE CELL WHERE THE RECEPTORS ARE AND WHAT WE WANT TO DO. THESE ARE GETTING IN THE BRAIN WITH THE IODINE STUDIES AND WE'LL TALK ABOUT MICRODIALYSIS BUT IN THE EARLY DAYS WE COULD LOOK AT INTRINSIC POTENCY BY ICV ADMINISTRATION OF DRUGS AND IN VIVO EFFICACY BY THE IV ADMINISTRATION. THIS CONFLATES ESSENTIALLY TO SERUM STABILITY AND BRAIN PENETRANTS BUT IT STILL GIVES YOU A GOOD WAY TO CHOOSE DRUGS LIKELY TO BE USEFUL. IF WE DISPLAY THAT SAME IV POTENCY THIS IS 55 DEGREES IN THE STUDY YOU SEE A U-SHAPED CURVE WHICH CONFIRMS THE NOTION THERE'S A SWEET SPOT INVOLVED. NOW, THE DAMGO COMPOUNDS ARE POTENT AND BY MAKING A COMPOUND WHERE WE'VE GOT THE NEW DELTA, THIS COMPOUND BINDS TO THE NANOMER RANGE AND THOUGHT THIS DRUG WOULD GO IN THE CLINIC AS A REPLACEMENT FOR MORPHINMORPHINE, A LESS SEDATING FORM. YOU CAN SEE THE NOCICEPTION IS GOOD IF YOU COMPARE MORPHINE TO LAC LAC LAC LACTOMORPHIN. YOU CAN SEE ON THE RIGHT THE MORE TEEN INDUCED TOXICITY OR DOPAMINERGIC EFFECTS OF THE DRUG THIS IS AN A90 DOSE AND SEE THE TAIL AND INCREASED MOTION AND AVOIDANCE OF THE OPEN FIELD ASSOCIATED WITH NARCOTIC INTOXICATION. ON THE OTHER HAND THE AFFECT OF DOSE OF THE LACTOMORPHIN WE GET NORMAL BEHAVIOR AND THERE'S AN ALL SUITE OF SIDE AFFECTS. AS I SAID WE THOUGHT THIS DRUG WOULD GO IN THE CLINIC AND HAD A FACTIONAL KILO GRAM AND READY TO DO STUDIES IN HYPOBULIMIC PICK WHEN THE STUDY WAS GIVEN TO THE ARMY. WE CONTINUED BY MAKING ENDORPHIN BASED DRUGS. WE HAVE THE SAME MESSAGE SEGMENT BUT WE HAVE AN ENDO PATHIC HELIX TO THIS MOLECULE TO BE ABLE TO MODULATE THE MEMBRANE AFFINITY. AND IN THE ABSENCE OF THE MEMBRANE YOU SEE RANDOM COILS AND IN THE PRESENCE OF A MEMBRANE OR MEMBRANE SURROGATE SUCH AS A MOUSE YOU SEE HIGHLY STRUCTURED COMPOUNDS. IN THE TRUE MEMBRANE YOU USING THE BILAYERS YOU SEE THE AFFINITY OF THE COMPOUNDS FOR THE MEMBRANE IS QUITE HIGH. SEVEN NANOMOLAR KDs IN THE ABSENCE OF A RECEPTOR SO THIS IS A BINDING JUST TO THE MEMBRANE. THIS IS PROBABLY TOO HIGH A BINDING BUT IF WE HAD A SECOND SUGAR YOU'D SEE WE'D GO TO THE MICROMOLAR REGIME WHICH GETS US TO AN AREA WHERE WE CAN DELIVER THE DRUG. IF THERE'S NO CARBOHYDRATE AT ALL, THE COMPOUNDS BECOME MEMBRANE DISRUPTIVE AND THIS IS ALL PREDICTED BY MELETIN AND HERE'S THE ARRANGEMENT WHERE YOU CAN SEE BY SIMPLY CHANGING THE PRESENCE OR ABSENCE OF THE GROUPS AND THIS IS GLYCINE OR THESE LISTED WE CAN AFFECT THE HELICITY. TO DIAL IN THE INTERACTION AND MODULATE THAT FURTHER BY GLYCOSYLATION. WE SEE LACTOCIDE -- LACTOSIDE SPENDS LESS TIME AND THIS FALLS OUT OF HELIX AND THIS DATA IS IN WATER. THIS HAD TO BE COLLECTED IN THE PRESENTATION OF SDS.HELIX AND THIS DATA IS IN WATER. THIS HAD TO BE COLLECTED IN THE PRESENTATION OF SDS.AND THIS DATA IS IN WA TER. THIS HAD TO BE COLLECTED IN THE PRESENTATION OF SDS. YOU SEE THE POTENCY IS DIFFERENT FROM THE TAIL FLICK AND WHAT IS INTERESTING IS NOT THE SHAPE OF THE CURB -- CURVE BUT THE AMOUNT REQUIRED AND THOUGH WE TRIPLE THE COMPOUND BELOW WHERE A KILO OF BINDING FOR THE DRUG. WE WANT TO CONTINUE WORKING WITH THESE COMPOUNDS BECAUSE WE KCAN MODULATE THE RECEPTOR PROFILE QUITE EASILY. AND THIS IS THE MORE TEEN OUT HERE. -- MORPHINE OUT THERE. THERE WAS INTEREST IN GLUTINIZING HORMONE AND WAS ABLE TO STABILIZE SUBSTITUTING FOR THE GLYCINE AND WE SAID WHY DON'T YOU TRY DECREASING AND WHEN THEY MADE THE LACTOSIDE WE HAD A HUGE GAIN IN SERUM STABILITY AND THE COMPOUND WAS ORALLY ACTIVE. THE FUN PART HERE IS TO SHOW THE COMPOUND HAS CLINICAL TRANSLATION POTENTIAL. I THINK EVERYONE'S FAMILIAR WITH ANGIOTINSIN AND PROBABLY WHAT'S MORE INTERESTING FOR US -- ANGIOTENSIN -- AND THERE WAS INTEREST IN DEVELOPING ANGIOTENSIN DERIVATIVES FOR TE BRAIN AND LET ME SPEND TIME TALKING ABOUT THAT. HERE ARE OUR ANTI-PATHIC STRUCTURE IS KIND OF U SHAPE AND THE LIPOTHELIC RESIDUES BINDS AND BY ADDING SUGARS WE CAN ADJUST THE AFFINITY FOR THE MEMBRANE ANDENGAGE IN MEMBRANE HOPPING. THIS IS EX VIVO AND GOING FROM ANGIOTENSIN TO THE SERRIN WE LOSE IT BECAUSE IT'S MORE EASILY CLEAVED BUT AS WE START ADDING THE CARBOHYDRATES YOU CAN SEE THE INCREASED STABILITY AND THE GLUCOSIDE IS MORE STABLE AND IF WE GO TO THE LACTOSIDE IT GETS MUCH MORE STABLE. AND TO FURTHER DEVELOP. THIS ISN'T THE ONLY COMPOUND IT WORKS WITH. WE HAVE THE OPIATE BASED PEPTIDES WE TALKED ABOUT SO OXYTOCIN COMPOUNDS THAT FORTUNATELY I WON'T BE ABLE TO TELL YOU VERY MUCH ABOUT AND THE COMPOUNDS WHICH ARE REPAIR PEPTIDES, IF YOU WILL, THAT'S TOO LONG A STORY TO TALK ABOUT TODAY BUT THE ANGIOTENSINS LOOK INTERESTING IN VIVO AND THE CONCENTRATION FOLLOWS MORE OR LESS THE EX VIVO WORK AND THE PENETRATION INTO THE CSF IS REASONABLY GOOD AND THE COMPOUNDS HAVE BEEN SYNTHESIZED ON A MULTI KILO SCALE AND DOING A FIRST IN MAN STUDIES FOR POST-SURGICAL DEMENTIA OR VASCULATURE DEMENTIA AND THE STUDIES ARE LOOKING PRETTY GOOD. AND FINALLY, I NEED TO SAY SOMETHING ABOUT FORMULATION OF THE COMPOUNDS. I GUESS IF YOU'RE USING CERTAIN TYPES OF ADMINISTRATION AND WE WANT INTRANASAL ADMINISTRATION AND HERE YOU CAN SEE AS WE TAKE OUR PNA5 YOU GET PARTICLES SOMEWHAT DELIVERABLE BUT YOU GET THESE CANDIES, IF YOU WILL, THAT ARE IMMENSELY DELIVERABLE VIA INHALATION. I'M TRYING TO SUGGEST TO YOU IT'S A GENERAL PROCESS. I CAN'T TELL YOU ABOUT THE OXYTOCINS OR GIVE YOU STRUCTURES FOR THE NEW AGONISTS WE'RE WORKING WITH TULANE BUT SUGGEST COMPOUNDS ON THE MARKET SIMILAR IN STRUCTURE. HERE'S SIMILAR TAIL FLICK DATA IN MALE AND FEMALE MICE. YOU CAN SEE NOT ONLY DO WE GET GOOD ACTIVITY BUT WE GET VERY GOOD DURATION OF ACTION HERE WE HAVE DATA AFTER FIVE HOURS AFTER ADMINISTRATION. THIS IS SUBCUE ADMINISTRATION. THIS IS THE SEX DIFFERENCE BETWEEN THE MALE AND FEMALE MICE BUT CLEARLY ALL THE WE SEE QUITE A BIT OF IMPROVEMENT FROM THE GLYCOSYLATION. I WANT TO BRIEFLY PLUG OUR NEW JOURN JOURNAL. WE'D LIKE TO CAST A WIDER NET FOR WHAT WE CALL NEUROLOGICAL DRUGS. I HOPE YOU'D BE WILLING TO SUBMIT PAPERS. IF YOU'RE NOT FAMILIAR THE INITIAL PROCESS IS BLINDED BUT IN THE END THE REVIEWERS NAMES ARE PUBLISHED ALONG WITH THE AUTHORS. THIS LEADS TO A MUCH FINER MATCHING OF THE EXPECTATION OF THE READER TO THE AUTHORS. THANK YOU VERY MUCH FOR YOUR TIME AND I'LL STOP HERE. >> THANK YOU VERY MUCH. ANY QUESTIONS? >> I HAVE A QUESTION AS WE'RE WAITING FOR THINGS TO COME IN FROM THE ONLINE AUDIENCE. I KNOW YOU CAN'T TALK ABOUT YOUR OXYTOCIN DRUG BUT ONE IS INJECTABLE PEOPLE USE ALL THE TIME, PETOCIN AND DIDN'T CROSS THE BLOOD BRAIN BARRIER. DOES YOURS? >> ABSOLUTELY AND THAT'S THE WHOLE POINT. THIS IS A GENERAL IF YOU LOOK AT PEPTIDE LIGANDS FOR PGCRs THEY HAVE DIFFERENT AFFECTS THAN PERIPHERALLY. WOULD A PREGNANT FEMALE BE USING OUR DRUG? IT'S NOT CLEAR THAT PATIENT POPULATION WOULDN'T BE EXCLUDED FROM USE OF THESE DRUGS. THE CENTRAL AFFECTS ARE DIFFERENT. WE SEE IN TERMS OF TAIL FLICK LOOKS LIKE MORPHINE FOR ABOUT 30 MINUTES. I DON'T THINK ANALGESIA IS SOMETHING YOU TYPICALLY ASSOCIATE WITH OXYTOCIN. AND EVERYBODY IS AWARE OF THE FACT THAT OXYTOCIN PROMOTES NEST BUILDING BEHAVIOR AND AFFECTION TOWARDS THE END GROUP AND THIS SORT OF THING. IT ALSO INCREASES AGGRESSION TOWARDS THE OUT GROUP. THE SIDE EFFECT WHEEZE WORRY ABOUT FOR THIS DRUG WOULD BE HYPER AGGRESSION. THIS A LOT OF WORK THAT NEEDS TO BE DONE BETWEEN MOUSE WORK AND CLINICAL WORK. BUT THE PANEL WAY FOR OUR DICOSYLATED VERSION AND THE OPIATE S I SHOWED YOU AND DIDN'T SHOW YOU I THINK ARE ALL VIABLE CLINICAL CANDIDATES. SOMETIMES THEY'RE PSYCHOLOGICAL. IF WE CAN GET A SPONSOR FOR OPIATE ANALGESICS WE'D CONSIDER IT. AND THE COMPOUNDS LIGANDS ALL SEEM TO HAVE THE SAME SORT OF AFFECTS YOU'D HAVE IN TERMS OF ADDICTION LIABILITY. ON THE OTHER HAND, WITH THE U PLUS DELTA YOU GET A DIFFERENT PROFILE THAT'S NOT SO ADDICTING. WE SEE THIS IN NOT JUST IN ANALGESIC PROPERTIES BUT AND WE CAN SEE VERY DIFFERENT AFFECTS ON THE LOCOMOTION. AND EMILY UNIVERSITY OF MICHIGAN HAS TRAINED A GROUP OF MICE TO REACT TO THE COMPOUND. IT'S NOT CLEAR TO SEE WHAT DRUGS THEY DON'T RESPOND TO. AND THIS IS I THINK TYPICAL OF ALL THE PEPTIDE BASED NEUROTRANSMITTERS. THEY ALMOST ALWAYS HAVE MULTIPLE RECEPTORS AND ALMOST ALWAYS DON'T HAVE SELECTIVITY. IF YOU LOOK AT THE ENDOGENOUS OPIOIDS YOU SEE U PLUS KAPPA OR DELTA BUT YOU NEVER SEE PURE GAPA OR GABA. >> SO IN ADDITION TO THESE COMPOUNDS BEING DRUGS DO YOU EXPECT THEM TO BE SIGNALLING DIFFERENTLY? FOR EXAMPLE, DO THEY HAVE ANY BIAS? >> I SEE WHAT YOU'RE SAYING. ONCE THEY BIND TO THE RECEPTOR, THE RECEPTOR'S INTERNALIZED AND THEN THE NORMAL PROCESS IF IT'S' PEPTIDE LIGAND RETURN TO THE SURFACE OF THE CELL. AND MORPHINE IS PATHOLOGICAL BECAUSE IT DOESN'T LET GO OF THE RECEPTOR. THAT RECEPTOR'S DISPATCHED TO A LYSOSOMAL COMPARTMENT WHERE IT'S DESTROYED. AND MORPHINE CROSSES TO BIND TO RECEPTORS THAT AREN'T AT THE CELL SURFACE YET. SO WE'RE INTERACTING WITH A DIFFERENT POPULATION OF RECEPTORS THAN WITH AN ALKALOID TYPE OF DRUG. >> THANK YOU. WE'RE ON TIME. THAT WAS A WONDERFUL PRESENTATION. >> THANK YOU SO MUCH. OUR NEXT SPEAKER IS DR. KIM JANDA. HE'S FROM THE SCRIPPS RESEARCH INSTITUTE WHERE HE'S PROFESSOR OF CHEMISTRY AND DIRECTOR OF THE WANG INSTITUTE FOR RESEARCH AND MEDICINE. TODAY WE'LL BE PRESENTING SOME WORK THAT IS VERY RELEVANT TO NIDA AND HAS INVESTED IN THIS AREA WHICH IS OPIOID VACCINES. DR. JANDA, PLEASE. >> I WAS ON A ZOOM MEETING THIS MORNING AND COULDN'T GET IT GOING SO THAT'S A BIG RELEECH THERE. -- BIG RELIEF THERE. HERE'S THE TITLE AND WHERE WE'RE AT AND WHO I AM. WE'RE GO FORWARD FROM THERE. I HAVE THE LECTURE OUTLINED IN FIVE DIFFERENT AREAS. SO BECAUSE I'M KIND OF A LITTLE BIT OF AN OUTSIDER HERE IN TERMS OF VACCINES I DON'T THINK ANYBODY'S GOING TO TALK ABOUT THAT, I'LL GIVE HISTORIC PERSPECTIVE ESPECIALLY WITH THE COVID STUFF GOING ON NOT IT'S WORTHWHILE AND TALK ABOUT HOW TO USE THIS FOR ADDRESSING WHAT WE'VE DONE FOR COCAINE AND FENTANYL AND A MONOCLONAL AND I LIKE TO CALL BITTER-SWEET AT THE END AND CONCLUSIONS. VACCINES HAVE A LONG HISTORY ORIGINALLY STARTING WITH THE CHINESE EMPLOYING TAKING DRIED PUSTULES AND INHALENTS AND NOT DIFFERENT THAN A LOT OF VACCINES NOW BEING USED FOR INHALATION. THE FATHER OF VACCINOLOGY I'M SURE PEOPLE HEARD ABOUT THE STORY OF THE YOUNG BOY WHO WAS ILL AND THE ASSOCIATION WITH WOMEN TO MILKING COWS AND HOW THEY WERE ABLE TO USE THIS TO PREVENT HIS DEATH. THE FIRST REAL VACCINE DONE IN THE LABORATORY DONE BY PATEUR AND ALUM IS PROBABLY IN 95% OF ALL VACCINES STILL BEING PRODUCED TODAY AND THAT'S ALMOST 100 YEARS OLD. AND A THEME IS TRYING TO COME UP WITH NEW ADJUVANTS IS IMPORTANT. PROBABLY ONE PERSON NO ONE'S HEARD ABOUT BUT PROBABLY SAVED MORE LIVES THAN ANYBODY IS HILLEMAN AT MERCK AND DISCOVERED VACCINES FROM MEASLES TO MUMPS AND IS KIND OF A FORGOTTEN GUY AND WE SEE THE MARCH OF DIMES STARTED BASED ON ROOSEVELT SAYING SENDING YOUR DIMES IN AND MILLIONS OF DIMES IS HOW IT STARTED. I'LL HAVE TO UPDATE THIS AT SOME POINT FOR THE COVID VACCINE BUT I'M NOT GOING TO YET BECAUSE I'M NOT SATISFIED WITH EVERYTHING GOING ON YET SO I'LL LEAVE THIS OUT. OUR VACCINES ARE A LITTLE BIT DIFFERENT THAN MOST IN THAT YOU'RE NOT GOING TO CAUSE AN IMMUNE RESPONSE. WE HAVE TO TRAIN THE IMMUNE RESPONSE TO SEE THESE AS FOREIGN. WE HAVE THREE AREAS ALL DEPENDENT ON EACH OTHER AND IF ONE IS FULLY DONE AND THE VACCINE FAILS. THE HAPTEN LINKER DESIGN AND THE CARRIER PROTEIN, SO THE EPITOPE AND ADJUVANTS. SO BACKGROUND I LIKE TO CALL THIS PROCESS IMMUNOPHARMACO THERAPY. IT'S A TERM I CAME UP WITH LIKE 30 YEARS AGO. AND IT'S DEVELOPING THE HAPTEN PROTEINS CONJUGATES AND SEQUESTER THE DRUG OF ABUSE. AND ADJUVANTS IN THIS CASES SHAPE THE OVERALL IMMUNE RESPONSE IN TERMS OF THE SPECIFICITY AND HOW MUCH ANTIBODY YOU GET. SO COMMON ADJUVANTS LIKE I TALKED ABOUT IS ALUM BUT OTHER ONES ARE CPG, MPLA THEY'RE ALL TOLL RECEPTORS AND PEOPLE ARE DEVELOPING THESE AND THEY'RE STRUGGLING TO GET INTO CLINICAL TRIALS BECAUSE A NUMBER OF ADVERSE SIDE AFFECTS. SO WITH THIS SAID, HOW DOES THIS OVERALL WORK? SO BASICALLY WE'RE GOING TO CREATE THE VACCINES TO BE TAKEN UP BY DENDRITIC CELLS TO BE PROCESSED AND PUT ON TOP OF THE MHC2 WHICH WILL THEN GENERATE T HELPER CELLS WHICH WILL THEN ACTIVATE EVENTUALLY B CELLS WHICH UNDER GO CLONAL EXPANSION WHICH GET MEMORY B CELLS AND IT'S VERY SIMILAR TO MOST VACCINES THAT ARE OUT THERE. IT'S JUST A LOT DIFFERENT HOW IT'S PUT TOGETHER. HOW DO WE FALL IN WITH WHAT I CALL THE ALFA -- ALPHA GAL. WE'RE TRYING TO TAKE ADVANTAGE OF NATIONALLY OCCURRING ANTIBODIES THAT EVERYONE HAS. SO ANTI-GAL CONSTITUTES 1% OF CIRCULATING IGG IN HUMANS AND ABSENT IN RODENTS. AND HOW ARE WE TAKING ADVANTAGE OF THIS? BASICALLY WITH THESE NATURALLY OCCURRING ANTIBODIES WE'RE GOING TO TRY TO DESIGN SOMETHING WE'LL SUCH THE ANTIBODIES BIND TO THIS AND THEY'LL SEEK OUT THE GAMMA RECEPTOR AND THIS IS GOING TO BE TAKEN UP INTO ANTIGEN PRESENTING CELLS AND GO THROUGH THE WHOLE PROCESS TALKED ABOUT. THIS IS IN A SENSE A WAY TO ADJUVANT USING OUR OWN ENDOGENOUS ANTIBODIES TO BASICALLY BRING THE VACCINE FORWARD. MOST OF THESE TYPES OF VACCINES DEVELOPED AND USED LIPOSOMES AND IN TERMS OF USING THE VACCINES WE DEVELOPED SO I SHOW A BIG ONE WE LOOKED AND NICOTINE AND TO THE RIGHT IS COCAINE. DOWN ON THE BOTTOM IS METH AMPHETAMINE AND OPIOID. WE'LL JUST FOCUS ON COCAINE AND THE OPIOIDS TODAY. PROBABLY THE BIGGEST HURDLE WAS GETTING A HOLD OF THE KNOCKOUT MICE. THE MICE THAT WERE BASICALLY DEFICIENT IN THE ENZYME. A GRAD STUDENT A NUMBER OF YEARS AGO WAS VERY PERSISTENT AND IT TOOK A YEAR TO GET THE MICE. ORIGINALLY THEY WERE DONE BY JOHN LOWE OUT IN MICHIGAN AND IN TRACKING THIS DOWN IT WAS ALL LICENSED OUT TO A COMPANY IN AUSTRALIA. TO GET THIS I THINK I HAD TO SIGN AWAY MY FIRST BORN, NO, JUST KIDDING BUT WE COULDN'T GET THE RIGHTS OUR OR LICENSE TO ANYBODY AND IT'S DIFFICULT TO GET A HOLD OF BUT WE'RE NOT ACTIVELY USING IT AT THIS TIME. I WON'T GO THROUGH THE SYNTHESIS. MORE ORGANIC CHEMISTS DON'T WANT TO WORK DOING SUGAR CHEMISTRY. WE DID GO ABOUT AND THERE'S LITERATURE PRECEDENCE HOW TO GO ABOUT THIS. SO WE WERE USING IN THE CASE THE THIRD RESIDUE HERE. THIS IS THE PREFERRED ANTIGEN BINDING FOR THESE ENDOGENOUS ANTIBODY. THE KEY PIECE THAT SIMPLIFIED THINGS IS WE USED THIS LINKER BASE AND ACID AND THIS ALLOWED US TO DO COUPLES WITH THAT AND DO CHEMISTRY MANIPULATION. WE'RE THE ONLY CHEMIST CHANGING THE PH. THIS IS A GOOD WAY TO PUT THIS ON. ONE WAS THE COMPOUND WE'RE USING THIS TO GENERATE AN IMMUNE RESPONSE FOR THESE KNOCKOUT MICE SO THIS TAKES A COUPLE MONTHS FOR DEVELOP AND THEN WE'LL CONJUGATE THAT AND IN THIS CASE YOU CAN SEE COCAINE FOR THE IMMUNIZATION PROCESS. WE USED A COUPTHESE ALONG WITH COMAIN AND THE YOU CAN SEE WE GOT RELATIVELY GOOD TIDERS AND THE KEY ASPECT WAS THE AFFINITY TO COCAINE. IT VARIED WIDELY YOU SEE THE NANOMOLS. YOU HAVE TO HAVE HIGH AFFINITY TO THE DRUG OR IT'S NOT GOING TO WORK AND WE GOT IT TO NUMBER SEVEN. WE'RE NOT ALLOWED TO SHOW MICE RUNNING AROUND SO WHAT WE LOOK FOR IN TERMS OF MONITORING THE ACTIVITY SO COCAINE'S A STIMULANT AND WE RECORDED THEIR MOVEMENT AND DISTANCE TRAVELED. SO WHEN WE GOT THE DOSE WE DIDN'T SEE MUCH DIFFERENCES FOR THE VACCINES BUT WHEN WE WENT TO 20 MIGS THE BEST WAS WITH THEO THEO -- THE OVA AND SOME OF THE BEST WE'VE SEEN WITH THE VACCINE. WE USED THIS FOR COCAINE. CAN WE USE IT FOR FENTANYL. THIS IS A PENNY SHOWING TO SCALE THE LETHAL DOSE AND YOU CAN SEE THIS IS A DERIVATIVE SO FENTANYL IS REMOVING THE THREE COMPONENTS AND WE CAN GET THESE TO DIFFERENT FENTANYL AND WHERE WE'VE DONE OTHER VACCINES AND HERE'S IT'S A BROADLY NEUTRALIZING VACCINE TO THE FENTANYL ANALOGS. ZAIR WE OF IN TERMS OF THE ANTIBODY RESPONSE IT DIDN'T MAKE A WHOLE LOT OF DIFFERENCE WHETHER WE DID THE FENTANYL GAL OVA OR AT SUBSEQUENT BOOSTS AND THE ANTIBODY RESPONSE WERE QUITE SIMILAR. WHERE WE SEE THE DIFFERENCES IS AFFINITY AND WE SAW THE BETTER AFFINITY FOR THE OVA ON ALL THREE VACCINES. WE HAD A NUMBER OF WAYS AROUND USED THE TAIL FLICK AND WARM WATER AND USED AN IMAGING. I WANT TO FINISH UP WITH WHAT YOU CALL THE BITTER SWEET CARFENTANIL ANTIBODY. WE'RE TRYING TO TRYING IT DEVELOP THE VACCINE VERSUS ADDICTION AND EVERY DOSE. -- OVERDOSE. I USE IT GET HUGE PUSHBACKS WITH OUR VACCINES BECAUSE IT TOOK MULTIPLE DOSES AND WE SEE WITH COVID IT'S NOT THE CASE AND NO ONE WILL TAKE MULTIPLE VACCINES SO THAT'S NOT TO THE CASE ANYMORE SO I DON'T WANT TO HEAR THAT. OVER TERMS OF THE ANTIBODY WE WOULD SEE THIS FOR TOXICITY OVERDOSE AND CARFENTANIL WHERE YOU USE NALOXONE WHICH CAN REVERSE THE AFFECTS NALOXONE HAS A SHORT SHELF LIFE AND THIS CAN STAY ON BOARD DEPENDING ON HOW YOU SET IT UP FOR HOURS, DAYS, WEEKS TO MONTHS. I'LL SHOWING THE DIFFERENT ANTIBODY MANIFOLD. THE NEXT IS THE SINGLE CHAIN AND I TALKED ABOUT IDG. AND DEPENDING ON HOW YOU ENGINEER THEM ONE OF OUR LEAD ANTIBODIES IS SHOWN HERE IS CALLED 1A4. THIS HAS AFFINITY TO CARFENTANIL AND FENTANYL 10 TO MINUS 13 AND WILL COMBINE DIFFERENT ANALOGS. WE'RE MOVING THIS FORWARD IN THE CLINIC AND WE HAVE A CRYSTAL STRUCTURE AND WE FOUND WE HAD A SUGAR ATTACHED TO AND DEVELOPING ANTIBODIES FOR CLINICAL TRIALS IS LIKE A NO-NO AND CAUSES A LOT OF TROUBLE WITH MANUFACTURERS. WE HAD TO SEE IF WE CAN GOT THIS OUT. HERE'S ANOTHER PICTURE AND YOU CAN SEE CARFENTANIL THERE. IT'S A DISTANCE AWAY BUT THIS MORE IN TERMS OF THE MANUFACTURING AND WE WOULDN'T HAVE KNOWN THIS WITHOUT THE CRYSTAL STRUCTURE. IT'S FORTUNATE WE WENT FORWARD TO DO THIS. THIS IS NOT SOMETHING THAT'S ISOLATED. BACK IN 2007 AMGEN REPORTED A SIMILAR SCENARIO YOU CAN SEE HERE AND WE NEEDED TO ACTUALLY TRY TO REMOVE THIS. SO WHAT WE DID WAS BASICALLY WE PUT THIS ANTIBODY INTO A DISPLAY SYSTEM AND WE BASICALLY MUTATED THAT RESIDUE WITH THREE OTHER RESIDUES. SO BEST ONE IS THE LYSINE AND WE HAVE A CRYSTAL STRUCTURE WITH THE LYSINE NOW REPLACING THAT AND THIS IS SHOWING WELL, THIS IS CARFENTANIL AND HIS SHOWS WHERE WE'VE REMOVED THE RESIDUE AND AND MANUFACTURE. CONCLUSIONS, THIS OVERALL LECTURE IS THAT USING THE DOUBLE CONJUGATION IS A GOOD WAY TO MOVE FORWARD IN TERM OF USING THE EPITOPE FOR CREATING THESE VACCINE CONJUGATES AND IT DIFFERS FROM MOST PEOPLE WHO USE LIPOSOMES. THE ANTIBODIES ARE PROTECTIVE IN TERMS OF DRUG EXPOSURE WITH REGARDS TO THE COCAINE VACCINE. THE VACCINE WAS NOT MUCH BETTER THAN THE ONES WE HAVE IN CPG. AND WE HAVE THIS UNWANTED SUGAR. AND WITH THIS SAID, THIS IS THE GROUP THAT DID THE WORK. AND IF THERE'S ANY QUESTIONS, I'D BE HAPPY TO TRY TO GET TO THEM OR LOOKS LIKE THERE'S A CHAT HERE OR SOMETHING. IS THAT RIGHT? >> I BELIEVE YOU HAVE BOTH NATURAL AND SYNTHETIC ADJUVANTS. IS THERE A PREFERENCE FOR EITHER? HAS ANYONE STUDY THE MECHANISM OF ACTION OR BIOMARKERS OF INTEREST? IN TERMS OF THE VACCINE, IN REGARDS TO ALL HUMANS HAVE THIS AND THOUGHT WE'D HAVE AN EASIER WAY OF DEVELOPING THE VACCINE. IT WORKED WELL. THE ANIMAL MODELS WE ARE USING IS LIMITED. IN TERMS OF ANTIBODIES WE'RE DOING IT WOULD REQUIRE USING THIS APPROACH. I FEED THAT IN BECAUSE WE HAVE THIS SUGAR RESIDUE AND BECAUSE IT'S GLYCOSCIENCE THIS IS A CASE WHERE WE DIDN'T WANT THAT TO COME INTO PLAY. WE'RE TRYING A LOT OF DIFFERENT APPROACHES IN TERMS OF TRYING TO DEVELOP IN TERMS OF MOVING FORWARD WHICH IS THE BEST APPROACH. IN TERMS OF NIDA AND WHAT THEY'RE DOING IN REGARDS TO YOU GUYS, IT DEPENDS IN TERMS OF THE COUNTER ACT, YOU WANT TO HAVE SOMETHING THAT'S GOING TO BE AS WE KNOW INJECT ABABLE I KNOW IT'S NOT AS APPROPRIATE WHERE WE ARE TRYING TO DO AN IM TYPE DELIVERY AND USING A SINGLE CHANGE WHICH ARE SMALLER, GET IN QUICKER AND IT DEPENDS ON WHAT YOU'RE LOOKING FOR AND HOW YOU WANT TO MOVE FORWARD. DOES THAT ANSWER THE QUESTION? >> YES. I THINK DR. WONG HAS A QUESTION. >> HI, VERY INTERESTING TALK. MY QUESTION COMES FROM THE CLINICAL SIDE IN TORONTO HERE IN CANADA I MOSTLY WORK IN EMERGENCY AND I SEE A LOT OF PATIENTS, OPIATE OVERDOSE AND STIMULANT PSYCHOSIS, MOSTLY AMPHETAMINE. A COUPLE GLYCO QUESTIONS WHEN DO YOU THINK THIS WILL BE WIDELY AVAILABLE FOR CLINICAL USE? THERE'S A COMPANY CALLED CESSATION THAT LICENSE THE TECHNOLOGY A COUPLE OF MY STUDENTS AND THEY HAVE PUT THIS INTO MANUFACTURE THROUGH A GRANT WITH NIDA IN TERMS OF DOING THAT. IF THEY CAN RAISE MONEY THEY SHOULD BE ABLE TO MOVE FORWARD BECAUSE WE TESTED OUT THE MANUFACTURED ANTIBODY AND IT WORKS PRETTY MUCH THE SAME. I THINK WOULD THINK THERE SHOULD BE THE NEXT THREE TO SIX MONTHS WITH AN IND IN THIS IF THEY HAVE ENOUGH MONEY AND THEN SHOULD BE ABLE TO GO FORWARD THEY'RE OVERALL APPROACH, I BELIEVE IN GOING FORWARD IS TO LOOK AT THIS FOR AN APPROACH FOR TOXICITY. THE REASON BEING IS THAT WE I THINK IN TERMS OF TRYING TO GET PEOPLE FOR CLINICAL TRIALS IT WILL BE DIFFICULT FOR OVERDOSE AND ASKED THE EFFICACY IN ANIMALS AND SHOULD BE ABLE TO MOVE FORWARD. I THINK THE REAL ADVANTAGE IN THIS AND YOU COULD PROBABLY BETTER ADDRESS THIS FROM THE CLINICAL STANDPOINT IS PEOPLE WHO DO HAVE AN OVERDOSE IN THE SYNTHETIC OPIOIDS IT'S NOT SOMETHING YOU CAN JUST PUT IN AND RELEASE RIGHT AWAY BECAUSE OF THE NARCOLYZATION THEY MAY HAVE TO BE KEPT IN THE HOSPITAL A COUPLE DAYS AND I HAVE SLIDES WHERE WE HAVE THE ANTIBODY DOESN'T HAVE THIS AND THE NALOXONE DOES AND THE ANTIBODY WOULD HAVE VALUE IN THAT CASE AND ONE ANTIBODY I SHOWED HAS A HALF LIFE OF TWO MONTHS. MOST ADDICTS ONCE THEY THINK THEY'RE OKAY ARE BACK USING SO THOUGH WE'RE USING THIS FOR AN OVERDOSE IT WOULD HAVE VALUE IN POSSIBLY TREATING SUBSTANCE ABUSE DISORDER. IF THERE'S MORE MONEY IT CAN MOVE FORWARD QUICKLY BECAUSE THE MANUFACTURING IS. >> WHAT'S THE NAME OF THE COMPANY AGAIN? >> CESSATION. C-E-S-S-A-T-I-O-N. YOU'LL FIND THE WEBSITE. >> AND WHAT DO YOU THINK OF THE SAME APPROACH FOR CRYSTAL METH AMPHETAMI AMPHETAMINE. THAT'S ALSO THE REAL SCOURGE IN OUR SYSTEM. THERE ISN'T A NIGHT THAT GOES BY WITHOUT SOMEONE COMING IN WITH METH-INDUCED PSYCHOSIS. >> IT'S MORE DIFFICULT TO DEVELOP IT AGAINST METH AMPHETAMINE BECAUSE IT ALMOST LOOKS LIKE AN AMINO ACID. IT'S NOT TOO SMALL IT'S HARD TO GENERATE IMMUNE RESPONSES SO WE'VE DONE TRICKS ON THAT AND WE ARE HOPING TO MOVE FORWARD WHICH I'M TRYING TO DEVELOP A VACCINE AGAINST METH AND COCAINE TOGETHER OR METH AND FENTANYL. UP WITH THING WE'RE LOOKING AT WHICH IS DIFFERENT, WE'VE DEVELOPED VACCINES AGAINST FENTANYL AND HEROIN AND WE CAN MIX THEM AND YOU'LL GET RESPONSE TO BOTH. TALKING TALKING TO THE FDA THEY WON'T ALLOW USING TWO VACCINE TO GO FORWARD. YOU'LL HAVE TO GET ONE PROVED AND THE OTHER ONE APPROVED BULL YOU CAN HAVE THEM TOGETHER. WHAT I'M TRYING TO DEVELOP AND TRYING TO MAKE WHERE WE HAVE BOTH DRUGS ON THAT SO WE'LL GENERATE IMMUNE RESPONSE IN ONE SHOT TO GET AWAY FROM THIS AND THAT'S NOT TRIVIAL TO DO BUT SOMETHING THAT CAN BE DONE. >> REALLY EXCITING RESEARCH. THANKS. >> I THINK WE HAVE TIME FOR ONE QUESTION. I SEE ONE HERE. WAS THERE A DIFFERENCE IN THE LOCOMOTOR ASSAY YOU'RE SEEING HIGHER IN THE VACCINATED MICE. >> WE'RE SEEING THE LOCOMOTOR ASSAY IN TERMS OF COCAINE? WE DO LOCOMOTOR ASSAY FOR OPIOIDS. THAT'S A LOWER DOSE. WE ONLY DO ANTI-NOCICEPTION IN CARDIOVASCULAR. THE THINGS WE'RE DOING REGARD TO OPIOIDS WE HAVE TO USE A LOWER DOSE. >> IF NOT, THANK YOU, DR. JANDA. >> GOOD AFTERNOON AND EVENING WHEREVER EVERYONE'S AT. >> THANK YOU. >> CHEERS. >> SO OUR NEXT SPEAKER FOR THIS AFTERNOON IS DR. ALBERT KENNEDY. HE'S A PROFESSOR OF CHEM I HAVE -- CHEMISTRY AND LOOKING AT NEXTGEN MICRODIALYSIS AND MULTIPLEXED IN VIVO CHEMICAL MONITORING WITH HIGHER SPATIAL AND TEMPORAL RESOLUTION. >> THANK YOU. AS THE TITLE INDICATES I'LL TELL YOU ABOUT WORK WE'VE BEEN DOING TO TRY TO IMPROVE SOME CHEMICAL MEASUREMENTS IN THE BRAIN. I HAVE TO SAY I'VE ENJOYED THE SESSION AND SYMPOSIUM WE DON'T DO ANYTHING WITH GLYCOSCIENCE AND DONE VERY LITTLE WITH NEURODEGENERATIVE DISEASE AND WHEN THEY ASKED I WASN'T SURE THEY HAD THE RIGHT PERSON FOR THE SESSION BUT HOPEFULLY THIS WILL GENERATE SOME IDEAS. BEFORE I GET INTO MY TALK, I WANT TO ACKNOWLEDGE MY STUDENTS WHO ARE LISTED HERE AND HAVE DONE THE WORK AND ALSO WANT TO ACKNOWLEDGE OUR COLLABORATORS, SOME OF THE WORK I'LL DESCRIBE THAT HELPED US INTO GETTING METHODS ON INTERESTING BIOLOGICAL QUESTIONS. THE GOALS ARE TO IDENTIFY THE CHEMICAL SIGNALS CHANGING IN THE BRAIN AND DRIVING THINGS LIKE BEHAVIOR AND CHANGES WITH DISEASE STATES. AND THIS IS SUPPOSEDLY NEUROTRANSMITTERS BUT I THINK THERE'S INTEREST IN MEASURING OTHER COMPOUNDS AS WELL AND WE'RE TRYING TO EXPAND THAT. IT'S A HUGE TECHNICAL CHALLENGE THOUGH. WE KNOW THERE'S COMPOUNDS IDENTIFIED AS NEUROTRANSMITTERS OR NEUROMODULATORS. THEY'RE RELEASED IN SPATIALLY SPECIFIC REGIONS. AND THE BRAIN IS COMPLEX THERE'S LOTS OF CHALLENGES TO DO THOSE SORTS OF MEASUREMENTS. THERE'S BEEN SOME TECHNIQUES DEVELOPED THAT ACTUALLY CAN GET US THERE ONE IS TOMOGRAPHY I VIEW AS THE GOLD STANDARD METHOD BECAUSE IT CAN HELP YOU MEASURE THINGS LIKE NEUROTRANSMITTER RELEASE NONINVASIVELY AND IN HUMANS. IT'S A POWERFUL METHOD. AND THERE'S IMPLANTABLE SENSORS. THESE CAN BE MADE QUITE SMALL TO GIVE SPATIAL RESOLUTION. BE AN EXCITING NEW TECHNOLOGY IS GENETICALLY ENCODED SENSORS THAT CAN GET YOU BACK TO THE SPATIAL IMAGING CAPABILITY AND CAN HELP YOU MEASURE NEUROTRANSMITTER DYNAMICS AS WELL. AS GOOD AS THESE ARE THERE'S LIMITATIONS AND THE TECHNIQUES STRUGGLE WITH MEASURING BASAL CONCENTRATION AND QUANTIFYING CONCENTRATIONS. A AND WHAT WE SEE IS THE BASE OF DEVELOPMENT RELATIVELY SLOW FOR THE NEUROTRANSMITTERS AND THE FINAL ISSUE IS THEY TEND TO BE MEASURING ONE THING AT A TIME. THERE'S A WAY OF MEASURING TRANSMITTERS FROM METABOLITES TO GET A DEEPER PICTURE OF WHAT'S GOING ON. THE TECHNIQUE MANY ARE FAMILIAR WITH AND ROBIN POINTED OUT IS THE IDEA OF DOING SAMPLING IN THE BRAIN. MOST COMMONLY DONE BY MICRODIALYSIS SAMPLING. THE IDEA IS YOU CAN PUT THE MEMBRANE INTO THE PROBE INTO THE BRAIN. AND THEN MOLECULES RELEASED IN EXTRA CELLULAR SPACE DIFFUSE ACROSS AND FRACTIONALIZE THEM BY A TECHNIQUE AND THE POWER COMES FROM SEPARATING THE SAMPLING FROM THE ANALYSIS TO USE WHATEVER TECHNIQUE YOU NEED FOR ANALYSIS AND PROFESSOR HOLTZMAN I WAS DISAPPOINTED HE'S DONE FANTASTIC WORK MEASURING TAU TO MICRODIALYSIS SAMPLING AND FOR MEASURING LOTS OF COMPOUNDS INCLUDING THE PROTEINS. I MADE THIS RADAR PLOT WHERE WE THINK ABOUT SOME THREE FIGURES AND THE SENSORS YOU TEMPORAL AND SPATIAL RESOLUTION AND WE'LL TALK ABOUT WHY THAT IS AND HOW IT MIGHT GET BETTER AND THE SPATIAL RESOLUTION IS WORSE BECAUSE OF THE SIZE OF THE PROBES. AND YOU SEE HOW COMPLIMENTARY ALL THE METHODS FOR DOING BRAIN CHEMISTRY IN VIVO. WE WANT TO TALK ABOUT ON DOUBLING DOWN ON THE MICRODIALYSIS TO IMPROVE THAT AND ALSO SOME TECHNOLOGY WE'RE DEVELOPING TO TRY TO IMPROVE THE TEMPORAL AND SPATIAL RESOLUTION. I'LL START THE APPROACH BY HOW WE STARTED TO GET MORE INFORMATION FROM A DIALYSIS SAMPLE AND USING DIALYSIS FOR THE NEURO TRANSMITTERS AND THIS IS A SAMPLE FROM AN ANIMAL AND ASSAYS ARE USED FOR A FOCUSSED GROUP OF COMPOUNDS. PEOPLE DEVELOP LC WITH THE DETECTION FOR DOPAMINE, NOR EPINEPHRINE AND SEROTONIN AND IF THEY WANT TO USE AMINO ACIDS THEY USE FLUOROESSENCE -- FLUOROESC FLUOROESC FLUOROESCENCE AND I WON'T GO THROUGH AT THE SPECIFICS BUT IN YOU SEE WE CAN GET ACETYL CHOLINE AND SOME OF THE METABOLITES. WE CAN SAY THE PEPTIDES ARE A DIFFERENT STORY. AND WE HAVE A COLLABORATION WITH A GROUP AT THE UNIVERSITY OF MICHIGAN. AND YOU CAN TELL THE DIFFERENCE TWO A HIGH RESPONDER RAT IF YOU PUT IT IN AN OPEN CAGE IT WILL RUN AROUND A LOT AND YOU CAN SEE THE TRACK DOING A LOT OF EXPLORING AND THE LOW RESPONDER ONE TENDS TO STAY IN THE CORNERS. AN INTERESTING THING ABOUT THESE ANIMALS IS THAT THE HIGH RESPONDERS ARE MORE PRONE TO SELF-ADMINISTER COCAINE COMPARED TO THE LOW RESPONDERS. THEY'VE BEEN INVESTIGATED AS A MODEL OF VULNERABILITY TO DRUG ABUSE AND OTHER THINGS ABOUT THEM THAT CORRELATE TO WHAT WE KNOW ABOUT HUMANS ON THE LIST. THERE'S BEEN MANY STUDIES ON THE BEHAVIOR OF THESE ANIMALS AND DIFFERENTIATING THEM AND SO ON AND UNDERSTANDING THE NEUROTRANSMITTER DIFFERENCES BETWEEN THEM THERE'S BEEN VERY LITTLE AND NOT MUCH HAD BEEN FOUND TO DIFFERENTIATE THE ANIMALS. WE DID AN EXPERIMENT USING OUR METHODS WITH THE LAB AND WHAT WE DID WAS WE MEASURED WITH OUR DIALYSIS AND THE NUCLEUS CIRCUMBENT AND THIS IS KIND OF AN ANNOYING SLIDE TO LOOK AT POSSIBLY BUT I'LL DRAW YOUR ATTENTION TO THE NOR EPINEPHRINE WHICH WAS A BIG SURPRISE TO US. THERE WERE OTHER DIFFERENCES THAT SUGGEST OTHER THINGS GOING ON LIKE HISTAMINE AND A METABOLITE OF DOPAMINE BUT NOREPINEPHRINE IS WHAT WE KEYED IN ON AND WE FOUND IN THIS NOR EPINEPHRINE CHANGED IN RESPONSE O COCAINE. AND IF YOU BLOCK THE RESPONDERS YOU CAN MAKE THEM ACT LIKE A LOW RESPONDER AND IT SHOWS THE TRANSMITTER IS HELPING DRIVE THE BEHAVIOR. AND WE CAN MEASURE COMPOUNDS THAT ARE THOUGHT TO POTENTIALLY BE IN THE BRAIN. THERE'S MORE THINGS LIKE TRACE AMIN NEURO TRANSMITTERS AND MORE MOLECULES IN HERE AND GET A DETAILED CHEMICAL LOOK AT WHAT'S IN A GIVEN BRAIN REGION BY THIS APPROACH. BEFORE I LEAVE THIS SUBJECT, ONE THING WE'RE WORKING ON WITH THIS TECHNOLOGY IS TRYING TO OPEN UP TO MORE COMPOUNDS. REALLY ASK THE QUESTION WHAT'S IN THE BRAIN EXTRA CELLULAR SPACE AND HOW DOES THAT CHEMICAL COMPOSITION CHANGE IN DIFFERENT MENTAL STATES OR PHYSIOLOGICAL CONDITIONS. SO TO DO THIS WE'VE BEEN STARTING BY TRYING TO BUILD A DATABASE OF COMPOUNDS PRESENT IN THE GIVEN BRAIN REGION IN EXTRA CELLULAR SPACE. THE WAY THIS IS DONE IS ESSENTIALLY METABOLOMICS TYPE OF EXPERIMENT WE'LL TAKE THE SAMPLES WE COLLECT AND IT WILL GENERATE A LOT OF MASS SPECTRA COMING FROM THE INSTRUMENT AND CAN TRY TO MATCH THOSE TO FIRST APPROXIMATION AND A LIBRARY OF MASS SPECTRA OF KNOWN COMPOUNDS. THEN GO THROUGH A MORE RIGOROUS PROCESS BUT GETS YOU FAR IN TERMS OF WHAT YOU HAD. AND AND TENDS TO BE DILUTED AND YOU'RE NOT GETTING VERY BIG SAMPLES WHEN YOU COLLECT THEM. AND WE FIGURE OUT WAYS TO IMPROVE THE CHROMAING TO CHROMATEOGRAPHY. THESE ARE DIFFERENT SIGNAL WE DETECTED. IN OUR POLITICAL APPROACH AND YOU'RE PICKING UP LIPIDS AND A QUARTER WERE LIPIDS AND THERE ARE LIPID NEUROTRANSMITTERS. AND THERE'S OTHER MOLECULES AS WELL AND THE GOAL IS TO BUILD A DATABASE WITH THE COMPOUNDS AND THEN WE'LL HAVE AN ASSAY FOR ALL OF THEM AND WOULD LOVE TO TRACK THE METABOLOMICS ACROSS THE STATE AND SOMETHING WE'RE BUILDING TOWARDS WITH THIS METHOD. I WANT TO MOVE ON FROM THAT TOPIC AND TALK A LITTLE BIT ABOUT THE TEMPORAL AND SPATIAL RESOLUTION ISSUE. THE SENSOR DATA REVEALED THERE'S RAPID CHANGES IN THE BRAIN AND SOME THAT APPEAR TO BE SIGNIFICANT AND ALSO THAT IT CAN BE VERY LOCALIZED. SPATIAL AND TEMPORAL RESOLUTION IS IMPORTANT IN THE LEVEL OF DETAIL AND THE REASONS IS OBVIOUS WHEN YOU LOOK AT THIS THIS IS A PROBE SIZE. THE PROBES ARE 1 TO 4 MILLIMETERS LONG AND WE NEED SMALLER PROBES. AND ALSO THE TEMPORAL RESOLUTION IS NOT THAT GREAT. THESE PROBES ARE TYPICALLY OPERATED BY PROFUSING THEM AT 1 MICROMILLIMETER PER MINUTE AND IF YOU HAVE 5 WHICH ARE COMMON IT MEANS YOU'LL SEE CHANGES EVERY FIVE MINUTES WITH THIS APPROA APPROACH. YOU NEED MORE SENSITIVE TECHNIQUE AND SMALLER SAMPLES BUT IF YOU THINK ABOUT IT AND GET TO ONE SECOND SAMPLING YOU HAVE ANOTHER PROBLEM, HOW DO YOU COLLECT AND MANIPULATE THE SAMPLES YOU'RE GETTING OUT OF THE PROBE. I WANT TO TALK ABOUT HOW WE ADDRESSED BOTH OF THESE ISSUES. FIRST OF ALL, WE TURNED TO MICROFABRICATION TO BUILD SMALLER SAMPLING DEVICES. ELECTRICAL PROBES CAN BE MADE QUITE SMALL. THIS IS A POPULAR TECHNIQUE FOR ANALYZING CIRCUITS. WE TURNED TO A SAMPLING TECHNIQUE. INSIDE THE DEVICE WE HAVE CHANNELS WHERE WE'RE ABLE TO MOVE FLUID AROUND. YOU CAN SEE THE CHANNELS MADE IN THERE AND THE TIP WE HAVE THIS PORTS. WE'RE GOING TO DO PUSH/PULL PROFUSION. SO AT ONE CHANNEL WE'LL PULL FLUID IN FROM THE BRAIN AND ON THE OTHER SIDE WE'LL PUSH OUT FLUID TO KEEP THE FLUID BALANCED AT THE REGION. THESE PROBES ARE 60 MICROMETERS WIDE AND THE SUPPORT IS ON THE MICRON SCALE. AND HAVE SPATIAL RESOLUTION TO SOMETHING LIKE THE CARBON FIBER MICROELECTRODE AND YOU CAN SEE WHAT THE CHANNELS LOOK LIKE AND HOW THEY'RE BURIED INSIDE THE SILICON. TO DRIVE HOME THE POINT OF DIFFERENT SIZE AND THIS IS A PROBE WE PURCHASED FROM A COMMERCIAL VENDOR AND HERE'S SOME PROBES WE MADE IN OUR LAB. WE WERE TRYING TO MAKE THEM SMALLER AND THESE MICROFAB PROBES ARE A LOT OF SMALLER AND THE SAMPLING AREA WE RECKON IS AT LEAST 1,000 FOLD SMALLER AND 3,000 FOLD DEPENDING ON THE SIZE OF THE PROBE. SO IT MAY BE OBVIOUS TO A LOT OF YOU WHO DO IMAGING AND SO ON THAT SPECIAL RESOLUTION IS REALLY IMPORTANT BUT I WANT TO ILLUSTRATE THAT THE SIGNIFICANCE OF IT FOR US HERE WITH A SAMPLE. SO THIS IS A MOUSE BRAIN ATLAS PICTURE AND BLOWUP THE REGION AND THIS SIGNIFICANT FOR A LOT OF COLLABORATORS INTERESTED IN REWARD AND DRUG ADDICTION. IT'S A BRAIN REGION THAT NEURONS PROJECT IN THE NUCLEUS CIRCUMBENS AND YOU CAN SEE THE SCALE BAR IS 500 MICROMETERS SO IT COULD BE CHALLENGE IING AND WITH THE NEW SMALLER APPROACH WE'VE BEEN ABLE TO MATCH GRADIENTS ACROSS THE DIVIDE BETWEEN THE RED NUCLEUS AND RED BTA. THE BARS REPRESENT DIFFERENT AND YOU CAN SEE THE PROBES YOU COULDN'T SEE UP HERE AND THESE ARE JUST 100 MICROMETERS APART. YOU CAN MAP OUT CONCENTRATION MORE FINELY THAN YOU COULD WITH DIALYSIS. AND BECAUSE WE'RE USING THE LCMS METHOD WE CAN MAP THAT FOR LOTS OF COMPOUNDS ACROSS THE BRAIN REGIONS. THE METABOLITES ARE VERY DIFFERENT. THE OTHERS ARE SIMILAR AND GLUTAMATE APPEAR TO HAVE DIFFERENCES BETWEEN THE TWO BRAIN REGIONS AS WELL. GETTING AT THE IDEA WE HAVE SPATIAL RESOLUTION WITH A CHEMICAL MEASUREMENT. THE NICE THING ABOUT THE MICROFABRICATION IS YOU CAN START TO DO MORE IN TERMS OF MAKING PROBES. A COUPLE THINGS WE HAVE ON THE DOCKET IS MAKING PROBES AND WE CAN ETCH THESE DOWN THINNER. THIS MORE LIKE 20 MICRONS THICK AND IT DOESN'T AFFECT THE SPATIAL RESOLUTION OF THE MEASUREMENT BUT MAKES THE PROBE LESS INVASIVE AND THEREFORE SHOULD BE MORE I THINK MORE USEFUL AND BETTER FOR MAKING MEASUREMENTS IN THE BRAIN WITHOUT DISTORTIONS DUE TO TISSUE DAMAGE. THE MICROFABRY CAKESCATIONCATION MICROFABRICATIONS FOR DOING MEASUREMENTS AND CAN DO OPTICAL WAVE GUIDES TO DO WAVE GUIDE OPTOGENETICS OR RECORDING IN THE BRAIN AT THE SAME TIME. THE PROBES THE DATA I SHOWED BEFORE WE HAD TO COLLECT AT LEAST 20 MINUTES TO HAVE ENOUGH SAMPLE TO ANALYZE AND WE'RE TRYING TO IMPROVE TEMPORAL RESOLUTION AS WELL. WE NEEDED A WAY TO GENERATE AND COLLECT SMALL FRACTIONS I MEAN NANO LITER FRACTIONS AND WE KNEW IT WASN'T GOING TO GO IN A LITTLE VIAL THE WAY WE NORMALLY COLLECT FRACTIONS SO WE DEVELOPED THE MICROFLUIDIC DEVICE AND THE IDEA IS THE SPAM COMES FROM THE SAMPLING DEVICE AND PUT IN THE BRAIN AND COMES IN THIS FLUID AND USE FLUORINATED SAMPLING. AND THIS COULD BE ENCASED IN A WAY TO ALLOW YOU TO PUT IT ON A BRAIN ON A BEHAVING ANIMAL. I WANT TO SHOW YOU THE FLUIDICS. YOU SEE THE SAMPLE AND THE DROPLETS BEING FORMED AND ANOTHER FLUID FROM THE TOP CAN BE USED TO ADD AGENTS OR ADD AN INTERNAL STANDARD WHATEVER YOU NEED TO PROCESS THE SAMPLE FOR ANALYSIS AND THIS COULD BE DONE ON A RODENT'S HEAD. NOW I'LL GENERATE SAMPLES AND WOULD LIKE TO KNOW WHAT'S IN THEM. THE TECHNIQUE WE FIND TO BE VERSATILE I'M TRYING TO PUSH IS USE WHAT WE CALL DIRECT MASS SPECTROMETRY. IT'S VERY SIMPLE TO OPERATE. WE TAKE THE SAMPLES COLLECTED IN A TUBE AND USE A SYRINGE PUMP TO PUMP THEM THROUGH TO FORM THEM TO IONS AND INTO THE MASS SPECTROMETER AND LET'S YOU ANALYZE RAPIDLY WITHOUT DIALYSIS OR ANYTHING ELSE. WHEN YOU PUMP THE SAMPLES IN WHEN THE SAMPLES COME OUT YOU GET THE SIGNAL AND WE DON'T GET CARRIER FLUID OR ANY SIGNAL AND THE NEXT SAMPLE COMES IN WE GET THE SIGNAL. YOU LOOK AT THE EMITTER WITH THE ELECTROSCOPE YOU SEE THE PLUME WHERE THE IONS ARE FORMED AND WHEN THE OILS ARE COMING OUT IT DRIBBLES OFF THE EMITTER. SO TO PUT THIS ALL TOGETHER FOR DEMONSTRATION, DOING MEASUREMENTS FROM A PROBE JUST GOT THIS MICRONS TYPE SPATIAL RESOLUTION AND FLUORO RESOLUTION. WE'RE ABLE TO DEVELOP ASSAYS MEASURE THEM AND HAVE A PULSE INTO THE BRAIN REGION TO DEPOLARIZE NEURONS AND CAUSE A RELEASE AND WE'LL HAVE THEM FOR ALL THE COMPOUNDS. AND ONE THING I WANT TO POINT OUT IS IF YOU LOOK AT THE LITERATURE YOU'LL SEE SIMILAR THINGS DONE WITH SENSORS. HERE'S A NICE EXAMPLE FROM A LAB DEVELOPING A GLUTAMATE ENZYME SENSOR THAT'S MICROFABRICATED AND PUT ON A CERAMIC SURFACE AND DID A SIMILAR EXPERIENCE WITH POTASSIUM AND LOOKING AT THE MAGNITUDE OF THE RESPONSE AND IT'S NEARLY IDENTICAL TO WHAT WE SAW WITH THE SAMPLING DEVICE. THE DIFFERENCE IS WITH THE MASS SPECTOMETRY YOU'RE ABLE TO MEASURE COMPOUNDS BECAUSE OF THE COMPOUND METHOD AND WERE ABLE TO PICK UP THE CHOLINE AND GABA AND WORKING TOWARDS THAT. THIS IS LOOKING AT THE CHANGES IN GLUTAMATE ASSOCIATED WITH HUNTINGTON'S DISEASE FROM THE MODEL AND THERE'S THIS CAG REPEAT AND YOU GET THE SIMILAR AFFECTS FROM THIS NEURODEGENERATIVE DISEASE IN THESE MICE. IN A CONTROL MOUSE WE'VE PUD -- PUT THE STROBE IN AND STIMULATE FOR GLUTAMATE RELEASE AND ABLE TO RECORD THAT WITH A MEASURING SYSTEM SEE HERE. WHEN THE STIMULATION HAPPENS YOU GET A QUICK RIDES IN GLUTAMATE AND GOES DOWN QUICKLY MOSTLY DUE TO THE GLIA TRANSPORTERS AND THIS WILL ALSO GIVE A GLUTAMATE RELEASE BUT THE RETURN TO BASELINE IS MUCH SLOWER WHICH IS LOWER WITH THE GLUTAMATE TRANSPORTER ASSOCIATED WITH THIS MODEL. THERE'S BEEN A LOT OF THEORIES HOW GLUTAMATE SIGNALLING COULD BE ALTERED IN THE ANIMALS AND POTENTIALLY CONTRIBUTING TO SOME OF THE SYMPTOMS SEEN IN THE ANIMAL. THEN WHAT'S FUN TO SEE IS THERE'S THIS LITERATURE THAT ANTIBIOTICS CAN INCREASE TRANSMISSION OF THE TRANSPORTERS SO IF WE PUT THAT CEFTRIAXONE WE LOWERED THE BASAL CONCENTRATION AND SAME ELECTRICAL SIMULATION RESEMBLE THE ANIMAL IN RETURNING BACK TO BASELINE. THAT'S A COLLABORATION TO EXAMINE THAT AFFECT. THIS ILLUSTRATES POTENTIAL FOR USING THIS IN ONE MODEL FOR EXAMINING CONCENTRATION DYNAMICS. SO IF I CAN COME BACK TO OUR RADAR PLOT, I WANT TO SHOW THE PULL/PULL PROFUSION STARTS TO GET TEMPORAL RESOLUTION CLOSE TO WHAT THE SENSORS CAN DO IN MONITORING NEUROTRANSMITTERS AND OTHER COMPOUNDS AND THE SMALL SIZE OF THE PROBE IS COMPARABLE TO THE SENSORS AND GOING INTO SMALL REGIONS AND NUCLEI AND WE RETAIN THE ABILITY TO HAVE INFORMATION AND SO WE THINK IT WILL HOPEFULLY BE A USEFUL TECHNIQUE FOR EXAMINING BRAIN CHEMISTRY. TO CLOSE I WANT TO SUMMARIZE THIS LCMS IS POWERFUL AND WE CAN EXPAND OUR VIEW BEYOND THE COMMON NEUROTRANSMITTERS AND TO TALK ABOUT THE SMALLER PROBES. I WANT TO CLOSE BY ACKNOWLEDGING MY GROUP WHO HAS DONE THIS WORK AND CHEMISTS WHO SOMETIMES LEARNED TO DO BRAIN SURGERY AND OUR FUNDING. THANK YOU. >> THANK YOU. ANY QUESTIONS FROM THE PANEL? FROM THE AUDIENCE? >> HAVE YOU LOOKED AT THE PEPTIDES. >> WE'VE BEEN ABLE TO MEASURE A NUMBER OF PEPTIDES IN DIALYSIS WITH A METHOD WHERE WE USE SMALL COLUMNS TO CONCENTRATE THE SAMPLES WE GET AND MEASURE THEM. THE CHALLENGE WE FOUND WITH THE ENDOGENOUS PEPTIDES IS THEIR CONCENTRATION IS GOING TO BE TENFOLD LOWER THAN EVEN THE LOWEST TRANSMITTER. DOPAMINE YOU MAY FIND INTENSE OF NANOMOLE AND SOME ARE PRESENT EVEN LOWER THAN THAT. SO IT'S THE DIRECT MASS SPECTROMETRY METHOD IS NOT THAT SENSITI SENSITIVE. IT'S GOOD FOR LOW NANOMOLE AND I WANT TO TRY BECAUSE I GET THE IDEA ONE OF THE PROBLEMS MAY BE NEURO PEPTIDE SECRETION SITES MAY BE SPARSE AND WHEN YOU PUT A PROBE IN YOU MAY DAMAGE THEM AND MAY MAKE IT HARDER TO MEASURE THE ENDOGENOUS PEPTIDE. A SMALLER PROBE MAY BE MORE SUITABLE. ALSO WITHOUT THE MEMBRANE WE CAN GET BETTER RECOVERY. IT'S SOMETHING TO TRY TO LOOK AT WITH THOSE ENDOGENOUS PEPTIDES. I USED TO ALWAYS JOKE WE JUST WANTED TO MEASURE DOPAMINE AND WE HAVE A LOT OF DRUG ABUSE PEOPLE IN MICHIGAN IS PROBABLY WHY AND NOW ALL THEY WANT TO KNOW ABOUT IS OPIOID PEPTIDE SO MAYBE THAT'S THE OTHER ONE TO GET AT. >> AND ALSO DOES THE SIZE OF THE ANIMAL MATTER LIKE MOUSE VERSUS RAT? >> THAT'S ANOTHER REASON WE WANTED TO MAKE THE SMALLER PROBES. THE MOUSE BRAIN IS SMALLER THAN A RAT BRAIN. THE PROBE SIZE A DIALYSIS PROBE GOING IN A MOUSE BRAIN WILL HAVE LESS BRAIN REGIONS IT CAN REASONABLY FIT IN TO MAKE MEASUREMENTS WHEREAS WITH THE SMALLER PROBES THEY'RE MORE SEUSS -- SUITABLE FOR GOING IN A MOUSE AND THERE'S LOTS OF INTERESTING MODELS BEIDEVELOPED ON THE MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE.IDEVELOPED ON THE MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE.BEINIDEVELOPED ON THE MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE.GIDEVELOPED ON THE MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE.DEVELOPED ON THE MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE.DEVELOPED ON THE MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE.MOUSE AND IT'S GREAT TO HAVE THE CHEMICAL CAPABILITY AS A TOOL TO EXAMINE THE MICE. AND WE'VE DONE WORK ON MICE TO ILLUSTRATE THAT POINT. >> WE CAN SQUEEZE IN ONE MORE QUESTION. YOU DISCUSSED COMING UP WITH LIPIDS IN MICRODIALYSIS AND THE USE TO PICK UP PROTEINS. IN YOUR CURRENT TECHNOLOGY, YOU'RE LOOKING AT MOLECULES THAT ARE UNDER OR AROUND 1,000 MOLECULAR WEIGHT? WHAT ARE THE LIMITATIONS. >> GREAT POINT. THE BEAUTY OF LCMS IS HOW GENERALIZABLE THAT TECHNOLOGY IS IN TERMS OFS? IN TERMS IN TERMS OF THE TY PE OF MOLECULE AND THE SAME INSTRUMENT CAN DO ALL THOSE THINGS. I WAS LISTENING TO THE VARIOUS TALKS YESTERDAY AS TO WHAT RAISES GLYCOSYLATION AND ALL THOSE SHOULD BE ADDRESSABLE TO BE MEASURED BY LCMS. GOOD. THANKS. >> THANK YOU. OUR FINAL SPEAKER FOR THE DAY IS DR. CHRISTINA WOO. SHE'S AN ASSOCIATE PROFESSOR AT BIOLOGY AT HOWARD UNIVERSITY. SHE HAS BEEN A RECENT RECIPIENT OF AN AWARD AND LOOKING AT WRITING AND ERASING OGLCNAC AND FROM TARGET PROTEINS IN THE BRAIN. >> I'LL TELL YOU WHAT WE'VE BEEN WORKING ON IN OUR GROUP FOR ERASING A TYPE OF SUGAR CALLED O GLCNAC AND TRY TO MAKE CONNECTIONS SO WHAT WE'VE DONE IN OUR GROUP. WE'VE STUDIED THIS PARTICULAR PROTEIN. IT'S A SUGAR MOLECULE SHOWN IN RED FOUND IN AMINO ACIDS WITHIN THE CELL. IT'S COMPARED TO OTHER TYPES OF GLYCOSYLATION WE'VE BEEN TALKING ABOUT TODAY. THIS SUGAR MOLECULE IS THOUGHT TO ACT AS A NUTRIENT SIGNAL ON NUCLEAR CYTOPLASMIC AND KINO PROTEINS AND THIS TIES TOGETHER MAJOR METABOLIC PATHWAYS IN THE CELL TO DEVELOP AND CREATE THIS MOLECULE. THIS MOLECULE THE DONOR UDP O-GLCNAC IS TRANSFERRED BY OGT AND THE SUGAR COMMONLY SYMBOLIZED BY THE BLUE SQUARE. THIS IS INSTALLED BY THE ODT TRANSFERASE AND REMOVED BY OGLCNAC AND THE CYCLING BETWEEN THE TWO ENZYMES THAT HAS INSTALL INSTALLS AND REMOVES THE SUGARS FOR AVAILABLE AND SIGNAL THE MODIFICATION. CORRELATED WITH A NUMBER OF HUMAN DISEASES A NUMBER OF WHICH CONNECT THROUGH THE BRAIN AND KEY CONNECTION TO CELLULAR DEVELOPMENT AND MITOSIS AND YOU NEED O-GLCNAC AND BOTH FOR PROPER EMBRYONIC DEVELOPMENT IN MAMMALS. ANOTHER EXAMPLE OF THE CONNECTION TO THE POSTMODIFICATIPOST POSTMODIFICATIPOST POSTMODIFICATIPOST MOOF POST-MODIFICATION OF OTHER DISEASES LIKE PARKINSON'S AND IF THE PROTEIN IS NOT GLYCOSYLATED IT HAS HIGHER AGGREGATION AND EVEN ONE CAN RESULT IN SOLUBLIZATION OF THE PROTEIN. SIMILAR, KNOCK OUT OF O-GLCNAC TRANSFERASE IN THE BRAIN OF THE MOUSE ONE TYPE OF NEURON IN MOUSE RESULTS IN DIABETES AND O-GLCNAC TRANSFERASE HAS RECENTLY BEEN FOUND TO HAVE SPECIFIC CONNECTIONS TO INTELLECTUAL DISABILITY WHEN AS THE POINT MUTATIONS LIKELY AFFECTS WHAT TYPES OF PROTEINS ARE BEING MODIFIED. SO TO DATE, THERE'S RIGHT NOW A HUGE EXPANSION IN THE NUMBER OF PROTEINS THAT HAVE ABOUT MAPPED THAT CARRY THIS MODIFICATION OVER 5,000 DIFFERENT PROTEINS INSIDE OF OUR CELLS HAVE BEEN MAPPED TO CARRY THE MODIFICATION THAT INCLUDES A NUMBER OF TRANSCRIPTION AND KINASES AND LIGASES AND ONE QUESTION WE ASKED IN MY GROUP AND I THINK THE FIELD IS BEGINNING TO ASK IS HOW AND WHERE CAN WE USE O-GLCNAC TO MITIGATE HUMAN DISEASE? AND PARTICULARLY RELEVANT TO THIS MEETING AND SOME OF THE TALKS THAT WERE OCCURRING IN THE MORNING IS O-GLCNAC HAS NOW BEEN TIED STRONGLY TO ITS CONNECTION ON TAU AND ALZHEIMER'S DISEASE AND RIGHT NOW THIS TARGET O-GLCNAC TRANSFERASE IS BEING USED AS A POTENTIAL TARGET FOR TAUOPATHY. THE MODEL HERE IS WHAT'S BEEN FOUND IS TAU WE HEARD ABOUT SOME OF THE AGGREGATES THAT TAU CAN FORM, THESE AGGREGATES ARE PROMOTED BY PHOSPHORYLATION AND DIFFERENT TYPES OF POST-TRANSLATION MODIFICATION. THIS PHOSPHORYLATION LEADS TO PROMOTION OF AGGREGATION AS SHOWN HERE. HOWEVER, GLYCOSYLATION WHICH CAN FORM ON PHOSPHORYLATION THIS CAN ENHANCE THE STABILIZATION OF TAU AND DECREASE THE AGGREGATION OF THE PROTEIN. RECENTLY CO-WORKERS AND MERCK HAVE REPORTED A NEW MOLECULE THAT CAN INHIBIT AND TARGET OGA WHICH SHOULD ENHANCE O-GLCNAC IN THE BRAIN AND ARE EXPLORING NIS IN THE CLINIC AND INHIBITERS OF OGA AND OGT HAVE BEEN WIDELY DESCRIBED AND STARTING TO BE TRANSLATED TO THERAPEUTIC OPPORTUNITIES. WE LOOKED TO CREATE A MORE PRECISE UNDERSTANDING OF THE MECHANISM OF O-GLCNAC ON THE TARGET PROTEIN AND WITH THE IDEA TO EVENTUALLY CHARACTERIZE WHAT THIS DONE ON A FUNCTIONAL LEVEL POTENTIALLY FOR THERAPEUTIC PURPOSES. THE MODEL WE CAME UP BETTER HOW TARGET PROTEIN GLYCOSYLATION IS AFFECTING THE FUNCTION OF THE PROTEIN, WE ENVISION A METHOD TO DIRECT THE TWO ENZYMES THAT WRITE AND ERASE THE POST-GLYCATION MODIFICATION AND IN LOOKING AT WHAT THESE MIGHT LOOK LIKE WE WERE VERY INTERESTED IN USING AN ANTIBODY BASED APPROACH AND ANTIBODIES ARE EFFECTIVE AT RECOGNIZING SIGNALS SUCH AS THE CASE OF OPIOID ABUSE AS WELL AS OTHER TARGET PROTEINS. HOWEVER, ANTIBODIES THEMSELVES ARE LARGE AND EFFECTIVE AS TARGETING PROTEINS ON THE CELL SURFACE AND NOT WHERE O-GLCNAC IS FOUND ON. A GAIN OF RESEARCH IN THE ANTIBODY FIELD HAVE BEEN INVESTIGATING OTHER ANTIBODY COME FROM CAMELS OR SHARKS AND THESE CAN BE ENGINEERED TO AFFORD A NANO BODY. SO NANO BODIES CAN RECOGNIZE A TARGET PROTEIN INSIDE A CELL. THE TECHNOLOGY WE'VE BEEN DEVELOPING IN OUR GROUP IS ROUGHLY SHOWN HERE AND EMPLOYS TARGETING ONE OF THESE 20,000 PROTEINS INSIDE OF OUR CELLS THAT MAY OR MAY NOT HAVE O-GLCNAC WITH AN ANTIBODY WITH TRANSFERASE IN THAT WAY THE NANO BODY TARGETS IT AND ENHANCING GLYCOSYLATION ON THE TARGET PROTEIN OR USING THE NANO BODY TO BE THE ERASER OF O-GLCNAC AND IN THIS WAY REMOVING AND DE GLYCOSYLATING THE CELL. SOME OF OUR FAVORITE NANO BODIES INCLUDES NANO BODIES THAT RECOGNIZE GFP OR THE LAG 6 WHICH ARE TWO DIFFERENT NANO BODIES. SO WE HAVE A TARGET PROTEIN WITH GFP. THIS SHOULD WORK READILY FOR YOU. WE'VE ALSO LOOKED AT NANO BODIES THAT TARGET BRAIN TARGETS SUCH AS EPEA NANO BODY AND TWO OTHERS THE BC6 AND BC2 ANTIBODY. MOST THE STUDIES I'LL SHOW YOU TODAY PRIMARILY USED THE USE OF OGT OR OTA AND THERE'S A TRANS TRANSFACTID AND DECOSYLATION AND AGAIN A GRADUATE STUDENT WHO RECENTLY GRADUATED DEVELOPED BY BY TAKING ODT AND TRUNCATING OFF HOW IT TARGETS THE ENDOGENOUS PROTEIN AND REPLACES THAT WITH THE ANTIBODY. WE HAVE JUN B RECOGNIZED BY THE NEPA ANTIBODY AND IF WE COME IN WITH A TARGET PROTEIN WHAT WE SEE IN THE O-GLCNAC LEVELS IS THAT THERE IS SOME GLCNAC FROM THE MISSING ANTIBODY BUT IT'S FURTHER ENHANCED BY THE TARGETED EPA NANO BODY QUANTIFIED HERE AND WE'VE SHOWN THIS FOR THE TRANSCRIPTION FACTOR IN THE BRAIN AND WHERE WE'VE SEEN THE PROTEIN FOUND WELL THE CELL WHICH IS SELECTIVELY GLYCOSYLATED. WE INVESTIGATED WHETHER WE CAN RECOGNIZE THIS. THEE EPA IS AN AMINO ACID TAB AND WE INVESTIGATED IN THE CONTEXT OF THE T3 CELLS WHETHER OR NOT IT WAS ENDOGENOUSLY THERE AND WE HAVE A PEG MEG 6 IS AN AND WE'RE LOOKING AT A FUNCTION OF THE GLYCOSYLATION BY ADDING A MASS TAG WE CAN SHIFT THE AMOUNT OF GLYCOSYLATION THE PROTEIN UPWARDS AND THIS IS REPRESENTING APPROXIMATELY ONE EQUIVALENT OF GLAC -- GLCNAC FROM THE BRAND AND WE HAVE AN OVERSUPPRESSION AND TARGETED SYSTEM AND EQUIVALENT LEVELS AND TO DETERMINE THE ACTIVITY WE'RE GETTING WE WANT TO LOOK AT THE GLOBAL O-GLCNAC AND HERE IT'S SHOWN BY THIS WESTERN BLOT WE SEE WITH THE OVEREXPRESSION WE GET HIGH LEVEL OF O-GLCNAC IN THE PRESENCE OF OUR TARGETED ANTIBODY OGT SYSTEM AND CAN QUANTIFY THIS SHOWING WE CAN GET EQUIVALENT LEVELS OF GLCNACIZATION AND TARGET THE PROTEIN WITH THIS METHOD. ON THE OTHER HAND, WE ALSO DEVELOPED AN ANTIBODY SPLIT OGA SYSTEM FOR DEGLYCOSYLATION AND WE HAVE A TARGET PROTEIN WITH THE NANO BODY SPLIT OGA AND REDUCING THE EVENT WITHOUT AFFECTING THE BROADER PROTEOME. THIS SYSTEM WAS FOUND THAT IF YOU TAKE ODA THE SINGLE ERASER OF O-GLCNAC AND SPLIT IT INTO TWO SPECIES CONSISTS OF A SOFT DOMAIN AND THE CATALYTIC DOMAIN AND THE DOMAIN SYMBOLIZED BY THIS CASE HERE IS INACTIVE IN THE PROTEOME. BUT ONCE YOU ADD THE ANTIBODY ON IN THE STOCK DOMAIN YOU RECONSTITUTE THE SYSTEM SPECIFICALLY AT THE TARGET PROTEIN OF INTEREST. LET ME SHOW YOU HOW THIS WORKS. WHAT WE'RE LOOKING AT HERE IS DIE GLYCOSYLATION AND -- DEGLYCOSYLATION AND IT'S GLCNAC RELATE AND IF YOU ADD IN ODA YOU GET DEGLYCOSYLATION ACTIVITY AND WE CAN RECONSTITUTE THE SYSTEM. SAME FOR THE TRANSCRIPTION FACTOR SP1 AND CAN DE GLYCOSYLATE IT WITH THE ODA SYSTEM AND LOOKED AT IT IN THE CONTEXT OF THE PROTEOME. THIS IS WHERE WE LOOK AT GLYCOSYLATED PROTEINS AND AS A FUNCTION OF THE DECREASE OF THE GLYCOSYLATION AND FIND ONLY THE SP1 IS BEING DEGLYCOSYLATED BY THE SYSTEM INDICATING IT'S HIGHLY SELECTIVE. SO WE'VE NOW SHOWN THIS WITH OTHER ANTIBODY TYPE PAIRS INCLUDING SOME I INTRODUCED EARLIER LIKE THE EPA ANTIBODY FOR THE NUCLEON. WE'RE EXCITE ABOUT OTHER BEING ABLE TO USE THIS SET OF TOOLS FOR TARGETING THEIR PROTEIN OF INTEREST AND IN PARTICULAR TO UNDERSTAND BETTER HOW O-GLCNAC IS TARGETING THE PROTEIN. HOW CAN I USE THE O-GLCNAC WRITER ON YOUR PROTEIN OF INTEREST? I SHOWED A FEW DIFFERENT EXAMPLES. ONE EXAMPLE INVESTIGATING THE ROLE OF O-GLCNAC ON THE TRANSCRIPTION FACTOR. YOU MAY ASK HOW THEY PLAY A ROLE IN THE PROTEIN HALF LIFE. USING THIS SYSTEM WE CAN SHOW GLCNAC IS RELATED TO STABILITY INSIDE OF CELLS. THE FIRST PIECE OF DATA WE ALWAYS WANT TO SHOW IS WHETHER OR NOT THE NANO BODY ODA SYSTEM IS DECREASING ISOLATION AND THE LEVELS OF GLYCOSYLATION ARE REPORTED BY SHIFTING THE PROTEIN UP IN MASS AND THEN IN THE PRESENCE OF OGA OR THE NANO BODY SPLIT WE CAN REDUCE THE LEVELS OF GLYCOSYLATION AND THE CATALYTICALLY FORM AFFECTS THE O-GLCNAC AND BY USING THE SYSTEM AND MONITORING THE PROTEIN HALF LIFE OR LIFE TIME, WE CAN SEE THE PROTEIN TARGET IS REDUCED EN ITS AVAILABILITY IN THE PRESENCE OF OUR NANO BODY OGA SYSTEM BUT RELATIVELY STABILIZED IN THE CATALYTICALLY INACTIVE FORM AND THE TARGET PROTEIN IS AFFECTING THE LIFE TIME OF THIS PROTEIN. ANOTHER EXAMPLE OF HOW WE CAN USE THIS SYSTEM TO LOOK AT WHETHER OR NOT O-GLCNAC IS PLAYING A ROLE ON TRANSCRIPTIONAL ACTIVATION. IN THIS CASE WE DECIDED TO LOOK AT ANOTHER TARGET PROTEIN CALLED CFOS. IT FORMS A HETERODIMER IN ACTIVATING CERTAIN GENES. WHAT WAS SHOWN IS DOWN REGULATION WITH AN INHIBITER FOR ODT. IT ACTIVATES AND WE GOT ACTIVATION IN THE PRESENCE OF GLYCOSYLATION WE ENHANCE IT MORE. THE QUESTION YOU MAY ASK IS IS IT PLAYING A ROLE IN THE ACTIVATION. YOU CAN APPLY THIS SYSTEM TO DEGLYCOSYLATE C FOS AND WE CAN SEE THERE ARE HIGH LEVELS WITH THE ODA SYSTEM AND WE CAN REMOVE THAT WITH THE GLYCOSYLATION OF INHIBITOR BUT IMPORTANTLY IF YOU LOOK AT THE O-GLCNAC LEVELS WE SEE SIMILAR GLCNAC LEVELS WITH THE NANO BODY SYSTEM AND AS IT INDEED DOING THAT GLOBAL DEGLYCOSYLATION EVENT. IF WE LOOK AT AP1 ACTIVITY WITH THE ODA SYSTEM COMPARED TO THE CATALYTICALLY INACTIVE FORM WE DON'T SEE A SIGNIFICANT DIFFERENCE AND SEE THE ENHANCE MANY OF TRANSCRIPTIONAL ACTIVATION AND THE C FOS DIRECTLY IS NOT REQUIRED FOR THE TRANSCRIPTIONAL ACTIVATION. WHERE WE'VE BEGUN TO GO INTRINSICALLY IS LOOKING AT WRITE AND ERASING TARGETS IN THE BRAIN AND DOING THIS IN COLLABORATION WITH THOSE OVER AT MGH MASSACHUSETTS GENERAL HOSPITAL. WHERE THIS COLLABORATION WAS SPARKED WAS FROM THIS RECOGNITION THAT NANO BODIES HAVE BEEN DEVELOPED AGAINST SEVERAL KEY TARGETS IN THE BRAIN. I'VE SHOWN YOU WORK FOR VIEWING THIS IN THE CONTEXT OF THE NUCLEON AND THERE'S BEEN ANTIBODIES DEVELOPED TO RECOGNIZE TAU AS WELL AS HUNTINGTON'S. WE ASKED CAN WE MODULATE THE EFFECTS IN VIVO. IF WE CAN THEY'VE BEEN DEVELOPED FOR DIFFERENT ISO FORMS OF THE DIFFERENT TAU OR AGGREGATES. WE CAN POTENTIALLY BE ABLE TO NARROW IN WHERE GLCNAC PLAYS A ROLE IN THE CONTEXT OF THE ABROGATION PROCESS AND CAN DO IT IN A MORE TARGETED MAN NER AND THERE'S A LAB TO HUNTINGTON'S WE PLAN TO EXPLORE. THE TAUOPATHY MODEL WHAT WE PLAN TO MONITOR IS BOTH THE ROUGH PHENOTYPE ON THE EYE THAT'S BEEN PREVIOUSLY OBSERVED IN OTHER STUDIES WHERE HIGH EXPRESSIONS OF TAU RESULTS IN THE VISUAL PHENOTYPE IN THE EYE AS WELL AS USING A MONITORING SYSTEM WHERE WE CAN MONITOR THE ACTIVITY AND NUMBER OF PASSES THAT THIS TAKES THROUGH A LASER. AND IN AND IN THE CIRCADIAN RHYTHM LOOKS LIKE THIS AND IN THE PRESENCE OF TAU THAT RESULTS IN HYPERACTIVITY PHENOTYPE. WE HOPE TO OBSERVE A SLIGHTLY DIFFERENT BUT SIMILAR PHENOTYPE. HERE AT THE HUNTINGTON'S DISEASE MODEL HAS DIFFERENCES IN TERMS OF SURVIVAL AND SURVIVAL HAS BEEN DECREASED AS A FUNCTION OF TIME AND EXPRESSION OF PROTEIN AND THE CLIMBING ASSAY AND THIS IS ALSO OBSERVED IN THE ACTIVITY MONITORING ASSAY WHERE WE SEE A DECREASE IN ACTIVITY. INTERESTINGLY WHAT'S BEEN FOUND FOR HUNTINGTON'S DISEASE IN THE CONTEXT OF GLCNAC IS THE LOWER LEVELS OF O-GLCNAC IN HUNTINGTON'S DISEASE FINDS BETTER MODELS THAN WHAT FOUND IN ALZHEIMER'S DISEASE FOR TAU AND HOPE THE SYSTEM AND THE NANO BODIES RECAPITULATES THIS IN VIVO. HOPEFULLY I'VE CONVINCED YOU THE METHODS HAVE BEEN EVOLVING AND MATURING IN THE FIELD IF YOU WANTED TO LOOK AT O-GLCNAC IN THE CONTEXT OF THE BRAIN THERE ARE NOW A NUMBER OF OPPORTUNITIES TO DO SO. THERE'S A NUMBER OF GLOBAL METHODS FOR INCREASING OR DECREASING O-GLCNAC FOR CHEMICAL INHIBITERS AND OVERSUPPRESSION OR KNOCK OUT OF THE ERASER. WE'VE STARTED TO FILL IN THE SELECTIVE METHODS IN THE FORM OF NANO BODY ODT AND SPLIT OGA SYSTEM I TOLD YOU ABOUT TODAY. AND THEIR ROLES HAVE BEEN SEVERAL SITE SPECIFIC LIKE GLYCOSYLATION AVAILABLE FOR MANIPULATING THE O-GLCNACINATION AND I WOULD LIKE THE PEOPLE IN THE GROUP WHO DID THE WORK AND DEVELOP THE O-GLCNAC WRITER AND ERASER. CONTRIBUTIONS FROM MANY WHO HELPED FROM THE WORK HELPING TO MAP GLYCOSITES AND FOR UPDATING THE WIKIPEDIA PAGE AND THOSE FULFILLING OUR DREAMS IN TERMS OF SEEING WHETHER THE SYSTEMS ARE AFFECTIVE IN VIVO. FINALLY, I'D LIKE TO THANK THE COMMON FUND FOR PUTTING TOGETHER THE MEETINGS AND SUPPORTING THE WORK. WE WOULDN'T BE ABLE TO DO SO WITHOUT THE WORK. I'M HAPPY TO TAKE ANY QUESTIONS. >> THANK YOU, DR. WOO. THAT WAS CHEMICAL BIOLOGY AT ITS BEST. ANY QUESTIONS? >> I CAN IMAGINE INHIBITING OR PROMOTING O-GLCNAC MIGHT HELP PREVENT TAU AGGREGATION BUT IN PHOSPHO TAU PATENTS ARE SET WHICH IS THE LIKELY SET OF DISEASE YOU'D TARGET THERAPEUTICALLY. THE PTMs ARE PRETTY STABLE ONCE THE AGGREGATES ARE FORMED. JUST OFFER A MODEL PERHAPS AREN'T THE BEST TO TEST THAT IN BECAUSE THEY HAVE FAIRLY LOOSE AGGREGATES. WHAT DO YOU THINK OF THAT AND WHAT'S THE POTENTIAL IN REAL DISEASE SITUATIONS? >> WE HAVE DIFFERENT FORMS OF TAU AGGREGATION. WHAT HAS BEEN SHOWN WITH THE CHEMICAL INHINT EERS -- INHIBITORS AND I THINK MARK HAS POTENTIALLY SOME EVIDENCE IT'S PREVENTIVE AS WELL AS POTENTIAL FOR ALZHEIMER'S OTHERWISE THEY WOULDN'T BE DOING THIS. I'LL LEAVE THE ANSWER OPEN ENDED. >> YOU MIGHT START THE PATHOLOGY AND YOU HAVE DISEASE PATHOLOGY ALREADY SET. >> PREVENTING THE PATHOGENIC ENDS. >> IT GOES TO ALL THE AGGREGATE FIELD. THEY ALL SEEM TO SHOW THE PRESIDENT OF PATHOLOGY. >> ANY MORE QUESTIONS? FROM HERE ON WE CAN QUICKLY MOVE TO THE NEXT SESSION WHICH IS THE PANEL DISCUSSION. I REQUEST EVERYBODY TO SWITCH ON THEIR CAMERAS. SO THE WAY WE'LL HAVE THIS IS YOU CAN KEEP IT QUITE INFORMAL. IF ANYONE HAS ANY SLIDE YOU'D LIKE TO BRING ATTENTION TO IF NOT WE CAN ALWAYS HAVE A DISCUSSION THAT ARE APPLICABLE TO THE FIELD AND FROM HERE I WOULD LIKE TO BRING IN MY CO-CHAIR DR. CATHERINE LEIMKUHLER GRIMES. >> IT'S GREAT WE HAVE PEOPLE ON CROSS DAYS SO FEEL FREE TO TURN ON YOUR CAMERA AND BE PART OF THE DISCUSSION. WHAT KIRAN AND I WERE HOPING IS THOSE TUNING IN FOR MAYBE PARTS SO TO INTRODUCE YOURSELF DISCUSS WHAT YOU TALKED ABOUT IN A SENTENCE OR SO AND IF YOU WANT TO SHOW A SLIDE YOU CAN BUT BRIEFLY SO PEOPLE UNDERSTAND WHO IS AT THE ROUNDTABLE. I CAN START AND WE CAN GO IN ORDER OF HOW WE'RE LISTED. I'M KATHERINE LEIMKUHLER GRIMES AT THE UNIVERSITY OF DELAWARE I HELP TO RUN THE BIOLOGICAL TOOL SUBGROUPS AND CHRISTINA IS PART OF THAT. IT'S GREAT WE MEET MONTHLY AND DISCUSS AREAS OF NEED. I LEARNED SO MUCH IN THE LAST TWO DAYS ABOUT THINGS NEEDED AND PROVIDED BY WHAT I HELP LEAD SO THANK YOU ALL FOR BEING HERE AND THE NEXT PERSON IN ALPHABETICAL ORDER IS AUSTIN YANG. >> I'M AUSTIN YANG WITH THE HELP OF THIS GROUP WE'RE INVOLVED IN THIS. >> AND KAREN DUFF WHO LED GROUP THREE. >> I WAS SPEAKING ABOUT PROPAGATION OF PATHOLOGICAL FORMS OF TAU THAT OCCURS IN ALZHEIMER'S DISEASE AND THE IDEA THEY'RE PROPAGATED THROUGH VESICLE TRANSFER AND USING SOME BIOINFORMATIC METHODS IN WHAT IS INVOLVED IN TAU SPREAD AND HOW THEY HAVE PROTEO GLYCANS AS A KEY GROUP THAT MAY BE INVOLVED BUT THERE'S TAU PATHOLOGY AND HOW THEY MIGHT BE INVOLVED IN SPREAD. THIS WAS -- I REALLY ENJOYED THIS SECTION. I'M TRYING TO PICK UP SOME INFORMATION THIS IS NEW FOR ME. WE LOOKED FOR PROTEO GLYCANS IN RESPONSE TO THE MEETING. THIS IS A TOP LEVEL HIT BUT I WAS INTERESTED TO BRING THIS FORWARD AND TRY TO GET MORE TRACTION ON IT AND THINK ABOUT IT MORE IN RESPONSE TO THE MEETING SO THANK YOU FOR MAKING ME DO THAT AND I WOULD BE HAPPY TO TAKE ON ANYBODY'S IDEAS TO WHAT OUR DATA MIGHT MEAN AND HOW WE MIGHT EXPLORE IT FURTHER. THANK YOU. >> I'M A PROGRAM OFFICER AT THE NATIONAL INSTITUTE ON DRUG ABUSE, MORE SPECIFICALLY THE DIVISION OF NEUROSCIENCE AND BEHAVIOR AND CHEMISTRY AND PHARMACOLOGY BRANCH AND WE'RE VERY INTERESTED IN THE CHEMICAL BIOLOGY ASPECTS OF THE MECHANISMS RELATED TO DRUG ABUSE AND THE ONE THING IDAHO LIKE TO CONVEY IF ANYBODY HAS NEW IDEAS, PLEASE FEEL FREE TO REACH OUT. AND I DON'T KNOW WHETHER I CAN MAKE IT AVAILABLE AND WE CAN DISCUSS AND OF COURSE WE HOPE THIS GENERATES INTEREST IN NEW APPLICATIONS AND I HAD TO THINK TWICE WHILE I WAS PLANNING THE MEETING WITH ALL OF YOU HOW TO BRING IN PEOPLE. HOW TO BRING IN NEUROSCIENTISTS AND THE GLYCO PEOPLE TOGETHER. WE WERE THING HARD EF HOW TO MAKE THE TWO WORLDS MEET AND I THINK WE HAVE ACHIEVED THAT OVER THE PAST TWO DAYS AND HOPEFULLY THAT TRIGGERS A LOT OF INTEREST AND IT WAS NICE TO SEE ESTABLISHED INVESTIGATORS AND I THINK THAT'S A REALLY NICE THING. YEP. >> GREAT. CAN YOU GIVE A SUMMARY? CAN YOU GIVE AN UPDATE? >> I'VE BEEN HERE 36 YEARS AND I USED TO HAVE HAIR BACK THEN AND YEAH, I WAS TALKING ABOUT HOW WE CAN USE GAL EPITOPE WITH THE XENOREACTIVE ANTIBODIES AGAINST THE DRUGS OF ABUSE I TALKED ABOUT COCAINE AND FENTANYL. >> GREAT, THANK YOU. I'M BOB KENNEDY AT THE UNIVERSITY OF MICHIGAN. I WAS TALKING ABOUT TECHNOLOGY FOR TRYING TO MEASURE DYNAMICS OF CHEMICALS AND EXTRA CELLULAR SPACE AND THINGS RELEASED FROM NEURONS AND GLIA AND TRYING TO MEASURE MANY OF THEM AT THE SAME TIME AND I WAS REALLY INTERESTED IN POTENTIAL APPLICATIONS OR CHALLENGES AROUND THINGS OTHER PEOPLE TALKED ABOUT. I'M LISTENING FOR OPPORTUNITIES TO TRY TO FIND NEW THINGS TO TRY TO MEASURE. >> THANKS. >> ALBERT WONG. YOU SPOKE SECOND THIS MORNING. THE CLINICIAN ON THE CALL. >> I'M A PSYCHIATRIST AND SCIENTIST IN TORONTO AND WORK AT THE UNIVERSITY OF TORONTO AND I SPOKE ABOUT ANIMAL MODELS FOR PSYCHIATRIC DISORDERS. >> CHRISTINA, YOU JUST SPOKE BUT MAYBE JUST SAY HI AGAIN. >> I'M CHRISTINA AT HARVARD UNIVERSITY AT THE CHEMICAL AND BIOLOGY DEPARTMENT AND TALKED ABOUT METHODS TO WRITE AND ERASE O-GLCNAC. >> RON, DO YOU WANT TO SAY HI? RON SCHNAAR. >> MY INTEREST IN NEUROSCIENCES COVER TWO AREAS. ONE I SPOKE ABOUT WAS REGULATION OF MICROGLIA. GLYC ANAND GANGLIOSIDES IN THE BRAIN. I PARTICULARLY LIKE KENNEDY IN HIS AMAZING VIDEO OF MIXING OF OIL AND WATER TO SEPARATE THOSE USED MAZE AND BLUE LIQUIDS BECAUSE I GRADUATED INTEREST THE UNIVERSITY OF MICHIGAN AND I NOTICED THAT. THOUGH NOW I'M AT JOHNS HOPKINS HERE 40 YEARS. >> IT'S A MIXTURE OF GREEN. >> GO SPARTY. >> MY NAME IS STEVE FINKBEINER AND WE'RE PRIMARILY FOCUSSED ON NEURODEGENERATIVE DISEASE AND RELATED DEMENTIAS AND WAS HAPPY TO PARTICIPATE AND I WANT TO THANK AUSTIN FOR THE INVITATION. I THINK I LEARNED A TON. WE TRIED TO TALK ABOUT TOOLS AND CAPABILITIES THAT MAY BE USEFUL FOR THE GLYCOSCIENTISTS INCLUDING LONGITUDINAL SINGLE CELL ANALYSIS AND A TOOLBOX OF BIOSENSOR TO HELP THOSE UNDERSTAND WITH PERTURBATIONS. I BEGGED FOR HELP FROM THE GLYCOSCIENCE COMMUNITY TO HELP WITH SOME OF THE PROBLEMS IN NEURODEGENERATIVE DISEASE. WE COLLABORATE WITH KAREN AROUND TAU AND TAU TRANSMISSION AND INTERESTED HOW THAT PROTEIN GETS TAKEN UP. THERE'S ALSO LEWY BODY DEMENTIA AND WE'RE INTERESTED IN TEMPORAL DEMENTIAS WHERE WE SEE ROBUST NEURAL INFLAMMATION AND INTERESTED IN MICROGLIA FUNCTION AND THE ROLE THE GLYCANS MIGHT PLAY IN HELPING THE CELLS RECOGNIZE NEURONS AND PARTICIPATE IN THE NEUROINFLAMMATION PROCESS AND ALREADY HAVE E-MAILS AND HOPEFULLY COLLABORATE ON THIS. >> MATT MACAULY AND WE STUDY THE GLYCAN BINDING PROTEINS AND A TOLD YOU ABOUT OUR INTEREST IN A FLIEKAN BINDING PROTEINS CALLED -- GLYCAN BINDING PROTEINS CD33 AND HAVE MOUSE MODEL TO STUDY THE CORRELATION BETWEEN AD AND THE ISO FORMS. >> I'M A PROFESSOR AT CAL TECH IN THE DIVISION OF CHEMICAL ENGINEERING AND WE STUDY GLYCANS AND WE'RE INTERESTED IN THE DIFFERENT MOTIFS AND REGULATING PLASTICITY AND THE ROLE IN PSYCHIATRIC DISORDER AND HAVE BEEN DEVELOPING CHEMICAL TOOLS AND BE HAPPY TO COLLABORATE WITH ANYONE INTERESTING IN APPLYING THOSE TOOLS AND HAVE INTEREST IN GLYCAN ANALYSES AND NEURONS AND THANKS FOR BRINGING US ALL TOGETHER. >> LAST IS PAM MARINO. >> THIS IS PAM AND I'M A COORDINATOR FOR THE COMMON FUND PROGRAM AND A PLEASURE TO WORK WITH OTHERS AND THERE'S TOOLS AROUND DATABASES, INFORMATICS EFFORTS TO STUDY GLYCOSCIENCE ACROSS THE DISCIPLINES AND INSIGHT TO YOU LOOK AT THE COMMON FUND GLYCOSCIENCE AND YOU'LL SEE THE GRANTS FUNDED AND IT'S INCUMBENT ON THOSE WE GAVE GRANTS TO TO MAKE THE TOOLS AND REAGENTS AVAILABLE. AUSTIN IS OFFERING $250K SUPPLEMENTS. SEEMS A PERFECT OPPORTUNITY. >> IT SEEMS A PERFECT OPPORTUNITY TO FIND A GLYCOSCIENTIST TO HELP YOU OUT AND APPLY FOR A SUPPLEMENT. WE'RE RUNNING A NUMBER OF THESE MEETINGS AND WE'RE DOING IT BECAUSE GLYCOSCIENCE IS APPLICABLE TO ALMOST EVERY DISEASE PROCESS AND WANT TO BE SURE AS SEVERAL SAYS YOU DROPPED AREAS OF RESEARCH BECAUSE THE TOOLS WEREN'T AVAILABLE AND THAT'S WHAT WE'RE HERE FOR TO MAKE SURE YOU HAVE TOOLS AVAILABLE SO YOU DON'T HAVE TO DROP PROMISING AREAS OF RESEARCH. WE WANT TO BE ABLE TO MOVE THE FIELD FORWARD AND IT'S IMPORTANT TO MOVE FIELD FORWARD WHEN HAVE YOU NEW TOOLS AND EASIER. THANK YOU FOR TAKING PART IN THIS. WE REALLY APPRECIATE YOU MAKE THE EFFORT. I KNOW SEVERAL OF YOU SAID BUT I'M NOT A GLYCOSCIENTIST, WHY AM I BEING ASKED? YOU'RE BEING ASKED BECAUSE WE WANT TO INTERFACE THE FIELDS AND YOU'VE ALL DONE A WONDERFUL JOB OF BEING ABLE TO DO THAT. THANK YOU VERY MUCH. >> GREAT, FOR THOSE LISTENING ON THE WEBCAST FEEL FREE TO CONTINUE TO USE THE QUESTION CHANNEL TO PIPE YOUR QUESTIONS IN. THAT'S YOUR CONDUIT INTO THE DISCUSSION SO FEEL FREE TO BE PART OF THAT AS WELL. I THINK KIRRAN AND I HAVE QUESTIONS. THE FIRST QUESTION WE'LL ASK IS WHAT ARE THE MAJOR CHALLENGES THAT NEED TO BE OVERCOME TO BETTER ENABLE OUR UNDERSTANDING OF THE BRAIN AND GLYCO BIOLOGY OF THE BRAIN. WE MIGHT HAVE SEEN ONE THIS MORNING IN TALKING ABOUT VISUALIZING GLYCANS AT THE DEVELOPED LEVEL AND BETWEEN TO TALKS IS WHERE WE CAN KIND OF START HERE. WHAT DOES THE PANEL THINK ARE MAJOR CHALLENGES TO OVERCOME? >> IT'S I QUESTION I HAVE AND I WOULD LIKE TO PUSH A LITTLE BIT MORE GRANULARITY ON THE QUESTION. THERE'S SO MANY MOVING PARTS HOW DO WE GET THE SPECIFICITY FOR NOT ONLY THE INDIVIDUAL CELLS WE'RE INTERESTED IN AND PARTNERS THEY WORK WITH BUT THE COMPARTMENTS THAT ARE ALSO IMPORTANT HERE. AND METHODS WE NEED TO GET REAL GRANULARITY TO SEE THESE PARTICULAR GLYCANS IMPORTANT ON THE PARTICULAR COMPARTMENT OR THE CELL WE'RE INTERESTED IN. BIOINFORMATICS IS USEFUL OR IS IT A MESS? ARE THERE OTHER TECHNIQUES THAT PERHAPS WEREN'T REPRESENTED BUT GETTING AT REAL SPECIFICITY WHICH IS MY INTEREST IS TO BE SPECIFIC. IT MATTERS TO ME IS IT A PROTEIN AND I HAVE NEEDS BECAUSE MY THERAPEUTIC DEVELOPMENT FORMAT WILL DEPEND ON THAT SPECIFICITY. >> THIS IS A GREAT QUESTION WE'VE BEEN FACING IN THE FIELD AND I'VE BEEN HERE AT 40 YEARS AT HOPKINS AND THERE'S WAYS TO APPROACH IT. ONE WAY I'M BEGINNING TO FIND IS USEFUL IS LOOKING AT CHANGES IN WHICH GENES ARE EXPRESSED AT THE SINGLE CELL LEVEL BY DIFFERENT CELL TYPES IN DIFFERENT PARTS OF THE BRAIN. AND TELL YOU NOT IN GREAT DETAIL BUT TELLS YOU SOMETHING ABOUT THE GLYCANS PRODUCED. ANOTHER THING I THINK WE FAIL AT AS GLYCOSCIENTISTS IS WE ALWAYS START -- WELL, I DON'T, BUT HISTORICALLY WE'VE STARTED OUR REVIEWS BY SAYING IT'S NO COMPLICATED. THERE'S BILLIONS AND BILLIONS OF STRUCTURES. YOU MAY BE SURPRISED TO FIND THAT ANALYSIS SUGGESTS THAT THE NUMBER OF FUNCTIONAL GLYCAN STRUCTURES MIGHT BE FEWER THAN THE NUMBER OF PROTEINS. IT MIGHT BE 5,000 OR 10,000 STRUCTURES. RIGHT NOW IT'S NOT FEASIBLE BECAUSE WE CAN'T TAKE THEM APART AND LOOK AT THE PIECES LIKE THEY DID FOR PROTEOMICS. IT'S NOT FEASIBLE TO LOOK AT THOSE PARTICULAR STRUCTURES. SO AT THE SINGLE CELL LEVEL WE'RE KIND OF LIMITS. ONE WAY TO APPROACH THAT IS WITH TOOLS THAT BIND TO AND CAN BE USED TO TAG AND CELL TYPES OVER TIME IN DISEASE. AND THOSE ARE ONE OF THE AREAS THAT IS THE FOCUS OF NOT ONLY TECHNIQUES BUT BIOINFORMATICS AND HAS BEEN. PAM MARINO HAS BEEN DRIVING THIS ACROSS GROUPS OF GLYCOSCIENTISTS FOR TOOLS FOR SPECIFIC STRUCTURES. YOU CAN'T GET 4,000 ANTIBODIES AND WHICH ARE THERE, IF YOU HAVE AN IDEA OF WHAT IS THERE THERE MAY BE TOOLS COMING TO ALLOW YOU TO DO THAT. AND WHAT IS USEFUL IN TERMS OF IDENTIFYING MOLECULE INVOLVED AT THE CELLULAR LEVEL BUT WE HAD BECOME QUITE ADAPT AT MODIFYING WHICH STRUCTURES CAN BE EXPRESSED IN PARTICULARLY CELLS AND CULTURE BY PARTICULAR CELLS IN CULTURE BY MODIFYING THE GENES INVOLVED IN THEIR EXPRESSION. THOSE TO ME FROM A LONG PERSPECTIVE IN THE GLYCOSCIENCES IS WHERE WE ARE. >> I'D BE PARTICULARLY INTERESTED IN THE TAGS YOU DESCRIBED BECAUSE I THINK WITH TOOLS LIKE THAT WE CAN UNDERSTAND BETTER CELLULAR COMBINATORIAL LEVELS AND UNDERSTAND WHAT THINGS DRIVE CELL-CELL INTERACTIONS AND DO A LOT OF DIGITAL PATHOLOGY AND A.I. FOR ALZHEIMER'S DISEASE AS WELL AND THOSE TOOLS CAN ALSO BE APPLIED TO HUMAN BRAIN TISSUE AND THINGS LIKE THAT SO WE HAVE WAYS TO CONNECT MODEL SYSTEMS TO THE THINGS WE'RE TRYING TO STUDY IN PATIENTS. THAT WOULD BE SUPER VALUABLE FOR US ESPECIALLY LABELS THAT CAN BE USED IN LIVING SYMPTOMS. >> OH, YEAH. FOR SURE. >> HAVING TIES IN PATHOLOGY SECS ARE KEY BECAUSE WE HAVE YEARS NOT REPRESENTATIVE OF WHAT WE NEED TO BE LOOKING AT. IF ONE THING I'VE REALIZED IN DOING READING AND THINKING ABOUT THIS FIELD THE PROTEO GLYCAN FIELD THERE'S SPECIFICITY AND EVEN TRYING TO MAKE A CELL MODEL WITH A REGULAR NEURON YOU MAY BE MISSING THE KEY POINT. THOSE ARE RELEVANT TO HUMAN PATHOLOGY TO INTERROGATE SAMPLES SO WE KNOW WE'RE LOOKING AT THE RIGHT SETS OF GLYCAN CHANGES OR SPECIFICITY ON THE NEURON AND GLIA AND THE RIGHT TYPE AND IT'S REAL REALLY WE HAVE TO GET TO THE SPECIFICITY OF THE DISEASE. >> DO YOU THINK TOOLS FOR PATHOLOGICAL SAMPLES WOULD BE USEFUL? >> I'M IN THE SURE THERE'D BE SPECIFICITY BUT IT'S A GOOD PLACE TO START FOR. -- FOR SURE. >> SOME ARE DEVELOPING TOOLS TO SELECTIVELY TAG GLYCANS USING APPROACHES WHERE YOU CAN USE GLYCOTRANSFERASE TO TRANSFER NON-NATURAL GLYCANS TO KNOWN GLYCANS IN CELLS AND TISSUES AND THEN CHEMICALLY TAG THOSE GLYCANS. THOSE METHODS ARE GENERALLY SELECTIVE BECAUSE THE TRANSFERASE IS AN ISOTOPE AND THERE'S UNLIMITED SETS NOW DEVELOPED FOR TAGGING SPECIFIC GLYCANS. THAT IS WHERE WE'RE STARTING TO LOOK AT THE TRANSFERASES WITH THE HELP OF NIH TO OVEREXPRESS THE TRANSFERASES TO BE EASIER MANIPULATED AND STUDIES AND ON THE GLYCAN END. WHAT BOTHERS ME IS SOMETIMES THE SPECIFICITY IS NOT THAT WELL ESTABLISHED AND SOMETIMES IT'S PROMISCUOUS FINDING BUT ONE OF THE AREAS FOR TOOL DEVELOPMENT THAT'S IMPROVED IS THERE'S A LOT OF CHEMISTS WORKING ON EXPANDING THE SYNTHESIS OF WELL DEFINED GLYCANS AND WHAT IS BEING USED AND CHARACTERIZED BETTER FOR THE LECTIN TOOLS BEING USED FOR DETECTION. >> I COULDN'T AGREE MORE IN TERMS OF THE SPECIFICITY. THEY'VE BEEN SAID TO RECOGNIZE GALA GALA GALACTOES AND THAT'S WEEK. YOU MAY HAVE 1,000 NOT 4,000. A 1,000 WELL DEFINED STRUCTURED YOU CAN DEFINE THE BINDING OF A PARTICULAR REAGENT TO PICK THOSE AND THE COMBINATION OF TOOLS. I WANTED TO JUST CIRCLE BACK BRIEFLY TO KAREN'S COMMENT. THE BASIC PRINCIPLES OF PROTEINS HAVE BEEN CARRIED OUT ACROSS THE EUKARYOTIC PROTEINS AND THEY'VE DEVELOPED QUICKLY ESPECIALLY IN THE IMMUNE SYSTEM ESPECIALLY SINCE THEY BROKE OFF AND MICE ARE NOT ALWAYS MEN. MATT SHOWED SOME DATA ON THIS. BUT WE'LL LOOK AT STRUCTURES THAT ARE FUNCTIONAL IN A LOT OF CASESES WE HAVE TO LOOK AT HUMAN TISSUE TO BE ABLE TO IDENTIFY AT LEAST VALIDATE BETWEEN SPECIES. >> AND WE TALKED ABOUT IPSCs AND NEURONS. ARE THEY GOOD MODELS OR JUST MISSING THINGS. FOR ME TO GET INVOLVED IN THE FIELD I HAVE FEEL THERE ARE TOOLS TO NOT JUST GIVE ME OWN IN OWN THE ANSWER THAT REQUIRES IT'S DIFFICULT. >> LET ME GIVE YOU ONE THING COMING UP WITH ITS OWN LIMITATIONS BUT HAS SOME RESOLUTION ON FIXED TISSUES OR AT LEAST FROZEN, QUICK FROZEN TISSUES. PEOPLE HAVE BEEN RELEASING ENGLYCANS AND MAYBE THAT'S ANOTHER THING TO LOOK AT IN TERM OF CAN WE MAP THE STRUCTURES ACROSS CELLULAR SPACE. >> THAT'S MUCH OF WHAT YOU SAID DEVELOPING THE PIPELINE AND TO WHAT LINDA WAS TALKING ABOUT USING THE ENZYME TO NOT ONLY CUT OFF BUT ENRICH AND GIVE BETTER SIGNALS FOR THE GLYCANS THAT DON'T GIVE GOOD SIGNAL >> WE HAVE SEVERAL ENZYMES WHERE IT'S IN THE BINDING CAPACITY BUT NOT CATALYTIC. THOSE HAVE BEEN COMMERCIALIZED AT THIS POINT FOR THE SIALIC ACIDS. >> AND I THOUGHT ABOUT THE WORK AND BEING VIEWED ON THE ENZYME DIRECTLY. >> RICHARD DRAKE THE TECHNOLOGY AND PART OF A CONSORTIUM AT NCI. THEY BUILT THE INFORMATICS INTO THIS NOW. THEY'RE USING IT FOR BIOMARKER DISCOVERY AND WHAT NOT. IT'S FAIRLY FAR ADVANCED AT THIS POINT. YOU CAN TREAT THEM AND ANALYZE THEM AGAIN AND SEQUENCE OFF THE TISSUE AND TAKE CARE OF THE SIDE CHAIN ISSUE YOU WERE TALKING ABOUT PREVIOUSLY. >> WE ALSO HAVE MORE PORTABLE AGENTS THAT CHANGE THE CELL SURFACE. THE GLYC AN AND THAT REAGENT ONLY HAPPENS WITH TARGETS AND DOESN'T REQUIRE COMPLICATED MASS SPECTROMETRY. HAVING THE PORTABLE TECHNOLOGY AND ISN'T TOO COMPLEX AND REQUIRES POTENTIAL AND COULD BE HELPFUL FOR SOMEBODY CAN HAVE A TAG AND QUICKLY HAVE A LONGITUDINAL STUDY WITHOUT GOING WITH CELLS. IT IS POTENTIALLY HELPFUL. WE DON'T HAVE TO HAVE A CHARACTERIZE CELL BUT CLASSIFY THEM AND THAT COULD BE VERY USEFUL AND ENOUGH TO CHANGE THE CLINICAL PRACTICE. HAVING THE PORTABLE REAGENT PUT ON THE HAND OF ANY GRADUATE STUDENT OR TECHNICIAN IS IMPORTANT FOR THIS FIELD AS WELL. >> THERE'S WORK CONTINUING AND THERE'S HUGE LIBRARIES OF VERY SPECIFIC ANTIBODIES BUT THESE ARE LAM FREE ANTIBODIES. >> THE BASE CAN BE PRODUCED IN PARALLEL. YOU CAN BYPASS THE DINE AM RANGE ISSUE AS WELL. THE FIRST IS APPROACH IS SOMETHING WE'RE LOOKING AT AS WELL. >> DO YOU THINK O-GLCNAC IS A MASTER REGULATOR? >> IN THE BRAIN? >> IT'S INVOLVED IN MITOCHONDRIAL FUNCTION. IF YOU MESS UP THE ENERGY SOURCES IN THE CELLS -- >> I THINK O-GLCNAC WELL THE UDP O-GLCNAC DONOR HAS A NUMBER OF FUNNELS ONE IS O-GLCNAC INSIDE THE CELL AND THEN AS WELL AS GLYCOSYLATION ON THE CELL SURFACE AND GLYCOSYLATION THAT SOME OF THE OTHER GLYCANS WE'VE BEEN TALKING ABOUT. I THINK THE LEVELS OF THE POST-TRANSLATION MODIFICATION ARE DIYNAMICALLY CHANGING AND FOR O-GLCNAC IN THE BRAIN IN PARTICULAR I THINK THERE'S HIGH LEVELS OF OGT AND OGA SO I DO THINK THERE'S A NUMBER OF OPPORTUNITIES FOR CHARACTERIZATION PARTICULARLY IN THE CONTEXT OF DRUG ABUSE I DON'T THINK HAS BEEN CHEWED ON AT ALL IN THE O-GLCNAC FIELD. >> TARGETING THE SYNTHETIC PATHWAYS IS AMENABLE BUT IN THE INFORMATION SYSTEM MY CONCERN IS NEURONS ARE THESE SPATIALLY ELABORATED CELL TYPES THAT OFTEN SOLVE A LOT OF THEIR PROBLEMS BY COMPARTMENTALIZING DIFFERENT PARTS. HOW EASY IS IT INTERPRET KNOCK DOWN OF A WHOLE BIOSYNTHETIC PROCESS AS WELL AS GLYCOSYLATION SUBSTRATES IS STRAIGHTFORWARD INTERPRETATION OR MAYBE HITTING MULTIPLE TARGETS SO TO THE PHENOTYPE IS MAYBE COMPLEX? >> THE ANSWER'S BOTH WE'VE SEEN IN MOUSE MODELS AND CONGENITAL DISORDERS. CERTAIN PATHWAYS OF GLYCOSYLATION ARE QUITE SPECIFIC. THERE'S A HANDFUL OF MOLECULES THAT GET INFECTED AND WHEN THAT GETS KNOCKED OUT, YOU CAN MAKE SOME INTERPRETATIONS ABOUT THE FUNCTION OF DOWN STREAM MOLECULES. THAT'S THROUGH FOR THE LIPIDS AND THE GANGLIOSIDE WHERE THERE CAN BE SEVERE AND MOTOR BEHAVIORAL FACETS. OTHERS ARE SHARED. SOME GLYCOTRANSFERASE OR CHANGES IN PRODUCTION OF NUCLEOTIDE PRECURSORS WILL HAVE A MASSIVE AFFECT ACROSS MANY MOLECULES AND IT MAKES IT VERY DIFFICULT THEN TO DECODE THEM. AS WE MOVE DOWN AS WE KIND OF GET INFORMATION AND FOCUS IN THERE'S REALLY INTERESTING QUESTIONS. ONE THING THAT SURPRISED ME IS THAT O-GLCNAC PROVIDED US WITH A KNOCKOUT OF A SINGLE PROTEIN TO WHICH WE SHOWED THERE WAS A GLYCAN ATTACHED THAT'S FUNCTIONAL AND IT JUST DISAPPEARED. THAT WAS IN THE MOUSE BRAIN. I THINK IT'S GOING TO BE A VERY CONTE CONTEXTURAL ANSWER TO THAT -- CONTEXTUAL AND WE MAY BE ABLE TO FOCUS IN AND ASSOCIATE PARTICULAR STRUCTURES WITH PARTICULAR FUNCTIONS. GREAT QUESTION. >> YOU HAVE A QUESTION IN THE CHAT FROM THE AUDIENCE. >> SURE. I THINK THIS IS A GOOD QUESTION TO GO TO THE PANEL BECAUSE I THINK THROUGHOUT THE TWO SESSIONS WE TOUCHED ON THIS. SO THE QUESTION COMES FROM WHERE DO WE THINK GRLYCOSYLATION COMES INTO PLAY IN ALZHEIMER'S DISEASE OR DISTURBANCE IN GLYCOSYLATION OCCURS LATER ON OR LATER IN THE DISEASE PROCESS? I THINK FROM THE PAST TWO DAYS I THINK I HEARD MANY -- I THINK THERE'S MORE THAN ONE ANSWER TO THIS SO IT WOULD BE GOOD TO HEAR THE PANEL AND MAYBE REITERATE SOME OF WHAT YOU TALKED ABOUT. >> THIS HAD A LITTLE BIT OF WHAT I ASKED CHRISTINA ABOUT IF YOU'RE INTERVENING WITH GLYCOSYLATION WILL THIS BE IMPACTFUL IN AD? I PERSONALLY CAN'T ANSWER THE QUESTION BECAUSE I DON'T WORK ON GLYCOSYLATION OF THE AD PROTEINS BUT I THINK THE PTM MODIFICATIONS WE HAVE TO BE SURE ABOUT IMPACT AND THERAPEUTICS WHERE YOU WORK BACKWARDS. ETIOLOGY IS GOING FORWARD BUT YOU'RE TRYING TO WORK BACKWARDS. IT'S AN IMPORTANT QUESTION AS TO WHETHER IT'S CAUSAL, INTERVENE INTERVENABLE, REVERSIBLE IN A LATE STAGE AND I THINK CHRISTINA ANSWERED IN PART. >> BECAUSE IF YOU LOOK AT CANCER CELLS THE FIRST THING THAT HAPPENS IS THEY CHANGE THEIR GLYCOHELIX AS THEY TRANSFORM. >> WHAT WE'RE TALKING ABOUT IS GLYCOSYLATION PROTEINS THAT ARE VERY SET AND RIGID AND UNCHANGEABLE ONCE CHANGED. IF WARE LOOKING AT THE INTERACTION BETWEEN GLYCOSYLATION AND PHOSPHORYLATION FORMING AGGREGATION OR INVOLVED IN AGGREGATION, THE PROCESSES ONES THEY'RE SET, THEY'RE FAIRLY IRREVER IRREVERSIBLE. >> MAYBE MATT CAN CHIME IN TOO WITH YOUR STORY AND THINKING ABOUT MAYBE NOT NECESSARILY BUT THE PROTEINS RECOGNIZING THE GLYCAN GLYCANS IF YOU WANT TO CHIME IN. >> ACTUALLY, I WANT TO BRING IN SOMETHING ELSE AND SOMETHING ON MY MIND. RECENTLY WE WERE TRYING TO FIND SPECIFIC ANTIBODIES. AND THIS GETS BACK TO YOUR QUESTION ABOUT SPATIALIZATION BECAUSE THAT'S ONE WAY. THERE'S A SURPRISING NUMBER AVAILABLE AND IF I GET CD1 THROUGH 300 I GO TO THE BD CATALOG AND ORDER AND HAVE IT THE NEXT DAY. THERE'S A SURPRISING NUMBER OF SPECIFIC CARBOHYDRATE NEIGHBORHOODS AND THEY'RE ALL OVER THE PLACE. RON SAID WE HAVE FOUR GREAT GANGLIOSIDE ANTIBODIES AND I LOOKED THEM UP AND THEY'RE AT THIS NICHE COMPANY AND THE OTHER IS AT THE OTHER NICHE COMPANY. RON, DOES ANYONE HAVE THOSE CARBOHYDRATE ANTIBODIES CATALOG? THAT WOULD BE GREAT RESOURCE NOR COMMUNITY? >> WHEN THE CONSORTIUM FOR GLYCOPROTEOMICS THERE WAS A SCREEN FER SPECIFIC CATALOG NUMBER. I AGREE. IT WOULD BE GREAT TO HAVE A CATAL CATALOG. PAM, IS ANYONE DOING THAT? >> WE'VE DONE IT WITH LECTINS AND THEY'RE SCREENED. TWO DIFFERENT COMPANIES HAVE LECTIN CATALOGS BUT THEY HAVEN'T DONE IT UNIVERSALLY WITH ANTIBODIES. AND THERE'S BEEN A LOT OF TALK ABOUT SOME ANTIBODIES BEING NOT AS SPECIFIC OR SPECIFIC AS ADVERTISED. SO SOME ANTIBODIES HAVE BEEN SCREENED AND SOME HAVEN'T. WE'VE BEEN PUSHING PEOPLE -- I'VE BEEN TRYING TO GET SOME SPIR FOLKS INTERESTED IN DEVELOPING THE ALIBABA OF GLYCOREAGENTS INSTALLING THE GLYCANS AND THEIR SOURCES AND ENZYMES AND THEIR SOURCES BUT I HAVEN'T GOTTEN ANYBODY TO DO IT YET. IF YOU HAVE AN ENTERPRISING GRADUATE STUDENT OR POST-DOC WHO WOULD LIKE TO GET AN SPIR, THAT COULD BE A GREAT WAY TO GET THIS OFF THE GROUND. >> YOU DID POSE A QUESTION TO ME AND I SHOULD ANSWER IN TERMS OF READERS AND I WANTED TO TOUCH ON HUMAN OR MOUSE AND WE'VE STRUGGLED WITH THAT TO THE POINT WHERE WE HAD TO GENERATE NEW MOUSE MODELS FOR US AS A NEW LAB IT'S AN EXPENSIVE ENDEAVOR. I WANTED TO PUT IT OUT TO THE OTHER PEOPLE ON THE CALL ON WHAT LENGTHS YOU'VE GONE TO TO ESTABLISH SOME OF YOUR MOUSE SPECIFIC PHENOTYPES. THERE'S LOTS OF WORK IN HUMAN BRAIN ORGANOIDS AND THAT'S A HUGE BLOSSOMING FIELD. IS THAT A GOOD PLACE TO LOOK AT GLYCOSYLATION IN TERMS OF A SUBSTANCE FOR A MOUSE. WHAT ARE PEOPLE'S THOUGHTS? >> WE WORKED ON ORGANOIDS A LOT AND I WISH I COULD SAY HOW MUCH THEY RECAPITULATE HUMAN BRAIN VERSUS MOUSE BUT I DON'T HAVE INDEPENDENT DATA. >> THE HUMAN BRAIN IS SO COMPLEX FOR THESE DISEASES AND HAS TO BE VALIDATED AND MOVED TO A MODEL. IT'S HORRIBLE AND DIFFICULT AND WE NEED A LOT OF MONEY AND WHY IT'S PROBABLY FAILED. >> THANKS TO DR. WANG ON HIS DISCUSSION AND CAN ANYONE TALK TO THE ASSOCIATION OF MULTIPLE GLYCOSYLATION ENZYMES WITH SCHIZOPHRENIA THROUGH THE GWAS STUDIES THUS SUGGESTING A POSSIBLE PATHWAY TO THIS PATHOGENESIS. >> I DON'T KNOW OF THE TRANSFER ASSESSES IN TERMS OF SCHIZOPHRENIA. >> IT'S POSSIBLE. THIS VALUABLE FROM GEN GENTLE OR FAMILY CONGENITAL SUSCEPTIBILITY SUSCEPTIBILITIES AND THAT'S A GREAT JUMP OFF POINT BUT I DON'T KNOW IF ANYBODY IS DOING THEM CONSISTENTLY. >> WHAT ARE THE LEVELS COMPARED TO THE BRAIN? I GUESS THEY COULD BE STUDIED THROUGH BLOOD SAMPLES FROM LIVING SUBJECTS FOR THE POSTMORTEM BRAIN? >> I THINK TO GIVE CONTEXT FOR THE MARKERS OF SCHIZOPHRENIA FOR GWAS WHERE THE WORK IS COMING OUT AT LEAST WHAT BOBBY IS CITING IS GWAS STUDIES ON SCHIZOPHRENIA AND THE ASSOCIATION BETWEEN DIFFERENT GENES AND THE RELATIONSHIP TO SCHIZOPHRENIA AND A NUMBER OF MARKERS OF GLYCOSYLATION TRANSFERASE HAS EMERGED FROM THIS AND SHOWED LEVELS OF METAL IONS THAT ALSO AFFECT GLYCOSAL TRANSFERASE ACTIVITY. EACH ONE WILL TAKE A DEEPER RESEARCH INTO CHARACTERIZING THE GLYCAN STRUCTURES AND MORE TARGETED STUDIES. I DON'T THINK THERE'S TOO MUCH ACTIVITY FROM THE GLYCOSCIENCE PROGRAM YET BUT I DO THINK IT'S A BIG OPPORTUNITY. >> YOU COULD START WITH POST MORTEM TO PROFILE THERE >> ARE THEY STABLE AFTER DEATH? SOME THINGS DON'T SURVIVE VERY WELL TALKING AT LEAST 24 HOURS. >> CERTAINLY ACIDIC CONDITIONS WILL RELEASE SIALIC ACIDS OTHERWISE THEY'RE TOUGH AS NAILS AND IN TERMS OF TECHNOLOGY YOU CAN FIX TISSUE BECAUSE YOU'RE FIXING PRE AMINO GLUTS AND RELEASE THE GLYCANS AND STUDY THEM. YOU'LL PUT UP STORAGE AND I THINK THAT MAKES AN INVITING TARGET. POSTMORTEM RELEASE WAS ONE OF THE MEASURES OF POSTMORTEM DELAY. >> PH. FOR ME IT'S A GREAT QUESTION ON POSTMORTEM TISSUES. >> WHAT I'M TALKING ABOUT IS PH280 DEGREES. WHEN WE TALK ABOUT ACID IN POST MORTEM TISSUE, WE'RE PROBABLY TALKING ABOUT PH5. BUT GOOD POINT. >> I THINK HARVARD HAS ITS OWN POST MORTEM SAMPLE. >> THEY GIVE THE NUMBER. >> USUALLY THE POST MORTEM BANKS GIVE A BUNCH OF INFORMATION AND IF THEY THE THEY'LL SAY. IT'S CERTAINLY FEASIBLE THEN. THERE'S NO NEED TO GO OUT AND COLLECT TISSUE. IT CAN BE OBTAINED USUALLY FREE OF CHARGE. >> IT MAY BE SOMETHING FOR NIH TO CONSIDER MAKING SURE POSTMORTEM TISSUES ARE WITHIN A RANGE AND UNLIKELY TO CAUSE ARTIFACTS. >> I HAVE A QUESTION FOR DR. WOO. I'M WONDERING IF THE DEGLYCOSYLATION HELPS IDENTIFY OR IMPLICATE IMMUNE BRAIN TARGETS OR STUDYING SIGNALLING AND PATHWAYS IN THE CONTEXT OF SUBSTANCE ABUSE OR MAYBE ACTION OF DRUG ABUSE. IS THAT A RELEVANT QUESTION? >> THANKS FOR ASKING THE QUESTION AND ONE I'VE BEEN ASKING MYSELF AND MAYBE LINDA CAN ALSO CHIME IN A BIT BECAUSE I KNOW SHE THOUGHT ABOUT SOME THINGS HERE AS WELL. I'VE PARTICULARLY BEEN THINKING ABOUT IT IN THE CONTEXT OF CERTAIN TRANSCRIPTION FACTORS THAT HAVE BEEN KNOWN TO MAINTAIN ADDITION SIGNALLING THAT ARE ALSO O-GLCNAC RELATED. RIGHT NOW THERE'S NOT MUCH PROFILING OF O-GLCNAC IN MODELS IF THAT WAS TO BE PUR SUVED. PURSUED. >> I AGREE IN LARGE NUMBERS OF THE O-GLCNAC FACTORS TO BE MODIFIED IT'S A GREAT PLACE TO START IN TERMS OF LOOKING AT REGULATION AND THEY CHANGE WITH NEURAL ACTIVITY. AND SEEING WHICH CHANGE IN DISEASE OR PERTURBATIONS AND COULD BE AND SEE HOW THEY CHANGE IN ADDICTION. >> THEY RESPOND TO STIMULI IN A NUMBER OF OTHER DISEASES BOTH IN THE CONTEXT OF THE BRAIN AND MORE BROADLY AND OFTEN INHIBITION OF THE CHANGES CAN ALSO INHIBIT THE SIGNALLING PROFESSION. I DO THINK THERE'S AN OPPORTUNITY THERE. >> IN LOOKING AT O-GLCNAC IS IT 10%, 20%? >> IT'S A GREAT QUESTION, AUSTIN. IT WILL DEPEND ON THE PROTEIN IN TERMS OF THE MODIFICATION AND RELATION TO THE FACT THAT WILL ALSO VARY DEPENDING ON WHAT THE FUNCTION IS. IN THE CONTEXT OF TAU AND SYNUCLEIN THEY'RE SHOWING THE PERCENTAGE OF GLYCOSYLATION IN THE CONTEXT OF ALPHA SYNUCLEIN THAT LEADS TO THE AGGREGATION AND BASICALLY YOU CAN SEE A LINEAR DECREASE IN AGGREGATION AS A FUNCTION OF INCREASING GLYCOSYLATION AND CAN SEE IT ON THE ALPHA SYNUCLEIN. DID THAT ANSWER YOUR QUESTION? >> I'M SURPRISED TO SEE THE LINEAR CHANGES. >> HE WAS MIXING TOGETHER THE PERCENTAGE OF GLYCANS AND MAYBE I'LL E-MAIL HIM AFTER THE COMMENTS AND SEEING IF YOU GLYCOSALATE AFTER MAN CAUSE FURTHER GLYCOSYLATION. >> WE HAVE FIVE MINUTES LEFT OF OUR ROUNDTABLE DISCUSSION. IF PEOPLE HAVEN'T HAD A CHANCE YET AN THERE'S SOMETHING BURNING ON YOUR MIND FEEL FREE TO SAY IT. I'LL ASK AS THE COMMON FUND TO AND ASK IF YOU'RE NOT A GLYCO GLYCOBIOLOGIST YOU SPREAD WHAT YOU LEARNED HERE TO YOUR COLLEAGUES AND DIRECT THEM TO US SO WE CAN DIRECT THEM TO THE EXPERT TO HEAR THE NEW NOVEL TECHNOLOGIES COMING OUT THAT ARE AVAILABLE. AS WE DO THIS WE HAVE TO CATALOG WHAT WE'RE DOING SO IT BECOMES READILY AVAILABLE LIKE CATALOGS OF ANTIBODIES THOUGH THEY'RE FROM DIFFERENT COMPANIES AND LECTINS AND THE CHEMICAL PROBES THAT WARE DEVELOPING. ARE THERE THINGS PEOPLE WANT TO SAY BEFORE WE SIGN OFF? >> I'D LIKE TO THANK EVERYONE AGAIN. EVERYONE DID A TON TODAY AND YESTERDAY. I THINK IT'S A GOOD MANGE OF GLYCOSCIENCE AND NEUROSCIENCE. THERE'S HUGE POTENTIAL HERE AND HOPE EVERYONE TAKES ADVANTAGE OF AUSTIN'S SUPPLEMENT PROGRAM. >> WE HAVE -- WILL TAKE THE REGULAR APPLICATIONS AS WELL. >> KAREN'S ALSO LOOKING FOR GLYC GLYC GLYCANS. >> ALSO SOMETHING NEW TO PITCH S&P >> I'D LIKE TO THANK ALL THOSE WHO WERE HERE AND NOT HERE AND FOR BEING PATIENT. I KNOW IT WAS A LONG DAY. THIS WOULD BE A WONDERFUL MEET MEETING TO DO ALL OVER AGAIN AFTER WE GET NEW DATA AND RESULTS. >> THANK YOU, EVERYONE. >> THANK YOU, ALL. >> BYE-BYE.