Welcome to the Clinical Center Grand Rounds, a weekly series of educational lectures for physicians and health care professionals broadcast from the Clinical Center at the National Institutes of Health in Bethesda, MD. The NIH Clinical Center is the world's largest hospital totally dedicated to investigational research and leads the global effort in training today's investigators and discovering tomorrow's cures. Learn more by visiting us online at http://clinicalcenter.nih.gov TODAY WE WELCOME OUR SPEAKERS, DR. DEREK NARENDRA AND CHIEF OF THE INHERITED MOVEMENT DISORDERS UNIT, AND DR. CLAIRE LE PICHON, DIVISION OF INTRAMURAL RESEARCH IN THE EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT. DR. NARENDRA EARNED HIS BACHELOR FROM COLUMBIA UNIVERSITY, AND MEDICAL DEGREE FROM THE UNIVERSITY OF MICHIGAN. THROUGH HIS GRADUATE RESEARCH, HE IDENTIFIED A NOVEL MITOPHAGY PATHWAY THROUGH THE COORDINATED ACTIVITIES OF PARKIN AND PINK1, MUTATIONS WHICH ARE THE LEADING CAUSE OF EARLY ONSET PARKINSON'S DEED. DISEASE. COMPLETED NEUROLOGY RESIDENCY TRAINING AT THE BRIGHAM AND WOMEN'S HOSPITAL AND MASSACHUSETTS GENERAL HOSPITAL HARVARD PROGRAM IN 2016, WITH ADDITIONAL FELLOWSHIP TRAINING OF MOVEMENT DISORDERS AT UNIVERSITY OF PENNSYLVANIA. IN 2017, DR. NARENDRA RECEIVED THE TRANSITION TO INDEPENDENCE AWARD AND JOINED NINDS AS A CLINICAL -- ASSISTANT CLINICAL INVESTIGATOR WITHIN THE NEUROGENETICS BRANCH. HIS LABORATORY FOCUSES ON MITOCHONDRIAL DYSFUNCTION AND STRESS RESPONSES IN DISORDERS SUCH AT PARKINSON'S DISEASE, ALS. IN 2020 HE RECEIVED A LASKER CLINICAL RESEARCH SCHOLARSHIP AND BECAME A TENURE TRACK INVESTIGATOR. DRTHE MOVEMENT DISORDER SOCIETY AND AMERICAN NEUROLOGICAL ASSOCIATION AND IN 2022, HE RECEIVED AN AWARD IN NEUROSCIENCE. OUR SECOND SPEAKER IS DR. CLAIRE LE PICHON. DR. LE PICHON EARNED HER BACCALAUREATE FROM UNIVERSITY OF CAMBRIDGE IN THE UNITED KINGDOM AND HER PH.D. IN BIOLOGICAL SCIENCES FROM COLUMBIA UNIVERSITY IN 2007. AT COLUMBIA, IN A LABORATORY OF DR. STUART FIRESTEIN, SHE DEVELOPED HER INTEREST IN NEURODEGENERATIVE DISEASE WHILE STUDYING THE FUNCTION OF THE CELLULAR PRION PROTEIN, PRPC. AFTER HER GRADUATE STUDIES, DR. LE PICHON JOINED GENERAL TECHGENENTECH,PRE-CLINICAL DRUGT FOR NEURODEGENERATIVE DISEASES. IN 2013, SHE JOINED THE NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE AS A SENIOR RESEARCH FELLOW AND IN 2016, SHE WAS INVITED TO BECOME A TENURE TRACK INVESTIGATOR IN NICHD. DR. LE PICHON'S LABORATORY EMPLOYS A MULTIDISCIPLINARY APPROACH USING MOUSE MODELS AND HUMAN IPSC-DERIVED NEURONS TO INVESTIGATE THE EARLY EVENTS UNDERLYING THE ONSET AND PROGRESSION OF NEURODEGENERATIVE DISEASE. DR. LE PICHON EARNED THE NIH'S DIRECTOR'S AWARD, THE KIRSCHSTEIN AWARD FOR MENTORING IN 2021, AND RECEIVED NUMEROUS NICHD SCIENTIFIC DIRECTORS AWARDS AND MERIT AWARDS. SHE IS A MEMBER OF THE AMERICAN SOCIETY FOR CELL BIOLOGY AND SOCIETY FOR NEURAL SCIENCE AND IS A NATIONALLY AND INTERNATIONALLY RECOGNIZED SPEAKER IN THE FIELD OF NEURODEGENERATIVE DISEASES. PLEASE WELCOME OUR FIRST SPEAKER, DR. NARENDRA, LESSONS FROM GENETICS. WELL, THANK YOU FOR THAT INTRODUCTION AND FOR THE OPPORTUNITY TO SHARE WORK THAT OUR GROUP HAS BEEN DOING OVER THE LAST FIVE YEARS FOCUSED ON MITOCHONDRIA AND HOW DYSFUNCTION CAN LEAD TO NEURODEGENERATION AND HOW THE CELERY SP CELL RESPO DAMAGED MITOCHONDRIA. THESE ARE MY DISCLOSURES, THAT I WON'T BE TALKING ABOUT TODAY. IT'S NOT PERHAPS SURPRISING THAT MITOCHONDRIAL FUNCTION CAN BE A DRIVER OF NEURODEGENERATION GIVEN THAT THE NEURONS AND MYOSITES HAVE VERY HIGH RG DEMANDS. THE WAY THEY'RE ABLE TO DO THAT IS THROUGH THESE MITOCHONDRIA THAT CAN PROVIDE ATP IN ORDER TO MAINTAIN THOSE CONTRACTIONS. THESE MITOCHONDRIA HAVE TO NOT ONLY BE PROPERLY FUNCTIONING BUT THEY ALSO HAVE TO BE PROPERLY SHAPED AND DISTRIBUTED IN THE TISSUE TO MEET THAT ENERGY DEMAND. SIMILARLY IF YOU THINK ABOUT THE NEURONS, SPINAL CORD AND ELSEWHERE, THEY HAVE VERY HIGH LOCAL DEMANDS FOR ATP SUCH AS AT THE SNAPS HERE. SYNAPSE HERE.THESE ARE ALSO MITD TEND TO ACCUMULATE MITOCHONDRIAL DAMAGE OVER THE NORMAL COURSE OF AGING, TO A GREATER EXTENT THAN MITOTIC CELLS THROUGH CELL DIVISION. THESE CELLS ESPECIALLY ARE DEPENDENT ON MECHANISMS THAT ALLOW THE CELL TO BE ABLE TO RECOGNIZE WHEN MITOCHONDRIAL DAMAGE HAS OCCURRED AND TO BE ABLE TO RESPOND TO THAT MITOCHONDRIAL DAMAGE. YOU MIGHT IMAGINE A COUPLE OF DIFFERENT STRATEGIES THAT THE CELL MIGHT USE. ONE MIGHT BE TO ALLOW THE CHANGE IN MITOCHONDRIA TO LEAD TO A CHANGE IN THE OVERALL CELLULAR STATE AND THEN SINCE THAT CHANGE IN CELLULAR STATE -- THIS SEEMS TO BE HOW THE CELE CELL RESPONDH AS IN ATP THAT ARE VERY DEPENDENT ON MITOCHONDRIAL FUNCTION IN THE CELL. YOU MIGHT IMAGINE THAT THERE MIGHT BE ANOTHER STRATEGY, THOUGH, IF THERE'S A SENSOR OF THAT MITOCHONDRIAL DAMAGE WITHIN THE MITOCHONDRIA ITSELF, THAT MIGHT SERVE AS A WAY OF RECOGNIZING WHEN THAT BECOMES DAMAGED AND ALLOW THE CELL TO RESPOND TO THAT DAMAGE, CONTAIN IT BEFORE IT LEADS TO A CHANGE IN THE OVERALL CELLULAR STATE. BUT THERE ARE MECHANISMS THAT ARE ABLE TO DO THIS, PERHAPS THE BEST DESCRIBED ONE OF WHICH IS THIS PINK1 PARKIN AUTOPHAGY PATHWAY. THE WAY THIS PATHWAY WORKS IS MY TMITOCHONDRIA UNDERGOES SOME SOT OF DAMAGE, NORMALLY IMPORTED TO THE -- IF THAT MITOCHONDRIA IS DAMAGED AND IMPORT PATH IS BLOCKED THEN PINK1 ACCUMULATES INSTEAD ON THE OUTER. PINK1 IS A KINASE AND IT WILL ACTIVATE PARKIN, WHICH WILL THEN EU BIC TI NATE PROTEINS ON THE OUTER MEMBRANE. THIS SERVES AS A WAY OF MARKING THIS DAMAGED MITOCHONDRION AND DELIVERY TO A LYSOSOME WHERE IT CAN BE DEGRADED. THIS ACTS AS A CLEAN-UP PATHWAY FOR DAMAGED MITOCHONDRION IN THE CELL AND BECAUSE RECESSIVE MUTATIONS ARE THE LEADING GENETIC CAUSE OF EARLY ONSET PARKINSON'S DISEASE, IT ALSO IMPLICATES THIS MITOCHONDRIA CONTROL PATHWAY IN PARKINSON'S AS WELL AS OTHER FORMS OF KNEW OWE DEGENERATION. IN ADDITION TO THIS MI TO HAVE -- INTO THEHEALTHY MITOCHONDRIE CELL. MITOCHONDRIA NORMALLY UNDERGO THESE SI CYCLES OF FUSION AND DIVISION AND THESE ARE MEDIATED BY A SET OF GTP ACES ON THE OUTER MEMBRANE, YOU GET FUSION OF THAT MEMBRANE AND THE INNER MEMBRANE IS MEDIATED BY FEW SHON -- IS MADE BY OMA1 THEN THE MY TOE COULMITOCHONDRIA -- CALLT ALLOWS THAT MITOCHONDRIA TO SPLIT AGAIN. THROUGH THESE CYCLES OF FUSION AND DIVISION THE CELL IS ABLE TO CHANGE THE SHAPE OF THE MITOCHONDRIAL NETWORK DYNAMIC FULLY IN RESPONSE TO CERTAIN DEVELOPMENTAL OR METABOLIC QUEUES AND IT ALSO ALLOWS THE CELL TO MAINTAIN QUALITY OF THE OVERALL MITOCHONDRIAL -- BUT IF THE M MITOCHONDRION UNDERGOES DAMAGE YOU DON'T WANT TO FUSE ANYMORE, IT DEGRADES THESE FUSION PROTEINS ON THE OUTER MEMBRANE, THEN CLEAVES THE OMA1 FROM THE INNER MEMBRANE. IT DOES THIS THROUGH AT LEAST TWO DIFFERENT MECHANISMS. ONE DEPENDS ON THE PINK1 PARKIN PATHWAY ON THE OUTER MEMBRANE, AND EVEN AT LOW LEVELS OF PARKIN YOU'LL SEE UBIQUINIATION AND DEGRADATION. THERE'S A PARALLEL PATHWAY THAT OCCURS ON THE INNER MEMBRANE OF THE MITOCHONDRION, WHERE INSTEAD OF PINK1 ACTING AS THE SENSOR OF MITOCHONDRIAL DAMAGE, YOU HAVE OMA1 WHICH IS ABLE TO SENSE DAMAGE OF THIS INNER MEMBRANE. IT BECOMES MORE ACTIVE AND WILL CLEAVE THE LONG FORM OF OPA1 TO SHORT FORM. THE SHORT FORM OF OPA1 CAN'T MEDIATE FUSION ON ITS OWN SO THIS INHIBITS FUSION SO YOU HAVE THESE TWO DIFFERENT SENSORS OF MITOCHONDRIAL DAMAGE, PINK1 WORKING ON THE OUTER MEMBRANE AND ONE THAT WORKS ON THE INNER MEMBRANE. THE OVERALL INTEREST OF OUR RESEARCH GROUP IS TO TRY TO UNDERSTAND THESE MECHANISMS OF CELL SENSING OF MITOCHONDRIAL DAMAGE AND THE ROLE THEY PLAY IN NEURODEGENERATION AND HOW THEY MIGHT BE MODULATED TO HELP TREAT NANEURODEGENERATIVE DISORDERS. OUR LAB FOCUS ON THIS PINK1 PARKIN PATHWAY AS SENSOR OF THE DAMAGE AND ITS ROLE IN PARKINSON'S DISEASE, AND THE SECOND IS TO REALLY FOCUS ON THIS OMA1 PATHWAY THAT WE FOUND IS VERY STRONGLY ACTIVATED BY MUTATIONS IN TWO OTHER MITOCHONDRIAL PROTEINS THAT LEAD TO DIFFERENT FORMS OF NEURODEGENERATION. AND THEN THE FOCUS OF TODAY'S TALK WILL BE ON A SPECIFIC MUTATION, CHCHD10 AND HOW THAT PARTICULAR MUTATION HAS LED US TO RECOGNIZE THE IMPORTANCE OF THIS OMA1 STRESS RESPONSE, PARTICULARLY IN VIVO. SO I'LL TELL YOU -- INTRODUCE THIS PROTEIN A LITTLE BIT MORE, THEY'RE SMALL PROTEINS WITHIN THE INTERMEMBRANE SPACE OF THE MITOCHONDRIA. SO MITOCHONDRIA HAVE THIS OUTER MEMBRANE AS WELL AS THIS INNER MEMBRANE AND THESE PROTEINS ARE LOCATED BETWEEN THOSE TWO MEMBRANES IN THE SPACE. INNER MEMBRANE WILL ALSO FORM MITOCHONDRIAL -- THESE PROTEINS WILL EXTEND DOWN INTO THOSE MITOCHONDRIAL CHRISTY AND IT SEEMS THAT THEY'RE IMPORTED FOR MAINTAINING NORMAL STRUCTURE AS WELL AS FOR MAINTAINING OPTIMAL CAPACITY OF THE MITOCHONDRIA. WE'VE SHOWN THESE PROTEINS ARE FUNCTIONING WITH ONE ANOTHER AND IF YOU KNOCK OUT EITHER ALONE IN THE MOUSE IT DOESN'T RESULT OF MUCH OF A PHENOTYPE BUT IF YOU FLOCK OUT BOTH OF THESE PROTEINS TOGETHER, THE MICE DEVELOP A CARDIOMYOPATHY. ADDITIONALLY IF YOU LOOK IN THE HEART TISSUE YOU SEE STRESS RESPONSE THAT I'LL GO INTO MORE DETAIL IN A MINUTE. WE KNOCK OUT BOTH OF THESE PROTEINS BUT YOU DON'T SEE IF YOU KNOCK OUT ONLY ONE ALONE SO THEY SEEM TO BE DOING SOMETHING TOGETHER ON THE CELL. DOMINANT MUTATIONS IN CHCHD10 CAUSE NEUROMUSCULAR DISORDERS. THIS RANGES FROM DEMENTIA, ALS, DESCRIBED WITH THE ORIGINAL MUTATION IN THE TWO FAMILIES THESE FAMILIES HAVE A VERY COMPLEX NEUROLOGICAL PHENOTYPE WHERE THEY HAVE INVOLVEMENT OF MUSCLE AS WELL AS SPINAL CORD AS WELL AS THE BRAIN. THERE'S A MUCH MORE LIMITED PHENOTYPE THAT CAN BE SEEN WITH ANOTHER MUTATION WHERE JUST THE LOWER MOTOR NEURONS IN THE SPINAL CORD ARE AFFECTED, CAUSING AN ADULT ONSET FORM OF SPINAL MUSCULAR ATROPHY. THIS IS RELATIVELY COMMON IN SOUTHEAST FINLAND. THERE'S ANOTHER MUTATION THAT'S PROBABLY THE MOST COMMON CHCHD10 MUTATION IN NORTH AMERICA WHERE IT CAUSES MORE OF A TYPICAL ALS PHENOTYPE, ALTHOUGH THESE PATIENTS ARE SLOWLY PROGRESSIVE. IN COLLABORATION WITH NCATS WHO IDENTIFIED THIS MUTATION, WE ARE DEVELOPING AN ANTI -- TO TRY TO TREAT THIS FORM OF ALS. WE SAW A COUPLE OF PATIENTS WITH THIS FORM OF ALS IN THE CLINICAL CENTER EARLIER THIS YEAR. THEN THERE'S ANOTHER MUTATION WHERE THE INDIVIDUALS WILL DEVELOP JUST A DOMINANT FORM OF MYOPATHY AND AS I'LL SHOW YOU IN A SECOND ALSO CARDIOMYOPATHY BUT THEY DON'T SEEM TO HAVE MOTOR NEURONS, THE SPINAL CORD SEEMS TO BE SPARED. IT'S INTERESTING CAN YOU HAVE TWO MUTATIONS THAT ARE NEXT TO EACH OTHER THAT CAN HAVE VERY DIFFERENT PHENOTYPIC MANIFESTATIONS. ONE THING THAT HAD BEEN UNCLEAR IN THE FIELD WAS EXACTLY WHICH MUTATION IS THE CAUSE OF THE MUTATION FOR THIS DOMINANT FORM OF MYOPATHY. THE REASON THAT WAS UNCLEAR IS THAT ALTHOUGH -- IN THE LAB AT NORTHWESTERN WAS ABLE TO IDENTIFY CHCHD10 AS THE CAUSATIVE GENE BY POSITIONAL CLONING, THEY FOUND OUT ONE VARIANT BUT ACTUALLY TWO VARIANTS. SO THEY COULDN'T SAY BASED ON GENETIC GROUNDS ALONE FROM THIS ONE FAMILY THESE TWO MUTATIONS WAS THE CAUSE OF THE MUTATION. SO THIS WAS IN THE BACK OF MY MIND WHEN I MET JEFF AT A CONFERENCE A FEW YEARS AGO WHO'S AN EXPERT IN MITOCHONDRIAL DISORDERS AT OXFORD. SHE MENTIONED SHE'D BEEN FOLLOWING FOR A NUMBER OF YEARS A FAMILY THAT HAD A FORM OF CARDIOMYOPATHY AND THEY RECENTLY HAD DONE SEQUENCING AND IDENTIFIED THIS MUTATION IN CHCHD10 WITHOUT THE OTHER MUTATIONS. SO THIS WAS QUITE INTERESTING BECAUSE IT SUGGESTS THAT THIS IS PROBABLY THE CAUSE OF THE MUTATION FOR THIS DOMINANT FORM OF MYOPATHIES AND WE RESOLVED TO HELP CHARACTERIZE THIS TOGETHER AS PART OF THE COOPERATION. SO THIS IS SEEN AT 8 YEARS OLD, DISTAL AND PROXIMAL MUSCLE WASTING, ALSO SOME MILD FACIAL LUPUS. THIS IS HIM 12 YEARS LATER, AND AT THIS POINT HE'S HAD PROGRESSION OF THE MYOPATHY, WHEELCHAIR-BOUND. HE ALSO DEVELOPED A CARDIOMYOPATHY SEVERE ENOUGH TO REQUIRE A HEART TRANSPLANT. UNFORTUNATELY BECAUSE OF THE IMMUNOSUPPRESSION, HE DEVELOPED LYMPHOMA AND PASSED AWAY. THIS IS HIS FAMILY, HIS MOTHER HAD A VERY SIMILAR PHENOTYPE AND ALSO DIED YOUNG, AT THE AGE OF 34, AND WE THINK THAT MOST LIKELY THIS WAS DUE TO A MUTATION IN HIS MOTHER. AT THIS POINT, PH.D. STUDENT IN THE LAB WAS INTERESTED IN TRYING TO BETTER UNDERSTAND THE PATHOGENESIS OF THIS DISORDER, SO WITH THE HELP OF NHLBI TRANSGENICS HE MADE A KNOCK-IN MOUSE MODEL WHERE HE INTRODUCED INTO THE MOUSE A GENE THAT WAS SEEN IN THE PATIENT. THESE HETEROZYGOUS MICE, DEVELOPED A PHENOTYPE THAT LOOKS VERY SIMILAR TO THE PATIENT'S PHENOTYPE, DECREASED SIZE AND WEIGHT, ALSO DECREASED STRENGTH AND MYOPATHY AND DECREASED HEART FUNCTION ON ECHO CARDIOGRAPHY. NOTABLY THIS PHENOTYPE IS DIFFERENT THAN WHAT WE HAD SEEN AND REPORTED FOR THE SINGLE KNOCKOUT MICE AS WELL AS THE DOUBLE KNOCKOUT MICE. AND THIS INDICATES THAT THIS MUTATION IS PROBABLY CAUSING DISEASE BY GAIN OF FUNCTION MOST LIKELY A TOXIC GAIN OF FUNCTION MECHANISM. IT ALSO HAS IMPLICATIONS FOR HOW WE'RE THINKING ABOUT TREATING THIS DISORDER BECAUSE IT MEANS IF YOU COULD LOWER LEVELS OF THIS TOXIC PROTEIN IT PROBABLY WOULD HAVE A THERAPEUTIC BENEFIT AND THE FACT THAT IT SEEMS TO BE TOLERATED AT LEAST IN MICE SUGGESTS THAT YOU CAN SAFELY LOWER EVEN -- LEVELS OF CHCHD10 AND NOS HAVE A TOXICITY RESULT. WHAT HAPPENS TO THIS PROTEIN WHEN IT HAS THIS MUTATION, IT TENDS TO FORM FOCI, HEART MUSCLE SHOWN HERE, AND WE THINK THAT THESE FO FOCI ARE AGGREGATES OF0 PROTEINS ACTUALLY LOCATED WITHIN THE MITOCHONDRIA. ALSO LOOKED AT NEIGHBORING MUTATION IN CHCHD10 AND OTHER KNOCK-IN MOUSE MODEL. THIS HAD BEEN PREVIOUSLY REPORTED TO ALSO HAVING A GATS TO HAVING AGATS THAT FORM IN THT MUSCLE. AGGREGATES WITH BOTH OF THESE MUTATIONS ARE DIFFERENT MORPHOLOGICALLY SO WITH THE G15R MUTATION THAT CAUSES THE SEVERE MYOPATHY, YOU HAVE THESE PUNCTATE-LIKE AGGREGATES WHEREAS YOU HAVE A DIFFERENT FORM OF AGGREGATE WITH A MILDER FORM OF MUSCLE INVOLVEMENT AND MORE SEVERE INVOLVEMENT OF MOTOR NEURONS. THE LOCALIZATION OF THESE AGGREGATES ALSO APPEARS TO BE DIFFERENT. SO WE CAN SEE THE BORDERS OF THE MITOCHONDRIA BY STAINING. THIS IS WHAT THEY LOOK LIKE IN THE HEALTHY WILD TYPE CONDITION, G58R, MAGENTA FOCI ARE LOCATED PREDOMINANTLY WITHIN THE MITOCHONDRIA, THE OTHERS ARE OUT OF THE MITOCHONDRIA. SO WHAT IS COMMON IS THEY'RE TOSSING C10 TO MISFOLD BUT PROBABLY IT FIST FOLDS INTO DIFFERENT TOXIC SPECIES AND PERHAPS THAT UNDERLIES THE DIFFERENT TISSUE VULNERABILITY TO THESE TOXIC PROTEINS. IT SEEMS THAT MISFOLDING OF CHCHD10 IS RELATED TO THESE TWO PROTEINS. TWO AUTOPSY CASES HAVE BEEN REPORTED, AND WHEN THEY LOOK IN THE AFFECTED REGION SUCH AS SUBSTANTIA NIGRA, INDICATING PROBABLY THIS PROTEIN IS MISFOLDING ALSO BECAUSE OF THIS MUTATION. SIMILARLY IN A CASE OF ALS THAT'S BEEN REPORTED DUE TO MUTATION IN CHCHD10, IN THE MOTOR NEURONS THAT ARE SURVIVING IN THE SPINAL CORD, YOU'LL FINDING A GATS OF CHCHD10 WITHIN THOSE MOTOR NEURONS THAT AREN'T TYPICALLY SEEN IN SPORADIC ALS. WE ALSO LOOKED ULTRA STRUCTURALLY AT THE MITOCHONDRIA TO SEE HOW THEY WERE AFFECTED BY THIS PROTEIN IN THE CENTER MEMBRANE SPACE. THE CHRISTY BEING VERY DISTORTED AS WELL AS THESE INTRACRYSTAL VESICLES THAT DEVELOP, SO WE THINK THIS MISFOLDED PROTEIN IS EXERTED STRESS ON THE INNER MEMBRANE -- MEMBRANE. AND IS LEADING TO WHAT'S ESSENTIALLY CHRISTYOPATHY, AND THE MILD DEFECTS WE SEE IN THE AFFECTED TISSUES ARE PROBABLY SECONDARY TO DAMAGE TO THIS INNER MEMBRANE. WE ALSO LOOKED AT THE OVERALL MORPHOLOGY OF THE MITOCHONDRIA IN THE HEART, USING A TECHNIQUE CALLED FIB SYN, THEY'RE PSEUDOCOLORRED AND YOU CAN SEE THE INDIVIDUAL MITOCHONDRIA, AND IF WE COMPARE THE CONTROL CONDITION AND WE FIND THE MITOCHONDRIA AND HEART ARE FRAGMENTED COMPARED TO THE CONTROL CONDITION AND OVERALL THE VOLUME OF THE MITOCHONDRIA IS ABOUT HALF OF WHAT WE SEE IN THE WILD TYPE CONDITION. SO THIS MITOCHONDRIAL FRAGMENTATION WAS REALLY DRAMATIC AND MADE US WONDER IF THIS MIGHT BE A -- PROCESS AND IN PARTICULAR IF IT MIGHT BE INDICATIVE OF THAT STRESS RESPONSE I INTRODUCED TO YOU AT THE BEGINNING WHEREBY DISRUPTIONS TO MEMBRANE -- IDENTIFY THE INNER MEMBRANES ARE DISTORTED DUE TO THE PROTEIN MISFOLDING IN THE CHRISTY, TO ACTIVATED TO CLEAVE THE LONG FORMS TO THE SHORT FORMS. IF YOU INHIBIT FUSION FRAGMENTATION OF THE MITOCHONDRIAL NETWORKS. SO WE LOOKED AT OMA1 PROCESSING OF THE SPECIFIC OMA1, THE C AND E SHOWN IN THIS WESTERN BLOT WITH THE AFFECTED TISSUES. SIM SKELETAL MUSCLE, HEART TISSUE, WE SAW THE SAME PATTERN INDICATIVE OF OMA1 ACTIVATION. SO WE HAVE THIS OMA1 STRESS RESPONSE ACTIVATED BY THIS PROTEIN THAT'S MISFOLDING WITHIN MITOCHONDRIAL CRISTAE. SO WHILE WE WERE DOING THIS WORK, A COUPLE OF VERY INTERESTING PAPERS CAME OUT, ONE FROM UCSF AND ANOTHER IN MUNICH. WHAT THEY FOUND IS THAT AT LEAST IN CULTURED CELLS THAT ARE EXPOSED TO MITOCHONDRIAL TOXINS LIKE CCP AND -- OPA1 HAS ANOTHER SUBSTRATE IN ADDITION TO OPA1 CALLED DELI1. IT COMMUNICATES THE M MITOCHONDRIAL SUPPRESS BY OMA1 TO THE CYTOSOL NUCLEUS TO SOMETHING CALLED THE INTEGRATED STRESS RESPONSE. SPECIFICALLY OMA1 WILL CLEAVE THE LONG FORM OF DELE1 INTO A SHORT FORM THAT WILL THEN ACTIVATE THIS EIFT ALPHA CLIE NAIS. WHEN THIS EIF2L ALPHA IS PHOSPHORYLATED, THIS TRANSLATION INITIATION FACTOR IS GOING TO LEAD TO THE INTEGRATED STRESS RESPONSE THAT HAS TWO MAIN COMPONENTS. THE FIRST IS TO PUT A HALT ON MOST PROTEIN TRANSLATION SO YOU GET DECROSED PROTEIN SYNTHESIS OVERALL IN THE CELL. BUT THEN CERTAIN TRANSCRIPTION FACTORS LIKE ATF4 AND 5 THAT HAVE THESE SPECIAL UPSTREAM -- ARE PREFERENTIALLY TRANSLATED AS YOU HAVE UPREGULATION OF THESE THAT CAN MEDIATE A TRANSCRIPTIONAL RESPONSE. IT'S CALLED THE INTEGRATED STRESS RESPONSE BECAUSE THERE ARE ACTUALLY FOUR DIFFERENT KINASES, EACH OF WHICH -- FOR INSTANCE, PERC AND EACH WILL PHOSPHORYLATE THE SAME RESIDUE LEADING TO THIS STEREOTYPED RESPONSE TO A VARIETY OF DIFFERENT CELLULAR STRESS. SO THIS HAD BEEN DEMONSTRATED VERY NICELY IN CULTURED CELLS BUT WE WONDERED CAN THIS ACTUALLY OCCUR IN VIVO IN A DISEASE RELEVANT MODEL SYSTEM. WE THOUGHT WE HAD A GOOD MODEL SYSTEM TO POTENTIALLY TEST THIS IDEA. SO WE FIRST ASKED IS THIS INTEGRATED STRESS RESPONSE ACTIVATED IN AFFECTED TISSUES OF OUR MOUSE MODEL. INDEED WE FOUND EIF2 ALPHA IS PHOSPHORYLATED BOTH IN THE HEART AND THE AFFECTED SKELETAL MUSCLE. WE NEXT ASKED IS THIS BEING SIGNALED BY OMA1 AND TO GET AT THIS QUESTION, WE KNOCKED DOWN OMA1 IN VIVO BY INJECTING THE ANIMALS AGAINST OMA1, WE ACHIEVED VERY GOOD KNOCK DOWN AT THE PROTEIN LEVEL IN THE HEART AND THIS SUPPRESSED THIS ACTIVATION OF THE INTEGRATED STRESS RESPONSE, INDICATING THAT IT IS BEING SIGNALED BY OMA1 IN VIVO. WE SAW VERY CONSISTENT RESPONSE IN THE TRANSCRIPTOMICS, WE SEE UPREGULATION IN THE -- HEARTS COMPARED TO THE WILD TYPE BUT TRANSCRIPTION FACTOR IS KNOWN TO BE PART OF -- AS WELL AS A NUMBER OF DOWNSTREAM TARGETS. IF WE LOOK AT MUTED HEARTS WE GET DOWN RG LAITION OF THE SAME TRANSCRIPTIONAL SIGNATURE INDICATING THAT. OMA1 IS RESPONSIBLE IN MUTED HEARTS. WE NEXT ASKED IS THIS RESPONSE PROTECTIVE OR DETRIMENTAL TO THESE ANIMALS? WE CROSSED OUR CROSSED ANIMAL WITH -- WHAT WE FOUND IS IF THEY CAN'T TURN ON THE STRESS RESPONSE MOST OF THE MICE WILL DIE IN THE FIRST EIGHT DAYS OF LIFE. ABOUT A THIRD OF THEM CAN SURVIVE LONGER, TO ABOUT 14 OR 15 WEEKS, BUT THOSE ANIMALS HAVE THESE MASSIVELY ENLARGED HEARTS. SO WE THINK THAT THIS STRESS RESPONSE IS HELPING THE CELL AND THE TISSUE COPE WITH THE MITOCHONDRIAL DAMAGE THAT'S BEING CAUSED BY THIS PROTEIN MISFOLDING WITHIN THE MITOCHONDRIAL CRISTAE. SO TO PUT THIS TOGETHER, WE HAVE THIS PROTEIN MISFOLDING WITHIN THE MITOCHONDRIAL -- AND IT'S ACTIVATING THIS TRANSCRIPTIONAL RESPONSE THAT LEADS TO A NUMBER OF METABOLIC CHANGES INCLUDING UPREGULATION OF PATHWAYS INVOLVED IN -- SYNTHESIS AND CARBON METABOLISM AND IS BEING SIGNALED BY OMA1 AND DELE1. THIS RAISES THE QUESTION, IS THIS A VERY GENERAL RESPONSE, IS IT ALSO SIGNALING OTHER FORMS OF MITOCHONDRIAL STRESS IN THE HEART AND OTHER ORGANS. AND A COUPLE OF PAPERS THAT HAVE BEEN PUBLISHED SINCE WE PUBLISHED THIS FINDING SUGGESTS THIS IS PROBABLY A MAJOR RESPONSE TO DIFFERENT FORMS OF MITOCHONDRIAL STRESS. THERE WAS A PAPER WHICH DEMONSTRATED THAT IF YOU INHIBIT CARDIO -- THERE IS IMPORTANT FOR THE INNER MEMBRANE OF MITOCHONDRIA WITH KNOCKOUTS OF EITHER OF THESE TWO PROTEINS YOU GET VERY SIMILAR TRANSCRIPTIONAL RESPONSE. THIS IS DEPENDENT ON THE -- AS WELL AS PHOSPHORYLATION AND VERY RECENTLY IT WAS DEMONSTRATED IF YOU CREATE DEFICIENCY IN THE HEART BY KNOCKING OUT THE SUBUNIT OF THE OXPHOS CHAIN ON COX10, YOU GET A VERY SIMILAR TRANSCRIPTIONAL RESPONSE, SIMILARLY DEPENDENT ON DELE SO WE THINK THIS IS GOING TO TURN OUT TO BE PREDOMINANTLY -- IN VIVO ORGANISMS. SO TO SUMMARIZE OUR FINDINGS, WE HAVE MUTATIONS IN C2 AND C10 THAT CAUSE NEUROTEE GENERATIVE DISORDERS WITH A CLEAR GENOTYPE-PHENOTYPE RELATION SHI. DOMINANT MUTATIONS IN C2 AND C10 CAUSES PROTEINS TO MISFOLD AND LICELY -- THE CHCHD2 -- LEADS TO MITOCHONDRIAL MYOPATHIES AND STRESS RESPONSE WITH LOCAL AND GLOBAL RESPONSES THAT PROTECT FROM MITOCHONDRIAL DAMAGE. I JUST WANT TO ACKNOWLEDGE A -- BIOLOGIST AND LAB MANAGER WITHIN THE LAB. AND WITH THAT, I'LL TURN IT OVER NOW TO CLAIRE, WHO WILL TELL BUT A DIFFERENT STRESS RESPONSE. >>THANKS, DEREK, AND THANK YOU, TOM, FOR THE NICE INTRODUCTION. SO TODAY I'M GOING TO TELL YOU ABOUT SOME OTHER TYPES -- ADDITIONAL TYPES OF STRESS RESPONSES THAT WE THINK ARE IMPORTANT IN THE CONTEXT OF NEURODEGENERATION. AND THOSE ARE AXON INJURY RESPONSES. AND SO IN MY LAB, IN NICHD, WE'RE REALLY INTERESTED IN HOW NEURONS RESPOND TO INJURY BY SPECIFIC STRESS RESPONSES IN PARTICULAR, AXON INJURY RESPONSES. I DO THINK THIS WORK WILL HELP US BETTER UNDERSTAND THE PATHOPHYSIOLOGY OF NEURODEGENERATIVE DISEASE, SO TODAY I'M GOING TO SHOW YOU A BRIEF INTRODUCTION ON ONE OF THE PATHWAYS THAT WE FOCUS ON, AND THEN SHARE WITH YOU THREE VIGNETTES OF WORK THAT WE HAVE DONE IN MY LAB TO KIND OF ILLUSTRATE THIS IDEA. JUST TO SHOW THAT I HAVE NO DISCLOSURES, AND TODAY IF THERE IS A LEARNING OBJECTIVE, I'D LIKE TO CONVINCE YOU THAT AXON DAMAGE SIGNALING IS A TYPE OF NEURONAL STRESS THAT'S RELEVANT TO MANY CONDITIONS THAT INVOLVE NEURODEGENERATION, INCLUDING SEVERAL DISEASES, STROKE, TRAUMATIC INJURIES TO THE BRAIN AS WELL. AND IN MY LAB, WE USE DIFFERENT TYPES OF MODELS, INCLUDING THE TYPES OF PRECISION MOUSE MODELS OF DISEASE THAT YOU HEARD FROM DEREK. I'M NOT GOING TO TALK ABOUT THOSE TODAY. WE ALSO HAVE MOUSE MODELS OF TRAUMATIC NERVE INJURY AND BRAIN INJURY, AND WE TRY TO LEVERAGE SINGLE CELL SEQUENCING TECHNOLOGY TO STUDY DIFFERENTIAL AND CELL TYPE-SPECIFIC RESPONSES TO INJURY AND DISEASE, AS WELL AS WE HAVE STARTED USING HUMAN IPSC-DERIVED NEURONS TO TRANSLATE AND HAVE A TRACTABLE SYSTEM FOR CELL BIOLOGICAL STUDIES. OKAY. SO IN VERY SIMPLE TERMS, NEURODEGENERATION CAN BE CAUSED BY A COMBINATION OF GENETIC PREDISPOSITION AS WELL AS ENVIRONMENTAL FACTORS EXPERIENCED DURING DEVELOPMENT AND IN THE LIFESPAN. IN MY LAB, WE'RE PARTICULARLY INTERESTED IN THE COMMON DOWNSTREAM PATHWAYS THAT LEAD TO NEURODEGENERATION. IN PARTICULAR, ONE OF THE PATHWAYS THAT WE FOCUSED ON QUITE A LOT SINCE I STARTED WORKING ON THIS WHEN I WAS AT GENENTECH IS DLK, AND MAPP. MAP3K12.THE IDEA IS IF WE CAN TT PATHWAYS LIKE THIS THAT ARE RELATIVELY I WOULD SAY A DOWNSTREAM PROCESS IN THE CONTEXT OF, YOU KNOW, THE STEPS LEADING TOWARDS LOSS OF NEURONS, THEN THIS COULD BE A POTENTIALLY VALUABLE WAY TO PREVENT NEURONAL CELL DEATH IN THE CONTEXT OF MANY DIFFERENT DISEASES. SO HOW WE CAME TO STUDY THIS WAS ACTUALLY THANKS TO THE FINDINGS THAT MY COLLEAGUES HAD MADE AT GENENTECH, WHICH IS THAT DLK IS ACTUALLY REALLY IMPORTANT IN DRIVING A PARTICULAR KIND OF NEURONAL CELL DEATH THAT HAPPENS DURING DEVELOPMENT, WHICH IS A NORMAL AND HEALTHY PROCESS. SO HERE I'M SHOW YOU ING YOU AN EXAMPLE. THESE ARE -- THE GREEN CELLS, THE GREEN CELLS ARE MOTOR NEURON NUCLEI FROM THE SPINAL CORD OF A DEVELOPING MOUSE, AND YOU CAN SEE THAT THERE ARE MANY MORE OF THEM, EMBRYONIC DATA -- BASICALLY DLK IS RESPONSIBLE FOR A WAVE OF NEURONAL CELL DEATH THAT OCCURS DURING DEVELOPMENT AS SHOWN HERE IN A KNOCKOUT ANIMAL, THIS FAILS TO OCCUR. AND SO THE VERY SIMPLE QUESTION WE ASKED WAS, WELL, IF THIS HAPPENS DURING NORMAL DEVELOPMENT AND DLK SIGNALS THE CELLS TO DIE, COULD THIS PATHWAY POTENTIALLY BE ACTIVATED AGAIN IN THE CONTEXT OF DISEASE IN A MATURE ORGANISM? SO TO DO THIS, I CROSSED A MOUSE MODEL OF, IN THIS CASE, ALS, SO IT'S A MOUSE THAT OVEREXPRESSES A MUTANT VERSION OF THE GENE THAT CAUSES EARLY ONSET ALS. AND SO YOU CAN SEE THAT IN THIS -- THE MOUSE MODEL FOR THIS DISEASE, THE MOTOR NEURONS THAT ARE LABELS BY THIS TRANS IF HE RACE STAIN ARE LOST. I CROSSED THESE MICE TO INDUCIBLE KNOCKOUTS OF DLK, SO THAT WE COULD BLOCK DLK SIGNALING IN THE MATURE ORGANISM. AND YOU CAN SEE HERE THAT THIS WAS ACTUALLY QUITE POWERFUL IN TERMS OF PREVENTING THESE CELLS FROM DYING. SO A LOT OF BASIC RESEARCH HAS UNCOVERED REALLY INTERESTING ASPECTS OF DLK SIGNALING, AND I'M GOING TO TRY TO SUMMARIZE THOSE IN THE FOLLOWING SLIDES. AND IT TURNS OUT THAT IN THE COURSE EVER THESE STUDIES, ONE OF THE BEST WAYS TO ACTIVATE DLK IS ACTUALLY BY INJURING THE AXON. SO IF YOU CAUSE AXON DAMAGE, YOU CAN TU TURN THE PATHWAY ON. WE LIKE TO USE THIS, THERE'S A L TO STUDY THE MODEL AND TO DRIVE IT. SO HERE'S THE SUMMARY. AXON DAMAGE CAN CAUSE THE ACTIVATION OF DLK, WHICH AS I MENTIONED EARLIER IS A MAP TRIPLE KINASE, RESPONSIBLE FOR PHOSPHORYLATING DOWNSTREAM SUBSTRATES. AND WHEN I SAY AXON INJURY, AND HERE'S KIND OF THE RELEVANCE TO NEURODEGENERATION THAT I HOPE TO GET ACROSS TODAY, IT'S NOT SIMPLY MECHANICAL INJURY OF AN AXON, BUT I MEAN THIS IN A VERY WIDE SENSE, SO WE CAN HAVE MECHANICAL INJURY, YOU COULD HAVE THE TYPES OF STRESSES THAT DEREK WAS TALKING ABOUT THAT MIGHT BE UPSTREAM OF THIS AXON TRANSPORT DISRUPTION AND PROTEOTOXIC STRESS. BECAUSE FOR EXAMPLE, IN A MOUSE MODEL OF ALS THAT I SHOWED YOU, THOSE ANIMALS ARE NOT UNDERGOING MECHANICAL INJURY, BUT SOME OTHER TRIGGER. SO THIS KINASE PHOSPHORYLATION SETS OFF THIS RETROGRADE SIGNAL THAT CAN RESULT IN A TRANSCRIPTIONAL RESPONSE SO WE HAVE AN UPREGULATION OF TRANSCRIPTION FACTORS AND THE ACTIVE FORM OF THIS TRANSCRIPTION FACTOR WHICH IS A PHOSPHORYLATED VERSION DIRECTLY DOWNSTREAM OF THIS CASCADE OF THIS KINASE CASCADE. NOW, THAT'S THE PART OF THE CELL THAT'S CONNECTED TO THE CELL BODY. AT THE OTHER END, WHAT WE CALL THE DISTAL AXON, THIS PART OF THE AXON UNDERGOES AXON DEGENERATION, AND THIS INVOLVES OTHER GENES THAT I'M NOT GOING TO DESCRIBE TODAY FOR THE SAKE OF TIME, BUT ONLY TO SAY THAT DLK ALSO CONTRIBUTES TO THIS TYPE OF WHAT'S KNOWN AT WALLERIAN DEGENERATION. IN ADDITION TO WHAT I'VE JUST DESCRIBED IN TERMS OF AXON DEGENERATION AND ACTIVATION OF A TRANSCRIPTIONAL RESPONSE, THIS RESPONSE CAN ACTUALLY LEAD TO MANY DIFFERENT OUTCOMES, INCLUDING THINGS LIKE THE NEURON CELL DEATH THAT I DESCRIBED EARLIER BUT ALSO IN SOME CASES TO REPAIR AND REGENERATION. AND THIS IS MOST NOTABLY BEEN SHOWN IN WORMS, IN FLIES AND IN MICE FOR PERIPHERAL NEURONS. OKAY. SO ONE THING MY LAB DEMONSTRATED WAS ACTUALLY THAT IN ADDITION TO ALL OF THESE THINGS THAT I DESCRIBED WHICH ARE REALLY INTRINSIC TO THE INJURED NEURON, THIS PATHWAY CAN ALSO SIGNAL TO THE OUTSIDE OF THE INJURED CELL, AND IN THIS CASE, THE VIGNETTE I'M GOING TO SHARE WITH YOU NOW IS THAT ONE OF THE TRANSCRIPTIONAL RESPONSES ACTUALLY CAN DRIVE NEUROINFLAMMATION. SO THIS WORK WAS DONE BY THIS TEAM IN MY LAB, AND WE PUBLISHED THIS JUST AS A KIND OF -- TO SITUATE YOU IN TERMS OF THE ANATOMY I'M GOING TO BE DESCRIBING. SO I'M GOING TO BE TALKING ABOUT THE SENSORY NEURONS THAT RESIDE IN THE DORSAL ROOT GANGLIA THAT LINE THE SPINAL CORD. AND I'M ALSO -- SO THESE ARE THE DRG NEURONS, AND THEIR CELL BODY PROJECTS AN AXON WHICH BIFURCATES AND ACTUALLY PROJECTS TO, ON THE ONE HAND, THE DORSAL HORN OF THE SPINAL CORD. AND AT THE OTHER END, TO THE PERIPHERY. AND THEN WHEN WE DO THE TYPE OF NERVE INJURY THAT I'M ABOUT TO DESCRIBE, WHICH WOULD BE, FOR EXAMPLE, CRUSHING OR CUTTING THE SCIATIC NERVE, WE'RE ALSO INJURING THE SPINAL MOTOR NEURONS AND THESE PROJECT THEIR AXON TO THE PERIPHERAL MUSCLE TO GENERATE MUSCLE CONTRACTION. SO WHAT I'M SHOWING YOU HERE IS ACTUALLY A MOUSE SPINAL CORD. THIS IS FROM AN ANIMAL WHICH HAS RECEIVED THIS TYPE OF SCIATIC NERVE INJURY ON THIS SIDE ONLY, AND WE HAVE STAINED THE WHOLE TISSUE TO REVEAL THE MICROGLIA IN THE SPINAL CORD. AND YOU CAN APPRECIATE THAT WE HAVE TWO CLOUDS OF MICROGLIA. ONE OF THEM IN THE DORSAL HORN WHICH IS WHERE THE SENSORY NEURONS THAT WE INJURED PROJECT TO, AND ANOTHER ONE IN THE VENTRAL HORN, HERE, WHICH IS WHERE -- WHICH IS SURROUNDING THE MOTOR NEURON CELL BODIES OF THE CELLS -- OF THE MOTOR NEURONS THAT WE INJURED. AND SO WE LOOKED TO SEE, AND HERE I'M SHOWING YOU BY THE TOP VIEW WHAT THIS WOULD LOOK LIKE IN AN ANIMAL THAT LACKED DLK. SO TO MODEL BLOCKING DLK. AND WHAT WE OBSERVED IS THAT THIS NEUROINFLAMMATORY RESPONSE WAS COMPLETELY PREVENTED, AND SO JUST QUITE RAPIDLY, I JUST WANT TO SHOW YOU EVIDENCE HERE THAT WHAT WE UNDERSTAND NOW, HOW THIS WORKS, IS THAT DLK DRIVES UPREGULATION OF THIS GENE CALLED CSF 1, WHICH IS ACTUALLY A CYTOKINE THAT'S NOT NORMALLY EXPRESSED IN NEURONS, AND YOU CAN SEE THAT THE UPREGULATION OF THIS GENE FAILS TO OCCUR IN THE DR DP.s, RGs, FOR EXAMPLE, OF K KNOCKOUT, SO WHAT WE THINK HAPPENS IS THAT UPON INJURY, THE DLK PATHWAY IS ACTIVATED, IT UPREGULATES CSF 1 IN THE SENSORY NEURONS AND THE MOTOR NEURONS. BY THIS RETROGRADE SIGNAL THAT I DESCRIBED EARLIER, AND THEN THE UPREGULATION OF CSF 1 AND THOSE NEURONS CAUSES LOCAL PROLIFERATION AND ACTIVATION OF THE MICROGLIA, WHICH EXPRESS THE RECEPTOR FOR THIS CYTOKINE. AND THAT'S WHY WE HAVE TWO CLOUDS OF MICROGLIA, HERE AND HERE. NOW, I'VE SHARED WITH YOU HOW DLK STRESS SIGNALING CAN UPREGULATE A GENE LIKE CSF 1 AND INVOLVE GLIAL CELLS IN A TISSUE-WIDE STRESS RESPONSE. AND WHAT WE REALLY WANTED TO FIGURE OUT WAS A WAY IN WHICH WE COULD KNOW THE ENSEMBLE OF ALL THE GENES THAT GET O UP OR DOWN REGULATED IN INDIVIDUAL CELLS AND WE SET OUT TO DO THIS FOR THE SPINAL MOTOR NEURONS. THE FIRST STEP WAS ACTUALLY JUST TO CHARACTERIZE THE MOTOR NEURON SUBTYPES IN THE HEALTHY ADULT MOUSE. AND SO FOR THIS, WE'VE USED SINGLE CELL SEQUENCING, AND THIS STUDY WAS PUBLISHED LAST YEAR BY THIS TEAM OF GRADUATE STUDENTS IN THE LAB, AND OUR ULTIMATE GOAL WOULD BE TO USE THIS TYPE OF PROFILING THAT I'M GOING TO SHOW YOU TO FOLLOW TRANSCRIPTOMIC ALTERATIONS IN INDIVIDUAL MOTOR NEURONS DURING DISEASE PROGRESSION OR INDEED AFTER INJURY. SO THE STRATEGY THAT WE EMPLOYED WAS TO USE A MOUSE LINE IN WHICH THE SPINAL MOTOR NEURONS ARE LABELED BY A NUCLEAR REPORTER, SO THESE ARE THE GREEN NUCLEI THAT YOU'RE SEEING HERE IN THIS CLEAR SPINAL CORD. THIS ALLOWS US TO ENRICH FOR THIS POPULATION WITHIN THE SPINAL CORD BECAUSE IT'S ONLY ABOUT 1% OF ALL OF THE NEURONS. -- SORRY -- ALL THE CELLS IN THE SPINAL CORD. AND SO IF WE DON'T DO THIS, WE FAIL TO ENRICH THIS POPULATION ENOUGH TO SEE SUFFICIENT NUMBERS OF THEM. WHAT WE DID WAS WE SEPARATELY SEQUENCED THREE REGIONS OF THE SPINAL CORD. SO THIS WAS OUR WORKFLOW. WE HARVESTED THE TISSUE, WE HOMOGENIZED THE TISSUE AND WE ISOLATED NUCLEI THAT WE FACT SORTED IN ORDER TO SELECT FOR THE ONES SELECTING GFP, WHICH AGAIN WAS UNDER THE CONTROL OF -- BASICALLY WHICH IS ONLY EXPRESSED IN THE CHOLINERGIC CELLS. AND JUST TO POINT OUT HERE THAT WHEN WE USE THIS APPROACH, WE'RE NOT JUST ENRICHING FOR THE SKELETAL MOTOR NEURONS WHICH WE WERE MOST INTERESTED IN, BUT WE ALSO WOULD BE PURIFYING INTERNEURONS THAT ARE CHOLINERGIC, AS WELL AS PREGANGLIONIC CELLS AS WELL AS MOTOR NEURONS. SO THE DATA LOOK LIKE THIS. THIS IS A REPRESENTATION OF SINGLE NUCLEUS TRANSCRIPTOMICS, AND EACH DOT IN THIS REPRESENTATION CORRESPONDS TO ONE NUCLEUS OF A CHOLINERGIC CELL THAT WE HAVE SEQUENCED. AND THEN THE PROXIMITY OF TWO DOTS TO ONE ANOTHER ON THIS GRAPH REPRESENTS THEIR TRANSCRIPTIONAL SIMILARITY, SO THIS IS BASICALLY MAPPING A RELATIONSHIP OF EACH CELL TO ANOTHER USING THE FULL REPERTOIRE OF GENES THAT ARE DETECTED IN EACH CELL. SO WHAT YOU CAN SEE HERE ARE THESE CLUSTERS THAT ARE COLOR-CODED WHICH WOULD CORRESPOND TO THE DIFFERENT SUBTYPES OF SPINAL CHOLINERGIC NEURONS THAT WE DISCOVERED. USING A COMBINATION OF A FEW EXISTING KNOWN MARKERS AND THEN SOME ITERATIVE LOOKING AT GENES THEY EXPRESSED AND GOING TO SEE IN THE TISSUE WHERE THESE WERE LOCATED, WE DETERMINED THAT THESE WERE THE POPULATIONS OF SKELETAL MOTOR NEURONS AND THAT THERE WAS QUITE A DIVERSE POPULATION OF PREGANGLIONIC CELLS, AND THEN THE REMAINING ONES WE HAVE HERE ALL CORRESPOND TO CHOLINERGIC INTERNEURONS WHICH WAS REALLY SURPRISING BECAUSE ONLY ONE OR TWO TYPES HAD EVER BEEN DESCRIBED BEFORE. SO WHAT WE CAN USE THIS KIND OF DATA FOR IS TO QUERY IT FOR WHICH THE TOP MARKERS OF EACH TYPE. SO ONE EXAMPLE I'M GOING TO SHOW YOU HERE, ON THIS GRAPH YOU HAVE A LIST OF GENES AND THEN THE SIZE OF THE CIRCLE SHOWS YOU WHAT PERCENTAGE OF THAT CELL TYPE EXPRESSES A GIVEN GENE, SO IN THIS CASE TNS1 WAS VERY WIDELY EXPRESSED IN SKELETAL MOTOR NEURONS BUT NOT IN THE OTHER TWO TYPES, SO TNS1 WOULD BE PREDICTED TO BE A REALLY GOOD MARKER. SO WHAT YOU CAN SEE HERE IS A DIFFERENT REPRESENTATION OF TNSL MOTOR NEURONS AND IF WE DO HYBRIDIZATION FOR THIS GENE, YOU CAN SEE THAT IT'S REVEALING MOTOR NEURONS IN THE VENTRAL HORN OF THE SPINAL CORD. NOW WE WANTED TO FOCUS IN ON THE ALPHA MOTOR NEURONS, WHICH ARE THE TYPE OF MOTOR NEURON THAT CONTROL MUSCLE CONTRACTION. AND IF WE JUST TAKE THAT SUBSET AND SUBCLUSTER THEM TO LOOK AT WHAT DIFFERENT TYPES OF THEM EXIST, WE COULD DETECT MULTIPLE TYPES. AND IN ORDER TO KNOW WHICH ONE CORRESPONDED TO WHICH, WE EMPLOYED A BACK LABELING METHOD IN WHICH WE WOULD INJECT A PARTICULAR MUSCLE WITH A TRASER. THE TRASER IS TAKEN UP AND WILL LABEL THE CELL BODY THAT WE COULD DETECT IN THE SPINAL CORD IN SECTIONS. THEN WE COULD OVERLAY ON TOP OF THIS IN SITU HYBRIDIZATION FOR ALPHA MOTOR NEURON MARKERS AND DIFFERENT MARKERS FOR THE DIFFERENT SUBTYPES OF ALPHA MOTOR NEURONS THAT WE DETECTED. SO ONE REALLY INTERESTING THING WE DISCOVERED WAS THAT THERE WAS A PARTICULAR SUBTYPE THAT WAS PRESENT ONLY IN THE CERVICAL REGION, BUT NOT REALLY IN THORACIC OR IN THE LUMBOSACRAL AREA, AND WE THOUGHT THIS WOULD BE A REALLY GOOD CANDIDATE FOR PHRENIC MOTOR NEURONS WHICH CONTROL THE DIE T TH THE DIAPHR, THEREFORE, CONTROL BREATHING. THE BEST MARKER FOR THIS CLUSTER THAT WAS NOT EXPRESSED IN OTHER CLUSTERS WAS THE GENE ERB4. WE HAVE CIRCLED TWO MOTOR NEURONS AS REVEALED BY THIS MARKER FOR ALPHA MOTOR NEURONS THAT WE HAD DISCOVERED AND WE DETECTED IN THESE CELLS THAT ERBB4, THE BEST MARKER FOR THIS CLUSTER NUMBER 7 WAS INDEED EXPRESSED WHEN WE HAD LABELED PHRENIC MOTOR NEURONS USING A TRANSTHORACIC INJECTION OF OUR BLUE MARKER HERE. SO IN ADDITIONAL WORK, WE KIND OF IDENTIFIED THIS OTHER CLUSTER AS INNOVATING DIGITS. AND SO WHAT WE THINK HERE IS THAT THE SUBTYPES OF ALPHA MOTOR NEURONS WE SEE CORRESPOND TO SPECIFIC MOTOR POOLS THAT INNERVATE SPECIFIC TYPES OF MUSCLES. AND THIS IS REALLY INTERESTING AND RELEVANT TO DISEASES SUCH AS ALS BECAUSE THIS NEURONAL POPULATION IS RELATIVELY RESILIENT IN DISEASE BUT THEN ULTIMATELY THE LOSS OF FRENK MOTOR NEURON FUNCTION LEADS TO THE DEATH OF THE PATIENTS. AN INTERESTING THING TO NOTE IS THAT RECENT STUDIES HAVE ACTUALLY DETECTED MUTATIONS IN THIS GENE ERBB4 AND SUGGESTED THAT THEY COULD CAUSE ALS, AND INTERESTINGLY, THE TYPE OF ALS THAT THEY CAUSE IS RESPIRATORY ONSET ALS, MEANING A TYPE OF ALS IN WHICH THE ABILITY TO BREATHE IS AFFECTED EARLIER AS OPPOSED TO LIMBS BEING AFFECTED FIRST. SO WE DEPOSITED THESE DATA HERE, SO JUST TO SAY WE HAVE A LOT OF ONGOING WORK IN THIS AREA IN TERMS OF NOW APPLYING THIS TECHNIQUE BECAUSE THIS WHOLE STUDY WAS JUST CREATING AN ATLAS OF HEALTHY NEURONS, AND NOW WE WANT TO APPLY THIS IN THE CONTEXT OF DISEASE AND AXON DAMAGE. SO I JUST WANT TO END ON A THIRD LITTLE VIGNETTE OF SOME WORK THAT WE HAVE ONGOING RIGHT NOW IN WHICH WE ARE STUDYING THE DLK PATHWAY BUT FOR THE FIRST TIME IN HUMAN NEURONS, SO RELATIVELY LITTLE IS ACTUALLY KNOWN ABOUT HOW CONSERVED ALL OF THIS IS IN HUMAN NEURONS. SO THE PLATFORM THAT WE'RE USING IS IPSC-DERIVED NEURONS, THANKS TO OUR COLLABORATOR MICHAEL WARD IN NINDS WHO'S PROVIDED US WITH THIS KIND OF IPSC NEURON THAT'S KIND OF PROGRAMMED TO EXPRESS NEUROGENIN2, AND THIS YIELDS A GLUTAMATERGIC TYPE OF NEURON THAT RESEMBLES A CORTICAL NEURON. AND THIS WORK IS BEING LED BY A POSTDOC IN THE LAB, JORGE GOMEZ GOMEZ-D EEEZA. I'M JUST GOING TO SHOW YOU AN EXAMPLE OF A VERY EARLY TRIAL WHERE JORGE KIND OF WAS REALLY EXCITED TO FIND A MICROSCOPE THAT WE COULD USE, AND TO DO THESE EXPERIMENTS, AND HE GOT REALLY A LITTLE OVERZEALOUS HERE, BUT YOU CAN SEE THIS IS HOW HE SHOOTS THE AXONS. NOWF WE HAVE THIS KIND OF MORE CAREFULLY CALIBRATED, BUT WE CAN DO THIS IN A REALLY RELIABLE MANNER, AND HE DOES THIS IN A DISH OF VERY SPARSELY LABELED NEURONS SO HE CAN SEE THE ENTIRETY, THE NEURONS ARE VERY DENSE IN THE DISH BUT HE CAN SEE THE ENTIRETY OF ONE WHOLE NEURON IF MAYBE ONLY A SMALL FRACTION OF THE NEURONS IN A DISH ARE EXPRESSING A FLUORESCENT PROTEIN. SO THAT'S WHAT I'M SHOWING YOU HERE IN GRAPHICAL FORMAT. YOU CAN ALSO IDENTIFY WHICH AXON BELONGS TO WHICH CELL BODY IN THIS SYSTEM, SO WE'RE VERY INTERESTED IN NOW OBSERVING WHAT HAPPENS IN THE AXON THAT'S CONNECTED TO THE CELL BODY, AS I REFERRED TO IN MY INTRODUCTION. AND WHAT HAPPENS IS SHOWN HERE, SO THE NEURONS ARE EXPRESSING A CYTOPLASMIC RED FLUORESCENT PROTEIN, AND YOU CAN SEE THAT THE ASTERISK MARKS THE SITE OF AXOTOMY, AND YOU CAN SEE THAT OVER TIME, THE RED FLUORESCENCE IS BEING LOST IN THE DIRECTION FROM THE AXOTOMY SITE TO THE CELL BODY. THESE CELLS ARE ACTUALLY UNDERGOING AXON DEGENERATION. THIS IS INTERESTING BECAUSE IN MOUSE NEURONS, IF YOU DOO THIS TO DO THIS TOMOUSE NEURONS IN CE PROXIMAL AXON DOESN'T UNDERGO DEGENERATION. HERE THE ARROWHEAD IS SHOWING YOU THE CELL BODY OF A NEURON WHOSE AXON WAS CUT, AND THIS CELL BODY HERE HAS AN AXON THAT WAS UNCUT. SO THIS IS WHAT HAPPENS. NOW I'M GOING TO PLAY THE VIDEO AND THIS IS REALLY SPED UP OVER A LONG PERIOD OF TIME, 15 HOURS, IN A FEW SECONDS, BUT YOU CAN SEE THAT THAT CELL ENDS UP DYING. WE'RE VERY EXCITED ABOUT THIS BECAUSE WE NOW HAVE A HUMAN NEURON MODEL THAT UNDERGOES AXON DEGENERATION AND CELL DEATH, AND I DON'T HAVE TIME TO SHOW YOU THIS WORK WHICH IS STILL UNPUBLISHED, BUT JORGE HAS DISCOVERED A NOVEL SIGNALING CASCADE DOWNSTREAM OF DLK THAT MEDIATES THIS. AND SO I JUST WANT TO FINISH WITH A BRIEF SUMMARY THAT I'VE SHOWN YOU THAT THERE IS A LINK BETWEEN THE AXON DAMAGE RESPONSE AND NEUROINFLAMMATION. WE THINK THIS COULD BE PARTICULARLY RELEVANT IN THE CONTEXT OF NEURODEGENERATIVE DISEASE IN WHICH WE KNOW THAT NEUROINFLAMMATION CAN PLAY IMPORTANT ROLES, AND SO AXON INJURY SIGNALING NOT ONLY IMPACTS THE NEURON ITSELF, BUT ALSO THE SURROUNDING TISSUE. I'VE ALSO SHOWN YOU HOW WE CAN USE SINGLE CELL TRANSCRIPTOMICS TO DEFINE CELL TYPES SUCH AS SPINAL MOTOR NEURONS, AND HOW WE PLAN TO USE THIS TECHNOLOGY TO INVESTIGATE HOW NEURON TYPES MIGHT DIFFERENTIALLY RESPOND TO INJURY. AND WE HAVE ALSO COMPLEMENTED WORK IN THE MOUSE MODELS WITH STUDIES IN HUMAN NEURONS WHICH CAN HELP ACCELERATE TRANSLATION OF BASIC RESEARCH TO CLINIC-USEFUL KNOWLEDGE. AND I REALLY THINK THAT FUNDAMENTAL KNOWLEDGE OF THE KIND OF BASIC RESEARCH THAT WE DO IS ESSENTIAL TO UNDERSTAND NEURODEVELOPMENTAL AND NEURODEGENERATIVE DISORDERS AND ALSO TO DEVELOP CLINICAL THERAPIES IN NEUROPROTECTION AND NERVE REPAIR. SO WITH THAT, I WOULD LIKE TO JUST ACKNOWLEDGE THE MEMBERS OF MY LAB WHO CONTRIBUTED TO THE WORK THAT I SHARED TODAY. I'VE UNDERLINED THEIR NAMES, AND ALSO THANK ME MIE COLLABORATO ME AT THE NIH INTRAMURAL PROGRAM. THANK YOU VERY MUCH FOR YOUR ATTENTION. >>THANK YOU, DR. LE PICHON AND DR. NARENDRA FOR SHARING YOUR FASCINATING BODY OF WORK IN OFFERING DEEPER INSIGHTS IN THE UNDERSTANDING OF CELLULAR MECHANISMS AND MODELS FOR NEURAL DEGENERATION. WE HAVE TIME FOR A FEW QUESTIONS. THE FIRST QUESTION IS ACTUALLY FOR DR. NARENDRA. SO, FANTASTIC LECTURE. DO WE HAVE A MECHANISM FOR CHRIS CRISTAE BECOMING -- OUTSIDE OF MITOCHONDRIAL MEMBRANE? IS THERE A MEMBRANE DISORDER OR MEMBRANE MITOCHONDRIAL DISRUPTION IN ALS OR PARKINSON'S-TYPE DISEASE? >>SO SORRY, I MISSED THE FIRST PART OF THAT QUESTION. >>DO WE HAVE A MECHANISM FOR THE MITOCHONDRIAL CRISTAE BECOMING FUSEOGENIC FROM THE MITOCHONDRIAL MEMBRANE, AND THE SECRETARY PART IS THERE SOME SORT SORT OF MEMBRANE MITOCHONDRIAL DISRUPTION IN ALS. >>SO I THINK TO TAKE THE SECOND PART OF THE QUESTION FIRST IN PARKINSON'S DISEASE AND ALS, I THINK THERE ARE OFTEN STRUCTURAL CHANGES THAT ARE SEEN TO THE MY TOE DONEMITOCHONDRIA THAT I SHOU BEFORE. I'M NOT SURE I ENTIRELY UNDERSTAND THE FIRST QUESTION. THERE'S AN OUTER MEMBRANE OF THE MITOCHONDRIA AND INNER MEMBRANE AND FUSION OF THE OUTER MEMBRANE IS MEDIATED BY THESE -- IT'S PROBABLY THE INNER MEMBRANE HAS CRISTAE COMPONENT AND ALSO THIS BOUNDARY COMPONENT, AND I THINK OPA1 IS KIND OF ON THAT BOUNDARY CLOSE TO THE CRISTAE JUNCTION, SO PROBABLY THE FUSION DOESN'T HAPPEN WHERE THE CRISTAE IS, PER SE, BUT PROBABLY HAPPENS SORT OF ADJACENT TO THE CRISTAE. >>OKAY, THANK YOU. FOR DR. LE PICHON, WHEN THE DLK STRESS PATHWAY IS TURNED ON IN NEURONS OF THE BRAIN, DOES IT MEAN THAT THOSE CELLS ARE COMMIT TODAY DIE? >>SO WE THINK BASED ON OUR -- WELL, SO I THINK SOME MORE RESEARCH IS NEEDED, BUT BASED ON WHAT WE HAVE SEEN SO FAR WITH THE PERIPHERAL -- THE PERIPHERY PROJECTING NEURONS AND THEN THIS -- SO THE DRG NEWER YONS AND MOTOR NEURONS, WHEN WE DO THOSE KINDS OF INJURIES IN THE MOUSE, THOSE CELLS DO NOT DIE AFTER AXON DAMAGE. HOWEVER, WE'RE REALLY CURIOUS ABOUT WHAT HAPPENS IN BRAIN CELLS BASED ON OUR EXPERIMENTS OF THE HUMAN NEURONS IN VITRO. SO I WOULD PREDICT YES, ONLY BECAUSE OF THE REDUCED CAPACITY OF CENTRAL NERVOUS SYSTEM NEURONS TO REGENERATE, BUT I DON'T REALLY KNOW. AND I THINK LOOKING AT HOW -- AT THE IPT PLAY BETWEEN ABILITY TO REPAIR AND A SURVIVAL IS ALSO AN INTERESTING QUESTION. >>THANK YOU. FOR DR. NARENDRA, ANOTHER QUESTION. YOU'D MENTIONED ABOUT THE DIVERSE PHENOTYPES IN INDIVIDUALS WITH CHCHD10 MUTATIONS. WHAT ACCOUNTS FOR THE DIVERSITY OF THOSE PHENOTYPES? >>WE DON'T FULLY UNDERSTAND IT, BUT MY SUSPICION IS THAT THESE MUTATIONS ARE CAUSING THE PROTEINS TO MISFOLD IN DIFFERENT WAYS, AND JUST LIKE IN DIFFERENT FORMS OF TAUOPATHY, YOU CAN GET ONE NEUROLOGICAL DISEASE VERSUS ANOTHER DEPENDING ON WE NOW KNOW DIFFERENT STRAINS -- DIFFERENT WAYS IN WICHITA IS MISFORMED. SAME THING SEEMS TO BE TRUE ABOUT ALPHA SYNUCLEIN THAT MISFOLDS IN PARKINSON DISEASE AND ANOTHER DISEASE CALLED MULTIPLE SYSTEM ATROPHY. I THINK PERHAPS IN THE SAME WAY THESE ARE CAUSING C10 TO MISFOLD IN DIFFERENT WAYS, AND IF IT MISFOLDS ONE WAY, IT'S MORE TOXIC TO NEURONS AND IF IT MISFOLDS ANOTHER WAY, IT'S MORE TOXIC TO MUSCLE CELLS. >>OKAY. THANK YOU. FOR -- >>SO FOR DR. LE PICHON, A QUESTION. HOW DOES KNOWING THE TRANSCRIPTIONAL ABILITY OF PARTICULAR NEURONS HELP ADVANCE UNDERSTANDING OF NEURODEGENERATIVE DISEASES? >>SO ONE THING THAT'S REALLY INTERESTING ABOUT DIFFERENT NEURODEGENERATIVE DISEASES IS, YOU KNOW, EACH ONE WILL HAVE A PARTICULAR CONSTELLATION OF SYMPTOMS, AND THAT'S PROBABLY BECAUSE DIFFERENT NEURON TYPES TEND TO BE MORE VULNERABLE THAN OTHERS TO THE DIFFERENT -- IN THE DIFFERENT DISEASE CONTEXTS. SO I THINK THAT UNDERSTANDING THE IDENTITY OF THE DIFFERENT NEURON TYPES AT THE GET-GO CAN KIND OF LAY THE GROUNDWORK FOR THEN UNDERSTANDING HOW ONE TYPE MIGHT BE MORE VULNERABLE TO A CERTAIN TYPE OF STRESS THAN ANOTHER, FOR EXAMPLE. I THINK THOSE ARE REALLY CLEAR EXAMPLE OF THAT IN PARKINSON'S E SUBSTANTIA NIGRA NEURONS, FOR EXAMPLE. AND I THINK, YOU KNOW, THE FIELD IS KIND OF MOVING IN THIS DIRECTION TO START EXAMINING THAT TYPE OF QUESTION. >>THANK YOU. SO FOR DR. NARENDRA, HOW -- IS THE OME1/DELE -- FOR MITOCHONDRIAL DYSFUNCTION? >>I THINK IT'S STILL NOT ENTIRELY CLEAR BUT IT SEEM LIKE IT IS PROBABLY GOING TO BE PRETTY BROAD. SO AT THE END THERE, I MENTIONED SORT OF THREE PAPERS THAT HAVE BEEN PUBLISHED THIS YEAR, INCLUDING OUR PAPER, SO WE HAVE KIND OF AN EXAMPLE OF A MISFOLDED PROTEIN STRESS THAT SEEMS TO ACTIVATE THIS PATHWAY. USING THE -- MODEL, WE SHOWED THAT IF YOU JUST CAUSE AN -- DEFECT, YOU ACTIVATE THE SAME PATHWAY. ANOTHER TWO MODELLS SHOWING IF YOU DISRUPT LIPID METABOLISM IN THE MITOCHONDRIA TO INTERFERE WITH KIND OF THE MEMBRANE, YOU GET THE SAME PATHWAY ACTIVATED. SO I THINK IT MAY TURN OUT THAT -- PREDOMINANT WAY IN WITS MITOCHONDRIAL STRESS SIGNALS TO -- RESPONSE IN VIVO. >>WELL, THAT ACTUALLY BRINGS US UP TO THE HOUR. AGAIN, I WANT TO THANK BOTH OF YOU FOR YOUR EXCELLENT TALKS OF YOUR INVESTIGATIONS TO OUR NIH COMMUNITY. I WISH ALL OF YOU A WONDERFUL AFTERNOON. THANK YOU.