Articles, Blog

Mitochondria: dynamic organelles critical for human health

November 10, 2019


IT’S A REAL PLEASURE TO HAVE PROFESSOR DAVID CHAN HERE. HE IS PROFESSOR OF THE DIVISION OF BIOLOGY AND ALSO AN INVESTIGATOR AT THE HOWARD HUGHES MEDICAL INSTITUTE AT CALIFORNIA INSTITUTE OF TECHNOLOGY. HE GOT HIS MD AND Ph.D. IN HST PROGRAM AT HARVARD AND MIT AND AFTER THAT, HIS POSTDOC RECALL WORK AT THE WHITEHEAD INSTITUTE. AND THEN JOINED THE CALIFORNIA INSTITUTE OF TECHNOLOGY BACK IN 2000 WHERE HE IS ASSEMBLED — HE IS WORLD-KNOWN AS AN EXPERT IN BOTH MITOCHONDRIAL DYNAMICS AS WELL AS MITOCHONDRIAL DNA PROTECTION AND PROCESSING. IT’S A FIELD THAT IS EXPANDING. WE WERE JUST CHATTING BECAUSE OF THE IMPACT IN CELL BIOLOGY AND POINT OF FACT, MUCH OF THE WORK THAT PROFESSOR CHAN HAS LED TO THE IMPORTANCE OF THIS ORGANELLE IN THE FUNCTIONING OF THE SELL. WE LOOK FORWARD TO YOUR PRESENTATION.>>SO I’M REALLY GRATEFUL FOR THE INVITATION TO COME AND SPEAK HERE. I HAD A GREAT TIME MEETING WITH INVESTIGATORS HERE AT NIH AND HEARING ABOUT THEIR VERY INTERESTING WORK. SO MY LAB IS INTERESTED IN THE DYNAMIC PROPERTIES OF MITOCHONDRIA. SO FIRST I WANT TO GO THROUGH SOME OF THE WAYS IN WHICH MIGHT QUANDARY ARE DYNAMIC — MITOCHONDRIA ARE DYNAMIC. MITOCHONDRIA UNDERGO CYCLES OF FUSION AND FISSION. YOU CAN HAVE TWO MITOCHONDRIA THAT COME ITSELF AND FUSE SO THAT THERE IS ONLY ONE MITOCHONDRIA AND AT THE SAME TIME, YOU VEHICLE THE OPPOSITE PROCESS WHERE A HEIGHT QUANDARYIA DIVIDES BY FISSION INTO TWO SMALLER ORGANELLES. ANOTHER WAY IN WHICH MITOCHONDRIA ARE DYNAMIC, IS THEY ARE TRANSPORTED TO SPECIFIC PARTS OF THE CELL AND THIS OCCURS ALONG THE CYTOSKELETON. SO, FOR EXAMPLE, YOU CAN HAVE MOVEMENT AWAY FROM THE CELL NUCLEUS AND RETRO GRADE MOVEMENT TOWARDS THE CELL NUCLEUS. AND IN CERTAIN CELL TYPES, THIS HELPS TO DISTRIBUTE THE MITOCHONDRIA IN THE CELL. BUT IN CERTAIN SPECIALIZED CELL TYPES LIKE NEURONS, THIS CAN REALLY LOCALIZE MIGHT MITOCHONDRIA TO SPECIFIC SUB CELLULAR SITES. SO FOR EXAMPLE N-NEURONS THIS TYPE OF ACTIVE TRANSPORT HELPS TO ENSURE THAT MITOCHONDRIA ARE WELL RESPECTED AT THE TERM IN THIS WHERE THEY PLAY IMPORTANT ROLLS IN ATP PRODUCTION AND CALCIUM. AND PARTICULARLY I THINK A DRAMATIC EXAMPLE IS SHOWN IN JEFF’S WORK AT HARVARD WHERE THIS IS A TRANSGENIC MOUSE THAT EXPRESSES A MIGHT MITOCHONDRIALY TARGETED MOLECULE AND YOU CAN SEE INDIVIDUAL MITOCHONDRIA IN THIS PART OF THE NERVE. WHEN THE MOTOR NEURONS TERMINATE AT THE MOTOR END PLATE, WHICH IS HIGHLIGHTED IN RED, YOU CAN SEE THAT THE MITOCHONDRIA ARE SO DENSE THAT YOU CAN’T MAKE OUT INDIVIDUAL ORGANELLES. SO THIS IS AN EXAMPLE OF HOW ACTIVE TRANSPORT CAN SERVE TO LOCALIZE MITOCHONDRIA TO SPECIFIC PARTS OF THE CELL. ANOTHER WAY IN WHICH MITOCHONDRIA ARE DYNAMIC, THEY UNDERGO CONDUCT RECALL CHANGES IN APOPTOSIS. SO THEY 81 TAIN TWO MEMBRANES. AN OUTER MEMBRANE AND A INNER MEMBRANE AND THE INNER MEMBRANE IS CONVOLUTED INTO THESE COMPARTMENTS. SO IT’S ARGUED THAT CONTENTS OF THE MEMBRANE ARE NOT DIFFUSIBLE AND YOU HAVE TO OPEN UP THESE CRICETID JUNCTIONS IN ORDER FOR THESE CONTENTS TO BE INTERACTING WITH EACH OTHER. AND DURING THE PROCESS OF APOPTOSIS, IN MANY CASES, THERE IS FRAGMENTATION OF MITOCHONDRIA SO THERE IS INDUCED MITOCHONDRIAL VISION, WHICH LEADS TO MITOCHONDRIAL FRAGMENTATION. THERE IS OPENING OF THE MITOCHONDRIAL OUTER MEMBRANES AND THERE IS ALSO CHANGES IN THE CRICETID JUNCTIONS SO THAT INTERMEMBRANE COMPONENTS CAN COME OUT AND THEY PLAY PRO-APOPTOTIC ROLLS. FINALLY, THE LAST DYNAMIC FEATURE OF MITOCHONDRIA I WANTED TO POINT OUT IS THAT THEY UNDERGO SELECTIVE DEGRADATION BY AWE TO HAVE GHEE. THAT IS A TERM — AWE TO HAVE GEE. THIS IS WORK FROM JOHN’S GROUP WHERE HE SHOWS USING MOUSE HEPATOCYTES THAT YOU CAN HAVE DAMAGE MITOCHONDRIAL UNDERGOING DEGRADATION. SO THESE RED SPOTS HERE ARE THE MITOCHONDRIA IN THESE HEPATOCYTES AND A LASER IS USED TO INACTIVATE THE MEMBRANE POTENTIAL IN SOME OF THESE MITOCHONDRIA AND THOSE MITOCHONDRIA ARE THE ONES THAT THEN ASSOCIATE WITH AN AWE TO HAVE GEE MARKER. LET ME GO BACK AND TALK ABOUT FUSION AND DIVISION. SO, INHERENTLY THIS IS A SOMEWHAT COMPLICATED PROCESS BECAUSE MITOCHONDRIA HAVE DOUBLE MEMBRANES. AND SO, DURING THE PROCESS OF MITOCHONDRIAL FUSION, THERE HAS TO BE COORDINATED FUSION OF FOUR LIPID BILAYERS, SO TWO OUTER MEMBRANES AND TWO INNER MEMBRANES AND THE NET RESULT OF THIS FUSION EVENT IS THAT THERE IS LIPID MIXING BETWEEN THESE TWO MEMBRANES AND CONTENT MIXING SO THAT THE CONTENT OF THE INTERIM MEMBRANE SPACE ARE MIXED AND THE CONTENTS OF THE MATRIX, THE INTERNAL CONTENT OF THE MITOCHONDRIA ARE MIXED. AND THIS PROCESS TURNS OUT TO BE DEPENDENT ON MEMBRANE POTENTIAL ACROSS THE INNER MEMBRANE. AND THERE ARE MANY FUNCTIONS OF MITOCHONDRIAL FUSION AND FISSION, WHICH I’LL TALK MORE ABOUT. BUT ONE OF THE FUNCTIONS IS THAT IT CONTROLS THE MORPHOLOGY OF MITOCHONDRIA. SO THE BALANCE BETWEEN FUSION AND FISSION CONTROLS MITOCHONDRIAL SHAPE, SIZE AND NUMBER. SO, THE MOLECULES THAT ARE INVOLVED IN MITOCHONDRIAL FUSION TURN OUT TO BE LARGE GTPPASES AND THERE ARE THREE IN MAMMALS. SO OUR WORK HAS FOCUSED ON MAMMALIAN CELLS AND THESE ARE SOME GROUPS THAT WORK ON SIMILAR PROBLEMS IN YEAST CELLS. SO IN MAMMALS, THERE ARE TWO SETS OF LARGE GTPPASES. THE FIRST TWO ARE MFM1 AND 2. THESE ARE OUTER MEMBRANE GTPPASES THAT HAVE A U-SHAPED TRANSMEMBRANE DOMAIN AND THEY ARE NECESSARY FOR MITOCHONDRIAL FUSION. SO MICE THAT ARE DEFICIENT FOR MFM1 OR 2 HAVE FRAGMENTED MITOCHONDRIA SO THE GREEN SPOTS OVER HERE ARE FRAGMENTED MITOCHONDRIA AND MFM DEFICIENT CELLS IN CONTRAST TO THE TUBULAR PRESENT IN WILDTYPE CELLS. AND AGAIN, THIS REITERATES THE FACT THAT WHEN YOU HAVE REDUCED MITOCHONDRIAL FUSION THERE IS STILL ONGOING MITOCHONDRIAL DIVISION AND THAT LEADS TO MITOCHONDRIAL FRAGMENTATION. SO THE BALANCE BETWEEN FUSION AND FISSION CONTROLS MITOCHONDRIAL SIZE, SHAPE AND NUMBER. IN ADDITION TO THE MIGHT FUSIONS LOCATED ON THE MITOCHONDRIAL OUTER MEMBRANE, THERE IS ALSO OPAL 1, A PROTEIN LOCALIZED TO THE MITOCHONDRIAL INNER MEMBRANE. THIS PROTEIN IS ALSO ESSENTIAL FOR MITOCHONDRIAL FUSION. SO CELLS THAT LACK OPAL 1 HAVE NO DETECTABLE MITOCHONDRIAL FUSION. AND BY USING MOUSE KNOCKOUS AND GENERATING CELL LINES FROM THOSE MOUSE KNOCKOUTS, WE CAN LOOK AT THE SELECTIVE ROLES OF MITOFUSEINS AND OPA1. SO BOTH OF THESE CELLS ARE DEFICIENT FOR FULL MITOCHONDRIAL FUSION BUT IF YOU LOOK MORE SPECIFICALLY AT OUTER MEMBRANE FUSION VERSUS INNER MEMBRANE FUSION, THEY HAVE A DIFFERENCE. SO MIGHT ON FUSEINS LOCATED ON THE OUTER MEMBRANE ARE DEFECTIVE FOR OUTER MEMBRANE FUSIONS. THEY DON’T UNDERGO THE FIRST STEPS. WHEREAS IN OPA1 DEFICIENT CELLS THOSE CELLS WILL UNDERGO OUTER MEMBRANE FUSION BUT THEY GET TRAPPED AT THIS INTERMEDIATE AGE AND SO THEY ARE UNABLE TO UNDERGO INNER MEMBRANE FUSION. SO WE CURRENTLY VIEW MITOCHONDRIAL FUSION AS A MULTI-STEP PROCESS WHERE OUTER MEMBRANE FUSION OCCURS FIRST. THIS DEPENDS ON MITOFUSE INS LOCATED ON THE OUTER MEMBRANE FOLLOWED BY INNER MEMBRANE FUSION WHICH IS DEPENDENT ON OPA1, WHICH IS EITHER — THERE IS AN ISOFORM ASSOCIATED WITH THE INNER MEMBRANE AND OTHERS THAT ARE IN THE INTERMEMBRANE SPACE. SO I MENTIONED THAT MITOCHONDRIAL FUSION CONTROL MITOCHONDRIAL MORPHOLOGY. AN EXAMPLE IS SHOWN HERE. SO IN THE NORMAL FIBROBLASTS, YOU HAVE NORMAL RATES OF MITOCHONDRIAL FUSION AND FISSION. HERE IS A FIBROBLAST. THE BLANK AREA HERE IS THE NUCLEUS AND YOU CAN SEE IT HAS TUBULAR MITOCHONDRIA. IF YOU KNOCKOUT PROTEINS INVOLVED IN MITOCHONDRIAL FUSION LIKE MFNs THERE IS LOWER LEVELS OF MITOCHONDRIAL FUSION WHICH NEEDS TOMMIED MITOCHONDRIAL FRAGMENTATION. YOU CAN DO THE OPPOSITE EXPERIMENT AND BLOCK FISSION IN THESE CELLS AND IN THAT CASE, YOU GET CELLS THAT HAVE OVERLY LONG AND INTERCONNECTED MITOCHONDRIA. YOU CAN ALSO SIMULTANEOUSLY BLOCK MITOCHONDRIAL FUSION AND FISSION AND WHEN YOU DO THAT, YOU CAN RESTORE MITOCHONDRIAL TUBUALS TO THESE CELLS THAT HAVE FRAGMENTED MITOCHONDRIA. THIS IS AN EXAMPLE OF HOW TO MANIPULATE THE MORPHOLOGY OF MITOCHONDRIA BY MANIPULATING THE BALANCE BETWEEN FUSION AND FISSION. NOW, ONE OF THE REASONS THAT WE ARE INTERESTED IN THESE PROCESSES IS THAT HUMAN GENETIC STUDIES INDICATE THAT THESE PROCESSES ARE CLEARLY IMPORTANT FOR HUMAN HEALTH. SO THE FIRST DISEASE THAT ILLUSTRATES THIS IS DOMINANT OPTIC ATROPHY. WHICH IS THE MOST COMMONLY INHERITED OPTIC NEUROPATHY CAUSED BY HETEROZYGOUS MUTATIONS IN OPA1. SO IN THIS DISEASE, THERE IS DEGENERATION OF THE RETINAL GANG RIA CELLS, WHICH HAVE CELL BODIES THAT ARE LOCATED IN THE RETINA AND THEIR PROCESSES ARE BUNDLED INTO THE OPTIC NERVE. AND SO THIS IS A BLINDNESS CAUSED BY DEFECT IN THE MITOCHONDRIAL FUSION GENE. THERE IS A SECOND DISEASE CALLED SHARKA TYPE II A CAUSED BY AGAIN HETEROZYGOUS MUTATIONS IN MFM2. SO MOST OR THERE ARE MANY FORMS OF CMT. THE MAJOR FORMS ARE DEMILEATING DISEASES. THEY ARE A DEFECT IN THE SWAN CELL. BUT IN TYPE II A, THIS IS AN EXKNOP THEE WHERE THE NEURON ITSELF IS DEFECTIVE. AND THING IS A QUITE INTERESTING DISEASE BECAUSE THIS EFFECTS MOTOR AND SENSORY NEURONSS WHOSE CELL BODIES ARE LOCATED NEAR THE SPINAL CORD BUT THEN INTERVENE THE EXTREMITIES. SO IN THIS DISEASE, PATIENTS HAVE WEAK HANDS AND FEET AND ALSO SENSORY LOSS. AND SO IN THIS DISEASE WHERE THERE IS A DEFECT IN THE MITOCHONDRIAL FUSION GENE, IT’S ONLY THE LONGEST PERIPHERAL NEURONS THAT ARE EFFECTED AND THE MORE PROXIMAL NEURONS ARE SPARED. SO TO LOOK AT SOME OF THESE ISSUES ABOUT WHY MITOCHONDRIAL FUSION SEEMS TO BE PARTICULARLY IMPORTANT FOR NEURONS, WE HAVE BEEN STUDYING MOUSE THAT HAVE KNOCKOUTS IN MFM1 OR 2 AND MUTATIONS IN EITHER ONE OF THOSE GENES WILL LEAD TO EMBYRONIC LETHALLALITY BUT WE MADE CONDITIONAL KNOCKOUTS TO HELP US LOOK AT THE POST EMBYRONIC ROLES OF THESE GENES IN THE CASE OF MFM2 KNOCKOUTS, IF WE BY PASS THE PLACENTAL DEFECT, THOSE SPLICE A VERY SEVERE ATAXIA AND IT IS ASSOCIATESSED WITH A SAY BELLA DEFECT. SO THIS IS AN EXAMPLE HERE. SO IN WILDTYPE ANIMALS, THIS IS THE CEREBELLUM SEVEN DAYS AFTER BIRTH. IT LOOKS RELATIVELY NORMAL IN THE MFM2 MUTANT BUT THE CEREBELLUM IS UNDERDEVELOPED. BUT IF YOU LOOK AT ANIMALS THAT ARE JUST ONE WEEK LATER, IN WILDTYPE ANIMALS, THERE IS EXTENSIVE CELL MIGRATION AND DIFFERENTIATION THAT OCCURS IN THE CEREBELLUM AT THIS TIME. BUT IN AN MFM2 KNOCKOUT, THERE IS ATROPHY OF THIS PART OF THE BRAIN. AND IT TURNS OUT THAT THERE IS A SPECIFIC NEURON THAT IS DEFECTIVE. SO, THIS IS JUST A HISTOLOGICAL SLIDE WHERE YOU CAN CONSIDER THIS TO BE THE OUTSIDE OF THE CEREBELLUM AND THIS IS CALLED THE MOLECULAR LAYER AND THIS IS THE INTERNAL GRAN LEGAL LAYER. THE GRANUAL CELLS ARE THE MOST ABUNDANT CELLS IN THE CEREBELLUM. AT THE INNER FACE A CELL TYPE CALLED THE PREKINSY CELL. THAT IS THE NEURON THAT IS DEFECTIVE IN THESE ANIMALS. SO IF WE LOOK USING A MARKER FOR PURCINCHY CELLS, THIS IS HOW THEY DEVELOP IN THE 50 TWO WEEKS OF LIFE. SO SIX DAYS AFTER BIRTH THERE IS A LAYER OF THESE CELLS HERE IN THIS PART OF THE CEREBELLUM. AND OVER TIME, THESE CELLS WILL EXTEND OUT THEIR DENDRITIC PROCESSES INTO THE MOLECULAR LAYER SO THAT BY DAY 15, YOU CAN SEE HOW EXTENSIVE THAT DENDRITIC LAYER IS. BUT IN ANIMALS THAT LACK MFM2, THEY START OFF WITH LAYER OF THESE CELLS BUT BY 10 DAYS AFTER BIRTH, YOU CAN SEE THERE IS A PRETTY SEVERE DEFECT. SO THEY HAVE THESE CELLS BUT THEY ARE DENDRITES THAT ARE MUCH SHORTER AND THEY HAVE THE REDUCED DENDRITIC SPINES. AND THESE CELLS WILL DIE OVER THE NEXT WEEK SO AT TWO WEEKS AFTER BIRTH, THERE IS VERY FEW PURKINJE CELLS IN THE CEREBELLUM AND THIS LEADS TO ATAXIA. SO IN STUDYING WHAT THE DEFECTS ARE IN THESE CELLS THAT LACK MITOCHONDRIAL FUSION, IT TURNS OUT THEY HAVE A VERY SEVERE RESPIRATORY DEFECT. AND THE BASIS FOR THAT IN PART, IS THAT THEY HAVE A DEFECT IN MAINTENANCE OF MITOCHONDRIAL DNA. SO, HERE WE ARE RETURNING BACK AGAIN TO FIBROBLASTS AND IN FIBROBLASTS WILDTYPE CELLS, THE GREEN HERE SHOWS THE MITOCHONDRIA AND THE RED IS A NUCLEAR STAIN. AND SO YOU CAN SEE THESE MITOCHONDRIA CONTAIN THESE COMPACT DNA CONTAINING STRUCTURES CALLED NUCLEASE. SO THESE ARE THE GENOMES OF THE MITOCHONDRIA. AND EVERY MITOCHONDRIAL TUBUAL HAS AT LEAST ONE EMPTY DNA NUKE LLOYD BECAUSE THE MITOCHONDRIAL GENOME ENCODES FOR ESSENTIAL COMPONENTS OF THE RESPIRATORY CHAIN. IN CELLS THAT LACK MITOCHONDRIAL FUSION, YOU CAN SEE THEY ARE FRAGMENTED. THEY DO RETAIN MITOCHONDRIAL DNA BUT YOU CAN SEE THAT A LARGE POPULATION OF THE MITOCHONDRIA LACK NIKE LLOYDS. THESE MITOCHONDRIA ARE RESPIRATORY DEFICIENT. AND SO OUR MODEL FOR WHAT THE FUNCTION OF MITOCHONDRIAL FUSION IS, IS THAT IT ALLOWS CONTENT EXCHANGE BETWEEN MITOCHONDRIA AND THIS CONTENT EXCHANGE IS IMPORTANT TO MAINTAIN THE FUNCTION OF THE MITOCHONDRIAL POPULATION. SO WE THINK THAT IN WILDTYPE CELLS, THERE IS A POPULATION OF MITOCHONDRIA THAT CONTINUOUSLY INTERACTS AND EXCHANGES CONTENT WITH EACH OTHER. AND YOU CAN IMAGINE THAT YOU CAN HAVE INDIVIDUAL MITOCHONDRIA THAT SPORADDICALLY DEVELOP A DEFECT. AND THERE COULD BE MANY REASONS FOR THIS. ONE REASON WOULD BE THAT THERE IS A DIVISION EVENT IN WHICH A MIGHT DONDRIA FAILS TO INHERIT A MITOCHONDRIAL DNA NUKE LLOYD. BUT THOSE DEFECTS CAN BE REPAIRED BECAUSE THAT MITOCHONDRIA CAN BE CONFUSED WITH A NEIGHBORING MITOCHONDRIA AND THEN OF SUBSEQUENT FUSION EVENT LEADS TO COMPUTATION OF THAT DEFECT. AND IN CELLS THAT LACK MITOCHONDRIAL FUSION, ONCE THESE DEFECTS OCCUR, THEY REMAIN OR PROLONGED OR PERMANENT. SO IN OUR SYSTEM WHERE WE LOOK AT THE DEFECTS THAT ARISE DUE TO A DEFECT OF MITOCHONDRIAL FUSION, WE ALSO SEE THE PROTECTIVE EFFECTS OF MITOCHONDRIAL FUSION. THERE IS ALSO A COMPLEMENTARY VIEW THAT WHEN DEFECTS ARE VERY SEVERE, IT MIGHT BE BENEFICIAL TO RESTRICT THE FUSION OF THOSE MITOCHONDRIA SO THEY CAN BE SEGGREATED AND DEGRADED BY AUTOPHAGY. SO FOR EXAMPLE, WE HAVE SHOWN WHEN MITOCHONDRIA LOSE COMPLETELY LOSE MEMBRANE POTENTIAL, THEY NO LONGER CAN FUSE WITH NEIGHBORING MITOCHONDRIA AND THOSE THEN BECOME SEGREGATED AND PERHAPS ARE DEGRADED BY AUTOPHAGY. SO IT CAN BE THAT DEPENDING ON THE SEVERITY OF MITOCHONDRIAL DYSFUNCTION, EITHER THOSE MITOCHONDRIA CAN BE REPAIRED BY CONTENT MIXING OR PERHAPS THEY ARE SEGREGATED WHEN THE DEFECT IS TOO SEVERE AND THEN DEGRADED. SO IN THE NEXT PART OF MY TALK, I WOULD LIKE TO SUMMARIZE SOME OF OUR STUDIES ON THE FUNCTION OF MITOCHONDRIAL FUSION IN SKELETAL MUSCLE. SO WE WERE INTERESTED IN SKELETAL MUSCLE BECAUSE OBVIOUSLY, MITOCHONDRIAL ARE VERY IMPORTANT IN THAT CELL TYPE. AND DEPENDING ON THE TYPE OF SKELETAL MUSCLE, FOR EXAMPLE, WHETHER IT IS OXIDATIVE OR — THE MITOCHONDRIA HAVE DIFFERENT DEGREES OF ABUNDANCE BUT IN GENERAL, THEY ARE HIGHLY — THEY ARE POSITIONED VERY PRECISELY IN THE CELL. SO, HERE YOU CAN SEE FOR EXAMPLE, THAT THERE ARE PAIRS OF MITOCHONDRIA THAT ARE POSITIONED ON THE TWO SIDES OF THE Z DISK IN THIS. SO BECAUSE THE MITOCHONDRIA AND SKELETAL MUSCLE ARE SO PRECISELY POSITIONED IN CONTRAST TO THE CASE OF FIBROBLASTS WHERE YOU CAN SEE THEM MOVE AROUND THE CELL CONTINUOUSLY, WE THOUGHT IT WOULD BE GOOD TO ASK WHETHER MITOCHONDRIAL FUSION IS IMPORTANT IN THIS CELL TYPE. SO WE KNOCKED OUT MITOFUSE INS IN SKELETAL MUSCLE USING THE MLC CRE DRIVER SYSTEM DEVELOPED BY SEVERE BURGEN. WHAT WE FOUND IS THAT THESE MICE HAVE SEVERE DEFECTS. SO THIS IS A MUSCLE SPECIFIC OF BOTH MITOFUSE INS. THEY HAVE LOW BODY WEIGHT, BLOOD TEMP AND GLUCOSE HIGH SERUM LACTATE THAT GETS WORSE WITH EXERCISE. THEY HAVE CHARACTERISTICS THAT ARE SUGGESTIVE OF A DEFECT. WHEN YOU LOOK AT THE MUSCLES FROM THESE ANIMALS, THE MUSCLES ARE DEEPER RED, WHICH MIGHT IS THAT THE THERE IS MORE OR HIGHER LEVEL OF MITOCHONDRIAL BINDING. AND THAT TURNS OUT TO BE THE CASE IN WHICH I WILL SHOW YOU IN A SECOND. SO WE DID SOME HISTOLOGICAL STUDIES OF THE MUSSEL FROM THESE ANIMALS. SO IN WILDTYPE MUSCLES, USING STAINING FOR COMPLEX TWO AND COMPLEX 4, WHERE THE COMPLEX 4 STAINING IS BROWN STAIN, YOU CAN SEE IN THE WILDTYPE ANIMALS, TRANSVERSE SECTIONS OF THE MUSCLE FIRST SHOW A HOMOGENEOUS STAIN MUSCLE SECTION THAT OVER THE FIRST TWO MONTHS OF LIFE DIFFERENTIATES INTO THIS CHECKER BOARD APPEARANCE. SO HERE IS A MUSCLE FIBER THAT HAS HIGH MITOCHONDRIAL FUNCTION AND HERE IS A MUSCLE FIBER THAT HAS LOW MITOCHONDRIAL FUNCTION. BUT WHEN WE LOOK AT ANIMALS THAT LACK THE MITOFUSE INS IN SKELETAL MUSCLE, WE SEE THAT THEY HAVE SMALLER DIAMETERS FOR THEIR MUSCLE FIBERS AND THEY HAVE THIS INTENSE BLUE STAIN, WHICH IS AN INCREASE IN COMPLEX 2. SO IN THIS TYPE OF STAINING, INCREASE IF COMPLEX 2 OFTEN INDICATES A MITOCHONDRIAL DYSFUNCTION BECAUSE OF THE DEFECT IN HEIGHT CONNED REAL DNA. I’LL EXPLAIN THAT IN A SECOND. BUT THESE ANIMALS DEVELOP RESPIRATORY DEFICIENCY IN THE MUSCLE FIBERS. SO WE COLLABORATED WITH MIKE McCAVEAT JOHN’S HOPKINS TO DO ON THESE MUSCLES. WE FIND IN SKELETAL MUSCLES, WE HAVE A CLASSIC NANCY WILDTYPE CELLS WHERE THERE ARE PAIRS OF MITOCHONDRIA AS I MENTIONED THAT FLANK THE Z DISK. BUT IN CONTRAST, IN ANIMALS THAT LACK MIGHT FUSE INS, YOU HAVE THIS OVER ABUNDANCE OF MITOCHONDRIA AND THEY PROLIFERATE AND FILL THE SPACE BETWEEN MYOFIBRILS AND IN ADDITION, WHEN YOU LOOK AT THE RESULT STRUCTURE OF THESE MITOCHONDRIA, YOU CAN SEE THEY ARE SMALLER AND THEY LACK THE INTERNAL STRUCKER R.URE THAT THE WILDTYPE HAVE — STRUCTURE. SO THERE IS A DEFECT IN THE STRUCTURE AND ABUNDANCE OF THE INTRAFIBULAR MITOCHONDRIA IN THESE SKELETAL MUSCLE CELLS. THIS IS ANOTHER PLACE IN SKELETAL MUSCLE WHERE MITOCHONDRIA ARE ABUNDANT THAT’S UNDER THE PLASMA MEMBRANE. SO THESE ARE THE SUBSARCOLEMALAL MIGHT QUANDARY — MITOCHONDRIA. IN MUSCLE THAT LACKS MITOFUSE INS, THERE IS A PROLIFERATION AND AGAIN THEY HAVE HETEROGENERATEY AND SWELLING AND ALSO A LOSS OF INTERNAL STRUCTURE. THERE IS A DEFECT IN THE ELECTRON TRANSPORT CHAIN AND MITOCHONDRIAL PROLIFERATION. SO THESE KINDS OF PROBLEMS ACTUALLY RESEMBLE THE PROBLEMS THAT YOU SEE IN THE HUMAN DISEASES THAT ARE CAUSED BY DEFECTS IN MITOCHONDRIAL DNA. SO THERE IS A CLASSIC HUMAN DISEASES CALLED IN SELF LOW MYOPATHIES, MATERNALLY INHERITED AND DUE TO MATERNALLY INHERITED MUTATIONS IN THE MITOCHONDRIAL GENOME. SO THIS IS THE CIRCULAR MITOCHONDRIAL GENOME, 16 KILOBASES IN LENGTH. AND THESE ARE THE LOCATIONS EVER VARIOUS MUTATIONS THAT GIVE RISE TO CLINICAL SYNDROMES. SO WE WANTED TO ASK IN THIS SKELETAL MUSCLE VISITS WE DON’T HAVE MITOFUSINS AND HAVE THE MITOCHONDRIAL DEFECT THAT RESEMBLES EN SEF LA MAYOPATHYS, IS THERE A DEFECT IN THE GENOME. SO WE LOOKED TO THE LEVELS OF MTDNA IN THE MITOCHONDRIA. IF WE LOOK AT WILDTYPE ANIMALS, THIS IS THE LEVEL OF MITOCHONDRIAL DNA COMPARED TO NUCLEAR DNA. THIS DEFECT IS DEPENDENT ON LOSING BOTH MITOFUSE INS. SO IF YOU LEWIS JUST MFN1 OR 2, THAT DOESN’T OCCUR. THERE IS A PROLIFERATION EVER MTDNA DURING THE FIRST FEW MONTHS OF LIFE. IF YOU TRACK THE LEVELS OF MT-DNA DURING DEVELOPMENT, WHAT WE FIND IN WILDTYPE ANIMALS IS THAT THE LEVELS INCREASED GREATLY IN THE FIRST TWO MONTHS OF LIFE. AND THIS IS ASSOCIATED WITH THE DIFFERENTIATION OF THE MUSCLE FIBERS AS I SHOWED YOU IN THE PREVIOUS SLIDES. BUT IN ANIMALS THAT LACK MFM1 AND 2, THEY HAVE REDUCED LEVELS AS EARLY AS ONE WEEK OF AGE AND THAT LEVEL DOESN’T INCREASE AS YOU SEE IN THE WILDTYPE ANIMALS. SO, AT 7 WEEKS OF AGE, YOU HAVE A VERY SEVERE DEFECT. SO LET ME SUMMARIZE THIS PART OF THE TALK. SO, WE WERE — WE ASKED WHETHER MITOCHONDRIAL DIE NAM 6 IMPORTANT FOR MTDNA STABILITY AND WE FOUND THAT IN THE CASE OF SKELETAL MUSCLE, IT’S IMPORTANT FOR MAINTENANCE OF MTDNA LEVELS AND I DIDN’T SHOW YOU THIS BUT THERE IS A DEFECT IN THE FIDELITY OF MTDNA. SO IN THE ABSENCE OF MITOFUSINS, THERE IS INCREASE IN POINT MUTATIONS AND DELETIONS. SO WE THINK THAT MITOCHONDRIAL FUSION PROBABLY PLAYS A PROTECTIVE ROLE IN THE PATH APOLOGIES THAT INVOLVED MTDNA. THIS IS ALSO AN ASSOCIATION WITH MITOCHONDRIAL FUSION AND THE ABILITY TO TOLERATE MTDNA MUTATIONS BUT I’M NOT SHOWING THE DATA FOR THAT TODAY. SO, WHAT THESE TYPES OF STUDIES AND ALSO HUMAN GENETIC STUDIES HAVE SHOWN, IS MITOCHONDRIAL FUSION FISSION ARE IMPORTANT FOR A WIDE RANGE OF TISSUES IN MAMMALS. SO FOR EXAMPLE, FOR MITOCHONDRIAL FUSION, FROM THE HUMAN DISEASES, WE KNOW THAT OPA1 IS IMPORTANT IN THE RETINOGANGLYIA CELLS IN THE EYE. MFN2 IS IMPORTANT IN THE NERVES. MFN2 IS IMPORTANT IN THE CEREBELLUM AND BOTH MITOFUSINS ARE IMPORTANT IN SKELETAL MUSCLE. ALSO WORK FROM OTHER LABS SO FROM SHOWING THAT MICE THAT LACK MITOCHONDRIAL FISSION ALSO HAVE NEURONAL DEGENERATION AND THERE IS ALSO ONE HUMAN CASE IN WHICH A DEFECT IN MITOCHONDRIAL FISSION RESULTS IN PERINAILS LETHALITY. A LOT OF EVIDENCE — PERINATAL LETHALITY. IMPORTANT IN CELLS NIPS MAMMALS. SO, BECAUSE OF THIS, WE DECIDED IT WOULD BE IMPORTANT TO BE ABLE TO BETTER STUDY MITOCHONDRIAL DYNAMICS IN TISSUES. SO, MOST STUDIES OF MITOCHONDRIAL DYNAMICS RELY ON CULTURE CELL LINES BECAUSE IT’S EASIER TO GET IMAGED MITOCHONDRIAL DYNAMICS AT HIGH RESOLUTION IN THOSE CASES. BUT MOUSE KNOCKOUT STUDIES AND ALSO HUMAN GENETIC STUDIES INDICATE THAT MITOCHONDRIAL DYNAMICS IS IMPORTANT TISSUES SO WE CONTINUING IS IMPORTANT TO DEVELOP SYSTEMS TO MONITOR MITOCHONDRIAL DYNAMICS IN INTACT TISSUES. AND SO THE WAY THAT WE DID THIS IS TO TRY TO DEVELOP SOME MOUSE MODELS WHERE WE CAN IMAGE MITOCHONDRIA MORE EFFECTIVELY. AND SO THIS WAS WORK THAT WAS DONE BY ANNE FAMILIAR WHO WAS AN MD-Ph.D. STUDENT IN MY LAB. AND SO WHAT SHE DID WAS TO DEVELOPE OR TARGET A FLORA FOR TWO MITOCHONDRIA THAT IS PHOTO ACTIVATABLE. WE TARGETED THE TEND DRA TWO TO THE MITOCHONDRIA AND WE KNOCKED IN THIS CONSTRUCT INTO THE UBIQUITOUSLY EXEXPRESSED ROSA 26 LOCUS AND WE MADE TWO VERSIONS OF THIS MOUSE. IN ONE VERSION, THIS CONSTRUCT IS UBIQUITOUSLY EXPRESSED AND IN THIS MOUSE, ESSENTIALLY ALL THE CELLS IN THE BODY CONTAIN FLUORESCENTLY-LABELED MITOCHONDRIA. IN THE SECOND VERSION, THE CONSTRUCT HAS A STOP SEQUENCE IN FRONT OF IT THAT IS FLANKED BY SITES, IN ORDER FOR THIS CONSTRUCT TO BE ACTIVE, YOU HAVE TO ADD CRE RECOMBINASE. SO WE CAN CONDITIONALLY ACTIVATE THIS MITOCHONDRIAL FLOR FORA SELECT ITCHILY AT DEVELOPMENTAL STAGES OR DIFFERENT TISSUES. FLUOROPHORE. ONE OF THE FEATURES IS IT IS CONVERTIBLE. IT’S NORMALLY GREEN BUT IF YOU ACTIVATE IT USING A LASER, YOU CAN TURN IT TO RED. AND THIS SYSTEM WORKS PRETTY WELL. SO HERE WE HAVE THE CONDITIONAL SYSTEM THAT I MENTIONED. SO WHEN WE ISOLATE FIBROBLASTS FROM THOSE MICE, THEY FIBROBLASTS DON’T HAVE FLORESCENT MITOCHONDRIA. BUT THEN WE CAN TRANSDUCE THOSE CELLS WITH A RETROVIRUS THAT CONTAINS CRE AND NOW THE MITOCHONDRIA IN THOSE CELLS ARE FLUORESCENTLY LABELED. SO HERE WE CAN SEE THE DIFFERENT MITOCHONDRIAL MOREOVERROLOGIES IN THE CELL SO THERE IS MITOCHONDRIA HERE AND A SHORT TUBIAL, LONG TUBIALS AND INTERCONNECTED TUBIALS. AND THEN WE CAN DO PHOTO ACTIVATION STUDIES TO LOOK AT THE FUSION EVER MITOCHONDRIA. SO HERE WHEN I UP THERE MOVIE, YOU WILL SEE PHOTO ACTIVATION. SO HERE SEVERAL REGIONS OF MITOCHONDRIA ARE PHOTO ACTIVATED. WHEN YOU LOOK OVER HERE, THERE IS A FUSION EVENT THAT LEADS TO CONTENT MIXING AND THEY’LL BE ANOTHER EVENT HERE FOLLOWED BY A THIRD EVENT OVER HERE. SO THESE — THIS MARKER IS LOCATED IN THE MATRIX OF THE MITOCHONDRIA. WHEN YOU GET CONTENT IT’S CHANGED AND THAT MEANS THERE ARE OUTER MEMBRANE AND INNER MEMBRANE FUSION THAT IS CURRENT. SO WHEN WE LOOK AT THE VARIOUS TISSUES, SO WHEN WE LOOK AT THE UBIQUITOUSLY EXPRESSED FORM OF THIS MOUSE, WE CAN FIND THAT THERE IS EXPRESSION OF THIS FLOR FORA IN MANY TISSUES. SO MANY TYPES OF NEURONS AND THE MYOCARD YUM, HEPATOCYTES IN KIDNEY CELLS. SO THESE ARE JUST FROZEN SECTIONS WHERE WE CAN QUICKLY GET A SENSE OF MITOCHONDRIAL MORPHOLOGY IN THE TISSUES AND ALSO LOOK IN LIVE CELLS. SOME OF THE LIVE CELLS THAT WE LOOKED AT ARE SPERM. SO THIS IS THE SPERM HEAD, THE TAIL, AND YOU CAN SEE THE PIECE OF THE SPERM THAT IS LIT IS WHERE THE MITOCHONDRIA DEN DRA IS AND WE CAN PHOTO ACTIVATE PART OF THAT. THIS IS FROM A LIGHT SKELETAL MUSCLE FIBER. AND AGAIN HERE ARE THE PAIRS OF MITOCHONDRIA, THE Z DISK IS RIGHT HERE AND THEN WE CAN ALSO ICE LATE THESE AND SEE THE MITOCHONDRIA ARE FLORESCENT. SO BY USING THE SYSTEM, WE CAN LOOK AT MITOCHONDRIAL DYNAMICS. SO HERE IS AN EXAMPLE. THIS IS SKELETAL MUSCLE. AND SO WE CAN PHOTO ACTIVATE A SUBSET OF MITOCHONDRIA AND THEN IF WE TRACK THE FLUORESCENCE IN A MOVIE, YOU CAN SEE THAT THERE CAN BE — SO IN THIS — THIS IS A LONGITUDINAL SECTION OF THE MUSCLE FIBER. SO IT IS RUNNING IN THIS DIRECTION. YOU CAN SEE THAT THERE ARE FUSION EVENTS THAT CAN OCCUR IN THIS DIRECTION AS WELL AS IN THIS DIRECTION. SO WE CAN USE THIS TO STUDY MITOCHONDRIAL DYNAMICS AND SKELETAL MUSCLE. WE CAN ALSO USE THIS SYSTEM TO BETTER UNDERSTAND THE CHANGES IN MITOCHONDRIAL SHAPE THAT OCCUR IN MOUSE KNOCKOUTS. SO REMEMBER I TOLD YOU EARLIER THAT IF WE KNOCKOUT MFN2 WE GET A DEFECT IN PRO KINSY CELLS. AN EXAMPLE OF THAT IS SHOWN HERE. IF WE KNOCKOUT MFN2 IN ADULT PURKINJE CELLS, WE CAN ALLOW THE CELLS TO DEVELOP. SO THIS IS A STAIN WHERE THIS IS THE CELL BODY OF THE PURKINJE CELL AND THESE ARE THE DENDRITES. AT THREE MONTHS OF AGE, YOU CAN SEE THAT MOST OF THE PURKINJE CELLS ARE GONE. SO LOOK AT THIS SYSTEM USING THIS MITODEN DRA MOUSE. AND YOU CAN SEE THAT IN WILDTYPE SECTIONS, SO HERE ARE THE PURKINJE CELLS. THESE ARE THE MITOCHONDRIA, AND WHEN WE KNOCKOUT MFN2, YOU CAN SEE THAT THERE IS MUCH MORE SPARSE MITOCHONDRIA. AND THAT IS PARTICULARLY EVIDENT IN THE DENDRITIC PROCESSES. AND IN THIS OR THESE SLIDES HERE, WE ARE GETTING FROZEN SECTIONS TO GET A QUICK SINCE OF THE MITOCHONDRIAL MORPHOLOGY. BUT WE CAN GET MUCH HIGHER RESOLUTION IF WE USE ORGANIC SLICES. SO THERE IS A TECHNIQUE WHERE WE SIMPLY TAKE A SLICE OF THE BRAIN AND THEN CULTURE THOSE SLICES INTO CULTURE AND THIS ALLOWS THESE SLICES TO SURVIVE FOR SEVERAL MONTHS AND IT PRESERVES THE CONNECTIONS BETWEEN SOME OF THE NEURONS SO WE CAN VISUALIZE MITOCHONDRIAL DYNAMICS USING CONFOCAL MY COSCOPEY AND WE CAN ALSO MAKE PERTY BATIONS. AND WHEN WE DO THAT, WE GET BETTER IMAGE OF THE CELLS THIS SILENT CELL BODY AND THE DENDRITIC — THIS IS THE CELL BODY AND THIS IS THE STAINING. YOU CAN SEE HOW DENSELY PACKED THE MITOCHONDRIA ARE IN THESE PROCESSES. IN A DIFFERENT PART OF THE ORGANIC SLICE, WE CAN SEE NEURONS IN THE MID BRAIN AND THESE ARE DOPAMINERGIC NEURONS PRESENT AND AGAIN WE CAN VISUALIZE THE MITOCHONDRIA IN THE CELLS. SO, WE CAN USE THIS SYSTEM TO LOOK AT DIFFERENT TYPES OF NEURONS IN THE BRAIN. SO IN THE LAST PART OF MY TALK, I WILL TALK ABOUT USING THE SYSTEM TO LOOK AT WHAT HAPPENS TO DOPAMINERGIC NEURONS WHEN THEY USE MFN2. SO, AS I’M SURE YOU ALL KNOW, SINCE RICHARD IS HERE, PARKINSON’S DISEASE HAS AN ASSOCIATION WITH MITOCHONDRIAL FUNCTION. SO PARKINSON’S DISEASE IS THE SECOND MOST COMMON NEURODEGENERATIVE DISEASE. IT’S A MOVEMENT DEFECT. AND FOR DECADES THERE HAS BEEN A LINK BETWEEN MITOCHONDRIAL FUNCTION IN PARKINSON’S DISEASE. AND THIS IS BECAUSE MITOCHONDRIAL TOXINS LIKE MPTP CAN CAUSE PARK INSONIAN SYMPTOMS IN MAMMALS AND IN HUMANS. MORE RECENTLY MORE DIRECT EVIDENCE THAT MITOCHONDRIAL FUNCTIONS IS INVOLVED IN PARKINSON’S DISEASE BECAUSE 10% OF PARKINSON’S DISEASE IS FAMILIAL AND THERE IS A NUMBER OF GENE THAT IS HAVE BEEN IDENTIFIED TO BE LINKED TO PARKINSON’S DISEASE AND TWO OF THEM ARE PARK IN AND PINK 1. THESE TWO GENES WORK IN A COMMON PATHWAY TO PRESERVE MITOCHONDRIAL FUNCTION. AND RICHARD HAS SHOWN THAT PINK ONE AND PAR IN ARE INVOLVED IN THE ELIMINATION OF THIS FUNGAL MITOCHONDRIAL. SO WHEN THEY BECOME DYSFUNCTIONAL, IT’S BEEN SHOWN THAT PARKIN LOCATES TO THOSE MITOCHONDRIA AND THAT SYSTEM CAN RESULT IN THE DEGRADATION OF THOSE DYSFUNCTIONAL MITOCHONDRIA. AND SO WE DID ONE STUDY WHERE WE LOOKED AT SOME OF THE CHANGE THAT IS OCCURRED IN THIS TYPE OF DEGRADATION. SO, WHEN MITOCHONDRIA BECOME DYSFUNCTIONAL IN THIS SYSTEM, PARKIN IS RECUTED ON TO THOSE DYSFUNCTIONAL MITOCHONDRIA AND IT IS A B3 UBIQUITIN LIGASE LEADING TO UBIQUITINATION ON THE MEMBRANES ON THE MITT CONNED REAL MEMBRANE AND LEADS TO THE UBIQUITIN PROTOSTOME SYSTEM CAUSING DEGRADATION ON MANY PROTEINS ON THE OUTER MEMBRANE AND THAT EVENT IS NECESSARY FOR THE DEGRADATION OF THOSE DYSFUNCTIONAL MITOCHONDRIA BY AUTOPHAGY. SO, IN PARKINSON’S DISEASE, THERE ARE GENES ASSOCIATESSED WITH THE DISEASE THAT SEEM TO EFFECT MITOCHONDRIAL DYNAMICS. AND IN ADDITION, IT’S ALSO BEEN SHOWN IN SOME ELEGANT FLY STUDIES THAT IF YOU PERTURB MITOCHONDRIAL DYNAMICS, YOU CAN PERTURB THE PHENOTYPE OF A PINK 1 OR PARKINKNOCKOUT. SO THERE HAS BEEN A NUMBER OF FLY LAB THAT IS HAVE SHOWN THIS ON HIGHLIGHTING WORK FROM THE LAB. SO HERE WE HAVE IN FLIES, A PINK 1 KNOCKOUT. IT LEADS TO APOPTOSIS IN THESE CELLS. BUT IF YOU THEN KNOCK DOWN THE FLY MITOFUSE IN, YOU CAN SUPPRESS THAT DEFECT OR IF YOU OVER EXPRESS DRP1, YOU CAN ALSO SUPRESS THAT. YOU CAN MODIFY BY MANIPULATING MITOCHONDRIAL DYNAMICS. SO JUST TO SUMMARIZE THE ARGUMENT. THERE ARE PARKINSON’S DISEASE ASSOCIATED MUTATIONS IN GENES THAT HAVE A LINK TO MITOCHONDRIAL DYNAMICS. SO FOR EXAMPLE, CELLS THAT LACK PINK 1 OR PARKIN CAN HAVE MITOCHONDRIAL MORPHOLOGY DEFECTS. IN ADDITION, THE EFFECT OF PARKINSON’S ASSOCIATED MUTATIONS CAN BE GREATLY MODULATED BY MITOCHONDRIAL DYNAMICS SO INCREASE FUSION OR INCREASED FISSION TO MODIFY DOSE EFFECTS. SO BECAUSE OF THAT, WE DECIDED TO ASK WHAT IS THE ROLE OF MITOCHONDRIAL DYNAMICS IN DOPAMINERGIC NEURONS AND PARTICULARLY IN THE NIAGRA BECAUSE THAT’S RELEVANT TO PARKINSONS DISEASE? SO THE WAY THAT WE DID THIS WAS TO AGAIN USE OUR CONDITIONAL MFN2 KNOCKOUT ANIMALS AND TO USE A MATING SCHEME WHERE WE USED THE DOPAMINE TRANSPORTER TO KNOCKOUT MFN1 OR 2 IN THE DOPAMINERGIC NEURONS AND INCORPORATED THIS MOUSE THAT ALLOWS US TO TRACK THE MIGHT CONNED REEL RIA. AND WHAT WE FOUND IS THAT IF WE KNOCKOUT THE MFN1, WE DON’T GET ANY PHENOTYPE AT ALL. BUT IF WE KNOCKOUT 2, WE GET ANIMALS THAT ARE RUNTED. SO THIS IS SHOWN HERE. SO HERE IS THE TRACE FOR WILDTYPE ANIMALS. JUST THE WEIGHT GAIN OVER TIME. AND YOU CAN SEE THAT IF THE ANIMALS LACK MFN2, THERE IS A PRETTY SEVERE DECREASE IN WEIGHT GAIN. AND IT TURNS OUT THAT THERE IS A PRETTY SEVERE MOVEMENT DEFECT IN THESE ANIMALS. I SHOULD POINT OUT THAT THESE ANIMALS, IF YOU JUST KEEP THEM IN NORMAL CAGES, THEY’LL DIE AT SIX WEEKS OF AGE DUE TO LACK OF FEEDING. BUT IF YOU PUT FOOD AND WATER AT THE BOTTOM OF THE CAGE, THEY’LL LIVE FOR OVER A YEAR. SO THAT ALLOWS US TO LOOK AT THE LONG-TERM PHENOTYPES OF THESE MICE. AND THIS IS AN OPEN FIELD TEST WHERE A MOUSE IS PLACED INTO AN OPEN FIELD AND THEN YOU SIMPLY TRACK THE MOVEMENTS IN THAT SPACE. AND SO THESE LINES INDICATE THE MOVEMENT OF THE MICE AND YOU CAN SEE THAT MFN2 DEFICIENT ANIMALS HAVE A MOVEMENT DEFECT THAT IS DETECTABLE AS EARLY AS FOUR WEEKS OF AGE AND PROGRESSIVELY GETS WORSE. AND FOR EXAMPLE, IF YOU SIMPLY CALCULATE OR MEASURE THE DISTANCE TRAVELED, THE DISTANCE THAT THESE MUTANT ANIMALS TRAVEL IS REDUCED AT FOUR WEEKS OF AGE AND IT SEEMS TO PLATEAU OUT AT EIGHT-11 WEEK OF AGE. AND THERE ARE SIMILAR RESULTS WHEN WE LOOK AT HOW FAST THESE ANIMALS MOVE. ALSO THEIR REARING AND THE TIME THEY STAY IMMOBILE. SO OBVIOUSLY BECAUSE WE ARE KNOCKING OUT MFN2 IN THE DOPAMINERGIC SYSTEM, WE WANT TO LOOK AT THE CONSEQUENCES FOR THE NEURONS IN THIS SYSTEM. SO THE FIRST THING THAT WE LOOKED AT WAS TO LOOK AT THE DISTAL PROJECTIONS OF THESE NEURONS. SO THE — THERE ARE DOPAMINERGIC NEURONS IN THE SUBSTANTIAL NIAGRA AND THEY PROJECT TO THE STRIATUM AND SO WE CAN LOOK AT THOSE TERMINALS AT THE STRIATUM BY STAINING FOR TYROSINE HYDROXYLASE. SO THIS IS THE STRIATUM OVER HERE. AND WE’RE SIMPLY STAINING FOR TH AND THAT TELLS US THE ABUNDANCE OF THE TERMINAL AT THE STRIATUM. AND WHAT YOU CAN SEE IS THAT EARLY AS THREE WEEKS OF AGE, THERE IS REDUCED STAINING IN THE STRIATUM SUGGESTING THAT THERE IS A DEFECT IN THE NERVE TERMINALS IN THIS PART OF THE DOPAMINERGIC SYSTEM AND WHEN YOU GO OUT TO 11 WEEKS, THE STAINING IS ALMOST ALL GONE. AND THIS CAN BE QUANTIFIED. THEN IF WE WORK BACKWARDS IN THE CIRCUIT ASK WE LOOK AT THE CELL BODIES WE FIND THAT AT THREE WEEKS OF AGE WHEN WE LOOK AT THE SUBSTANTIAL NIAGRA, AND WE LOOK FOR HT STAINING, THE CELLS BODIES ARE PRESENT. SO THERE IS A DISTAL DEFECT IN NEURONS. WITH WE LOOK AT THE CELL BODIES THEY ARE NORMAL BUT AT 11 WEEKS THERE IS DAY DEFECT AT 14 WEEKS. SO, THIS IS A QUANTIFICATION OF THAT RESULT. SO IN CONTRAST TOKING AT THE NERVE TERMINALS, WHEN WE LOCK AT CELL BODIES, AT THREE WEEKS THEY ARE NORMAL AND 8-9 WEEKS DURING NORMAL, AND ONLY AT 11 AT 10-12 WEEKS TO SEE THIS DEFECT IN THE CELL BODIES. SO WHAT HE WE THINKSHIPS A RETRO GRADE DEFECT AT THE NERVE TERMINAL AND THEN CELL DEGENERATION. AND THIS, AS NOTICED, BECAUSE IT DEALS WITH MFN2, HAPPENS TO INVOLVE A MITOCHONDRIAL DEFECT. SO USING THE ORGANELLE SLICED CULTURES THAT I MENTIONED IN CONJUNCTION WITH THE MITODEN DRA, YOU CAN SEE THAT WHETHER YOU LOOK AT PROXIMAL PROCESSES OR DISTAL PROCESSES OF THOSE DEEPA MA NERGIC NEURONS. THE KNOCKOUT SPLICE GREATLY REDUCED NUMBERS OF MITOCHONDRIAL. AND THERE IS ALSO A PRETTY SEVERE DEFECT IN THE TRANSPORT OF MITOCHONDRIA IN THESE NEURONS. SO HERE IS A CONTROL NEURON WHERE WE ARE LOOKING AT A DENDRITE AND ONE OF THESE DOPAMINERGIC NEURONS AND THE SLICE CULTURE AND HERE WE EFFECTO ACTIVATE A CLUSTER OF MIGHT MITOCHONDRIA AND THEN TRACK THESE OVER TIME AND SO THIS IS A GRAPH WHERE TIME IS IN THIS DIRECTION AND SO THESE VERTICAL — THESE DO IAGONAL LINES OVER HERE INDICATE MOVEMENT OF MITOCHONDRIA FROM THIS CLUSTER AS THEY TRAVEL ALONG THIS PROCESS. AND IN CONTRAST, WHEN WE LOOK AT MFN2 KNOCKOUT NEURONS, HERE WE PHOTO ACTIVATE A CLUSTER OF MITOCHONDRIA. YOU CAN SEE THERE ARE VERY FEW TRANSPORT EVENTS THAT COME OUT OF THIS CLUSTER. SO WE CAN QUANTIFY THIS AND FROM IS DEFECT IN BOTH THE NUMBER OF TRANSPORT EVENTS AS WELL AS THE VELOCITY OF THESE EVENTS. SO WE THINK THAT WOO TRANSPORT DEFECT IN MICE WHICH LEADS TO THIS RETRO GRADE DEFECT IN THE NEURONS. SO TO SUMMARIZE THIS LAST PART OF THE TALK, SO WE HAVE BEEN DEVELOPING MOUSE MODELS TO STUDY MITOCHONDRIAL DYNAMICS AND USED IT TO STUDY DYNAMICS IN DOPAMINERGIC NEURONS. — [ READING ] SO WE THINK THAT THIS MOUSE MODEL MIGHT BE A GOOD MODEL TO LOOK AT THE CELL BIOLOGICAL DEFECTS THAT OCCUR IN THIS TYPE OF DEGENERATION. SO LET ME THANK THE PEOPLE WHO DID THIS WORK. SO, THE WORK WITH THE MOUSE MODEL TO LOOK AT MITOCHONDRIAL DYNAMICS, AND DOPAMINERGIC NEURONS IS ANH PHAM AND LOOKING AT SKELETAL MUSCLE AND CEREBELLUM DEFECTS IS DONE BY HSIUCHEN CHEN, A SENIOR SCIENTIST IN THE LAB. THE WORK I MENTIONED ON DEGRADATION EVER MITOCHONDRIA OUTER MEMBRANE PROTEINS IN AUTOPHAGY IS DONE BY NICKIE CHAN AND SOME OF THE WORK ON THE DOPAMINERGIC NEURONS WAS DONE WITH HELP FROM ANDREW STEELE A RESEARCH FELLOW AT CAL TECH AND WORK HAS BEEN DONE FROM THE LONG TERM COLLABORATION WITH MICHAEL McCARY FROM JOHN’S HOPKINS. THANK YOU VERY MUCH. [ APPLAUSE ]>>THE TRANSGENIC MOUSE AND THE REPORTER PROTEINS WHICH IS INTERESTING THAT THE PICTURE YOU SHOWED IN THE SKELETAL MUSCLE, WAS THAT IN VIVO OR A MUSCLE REMOVED FROM THE ANIMAL? BECAUSE THAT WAS A HIGH RESOLUTION PICTURE.>>THAT IS A MUSCLE REMOVED. SO EITHER SO WE CAN DO IT BOTH WAYS. SON TO HAVE THE ENTIRE MUSCLE AND IMAGE IT AND THE OTHER WAY IS TO TEASE OUT MUSCLE FIBERS. AND IN BOTH CASES IT’S NOT IN THE IN TACT ANIMAL.>>IN THE MUSCLE, YOU SAW WHAT LOOKED LIKE SOME RED AND YELLOW AREAS CONSISTENT WITH FUSION. COULDN’T THAT BE THE FUSION FROM PARTIALLY ERATE A. RADIATED WHERE YOU HAVE SOME RED DYE AND GREEN DYE AND WHAT YOU’RE LOOKING AT IS THE FUSION LIKE IT SHOWED SOMETIME AGO?>>SO WHEN WE PHOTO ACTIVATE, DEPENDING ON THE DEGREES, YOU CAN GET ALL DIFFERENT FUSES. SO FOR EXAMPLE, MITOCHONDRIAL IS PARTIALLY PHOTO ACTIVATED. BUT IF WE MAKE MOVIES, WE CAN SEE CONNECTIONS BETWEEN MITOCHONDRIA IN WHICH WE TRANSFER AND SEE THOSE STEP-WISE TRANSFER ON TO ANOTHER MITOCHONDRIAL.>>FROM WHAT YOU SHOWED IT’S CLEAR IF YOU ALTER THE MITOCHONDRIAL FISSION AND FUSION MACHINERY THAT YOU CAN GET CHANGES IN THE MOREOVERROLOGY AND THE FUNCTION OF MITOCHONDRIA. BUT YOU ALSO SHOWED THAT CELL CULTURE PARTICULARLY THAT FISSION AND FUSION EVENTS ARE HELPING ON THE SECOND TIME SCALE WHERE AS IN THE SKELETAL MUSCLE MAY BE HAPPENING ON THE ORDER OF MINUTES OR MAYBE HOURS. SO WONDER FIGURE YOU HAD INSIGHTS INTO WHAT THE SIGNAL IS THAT TELLS THEM WHEN TO HAVE FISSION AND FUSION EVENTS?>>RIGHT. I AGREE THAT THE MITOCHONDRIAL FUSION AND FISSION EVENTS ARE MUCH — WE SEE MANY FOR IN CULTURE THAN IN SKELETAL MUSCLE. WE DON’T KNOW WHAT THE SIGNALS ARE AND THERE HAVE BEEN SOME INTERESTING FINDINGS. FOR EXAMPLE, MITOCHONDRIAL FISSION IS REGULATED BY CELL CYCLE. IT’S ALSO REGULATED BY NUTRIENT STATUS. AND THERE ARE OTHER TYPES — CELLULAR STRESSES THAT REDUCE MITOCHONDRIAL FUSION BUT I WOULD SAY IN THE NORM CELLS, WE DON’T KNOW WHAT THEY ARE.>>DIDN’T IT BECOME CLEAR TO ME WHY THE PARKINSON’S MICE THERE — DOPAMINERGIC NEURONS DIE. IS IT BECAUSE YOU HAVE REDUCTION IN THE NUMBER OF MITOCHONDRIA? OR CONFOUNDED BY THE FACT THEY HAVE OR IS IT MITOCHONDRIAL DNA MUTATIONS?>>I THINK THERE IS A CLEAR — I DON’T THINK THERE IS A CLEAR ANSWER TO THAT. SO, EVEN IN SPORADIC PARKINSON’S DISEASE, THERE ARE SOME STUDIES THATIGED KATE THAT THEY HAVE REDUCTIONS. SO THE SIMPLE ANSWER IS THAT MAYBE THOSE NEURONS ARE MORE SENSITIVE TO REDUCED MITOCHONDRIAL RESPIRATION ACTIVITY. AND THERE ARE SOME ARGUMENTS WHY THOSE NEURONS MIGHT BE. SO FOR EXAMPLE, IT’S BEEN ARGUED THAT THOSE NEURONS ARE METABOLICALLY HIGHLY ACTIVE SO THAT IS ONE POSSIBLY.>>DO YOU SEE ANY CHANGING OPTIC NERVE DEGENERATION? DO YOU FIND ANY OPTIC NERVE DEGENERATION IN MITOFUSION TO MICE AND ANY VISUAL BEHAVIOR DEFECTS? IN PART II, MUTATION VISIT PROBLEMS WITHS FUSION. SO IF YOU HAVE THIS OTHER MEMBRANE FUSION DEFECTS, YOU MAY ALSO SEE SOME SORT OF OPTIC NEUROPATHY. DID YOU HAVE A LOOK AT BEHAVIOR WITH MARRYING THESE MICE AND MFN2 KNOCKOUTS?>>YOU’RE ASKING IF THERE IS ANY OPTIC DEFECTS.>>NEUROPATHY OR VISUAL BEHAVIOR DEFECTS? DID YOU EVER CHECK THAT?>>SO WE HAVEN’T LOOKED AT OPTIC DEFECTS. SO WE DON’T KNOW. BUT I WOULD SAY THAT IN MY TALK, I MENTIONED THAT THERE IS ADDOOMED DOMINANT APATHY BETWEEN BLINDNESS AND THEN CM28. SO WHEN THIS WAS DISCOVERED, IT SEEMED LIKE THIS REALLY DISPARATE — DISPARATE TWO SYSTEM TAC HAVE THE CLINICAL PHENOTYPES. AND THAT IS THE CASE. BUT AS PEOPLE IDENTIFIED MORE AND MORE FAMILIES, THEIR FAMILIES WHERE THERE IS OVERLAP. SO THERE ARE CMT PATIENTS THAT HAVE OPTIC ATROPHY AND AT THE SAME TIME THERE ARE PATIENTS WITH OPA1 MUTATION THAT IS ALSO HAVE PERIPHERAL NEUROPATHY. SO THERE IS DEFINITELY OVERLAP BETWEEN THE TWO CLINICAL SYNDROMES.>>SO WHY DO YOU BELIEVE THAT THE OPA IS SPECIFIC TO THE EYE AND THE NERVES WHEN YOU SHOWED WHEN YOU DISRUPTED THE SKELETAL MUSCLE THERE IS A HUGE SKELETAL MUSCLE NERVE DEVELOPS.>>SCORE IN HUMAN DISEASES THERE IS A SUBTLE DEFECT. THESE ARE HETEROZYGOUS MUTATIONS. AND FOR EXAMPLE, WE DO HAVE — SO WE HAVE KNOCKED IN A COUPLE OF THESE ALLELES INTO MICE AND THERE IS NO DEED — DEFECT. SO IT’S A SUBTLE DEFECT.>>IN HUMANS IT’S EASIER TO DETECT ANY KIND OF PHENOTYPE AND IF YOU SEE ANY PROBLEMS, IT’S NOT THESE MICE CANNOT DO THAT.>>RIGHT.>>VERY NICELY CHANGES THAT OFFERED BETWEEN MITOCHONDRIAL DNA AND THE NUCLEUS DNA DO YOU HAVE A SIMILAR STUDY FOR BRAIN SLICES? AT WHAT TIME DO THEY LEVEL?>>WE HAVEN’T LOOKED AT MITOCHONDRIAL DNA CONTENT IN THE BRAIN.>>SO SOME OF THE REDUCTIONS HAVE BEEN SHOWN — [ INDISCERNIBLE ] DO THEY EFFECT OTHER PROTEINS?>>I DON’T THINK — I MEAN, THOSE TOXINS ARE PRIMARILY INHIBITING COMPLEX ONE SO I DON’T THINK THAT THERE IS A DIRECT EFFECT ON THE MIGHT FUSE INS OR OPA1 IN THAT CASE.>>SO THIS CONCEPT APPLIES NICELY. SO WHAT HAPPENS WHEN CELL BECOMES MANAGEABLE? [ INDISCERNIBLE ] YOU MEANING THE RELATIONSHIP OF DYNAMICS? THERE IS REALLY NOT MUCH INFORMATION ON THAT. I THINK THERE IS ONE STUDY THAT FOUND THAT IN A TYPE OF LUNG CANCER THERE WAS MITOCHONDRIAL FISSION. AS FAR AS I KNOW THAT’S THE ONLY STUDY RELEVANT TO THAT.>>BUT YOU CAN PREDICT THAT WHEN FISSION AND FUSION SYSTEMS MESS UP IT WILL BE DIFFICULT — [ INDISCERNIBLE ]>>YES. — . [ LOW AUDIO ]>>I DIDN’T UNDERSTAND THE FIRST PART –>>THERE ARE SOME PROTEINS WHICH ARE IMPLICATED WITH A MUTATION THAT LEAD TO ATAXIA — [ INDISCERNIBLE ] AND THEY ARE ALSO KNOWN TO — HOW MITOCHONDRIA DNA DAMAGE — . [ LOW AUDIO DAMAGE — LOW ODD.>>I THINK IN GENERAL, PEOPLE WHO TREAT CELLS WITH DRUGS THAT CAUSE INCREASE IN OXIDATIVE STRESS — I THINK IN THOSE CASES — WELL, IN MANY CASES WE SEE MITOCHONDRIAL DAMAGE. DEPENDING ON THE CELL SYSTEM AND OTHER SYSTEMS, YOU MIGHT FIND INCREASE IN MITOCHONDRIAL LEAPT BECAUSE THERE IS ALSO THIS OTHER SYSTEM THAT WORKS WITH CALLED STRESS INDUCED HYPERFUSION WHERE CERTAIN TYPES OF STRESS LEAD TO INCREASE IN MITOCHONDRIAL FUSION. SO I GUESS IT’S NOT CLEAR. BUT USUALLY INCREASING OXIDATIVE STRESS IS THOUGHT TO INCREASE LEVELS OF MITOCHONDRIAL MUTATIONS. I THINK THAT CERTAINLY CAN LEAD TO DEGENERATIVE DEFECTS.>>SO I THINK WE CAN CONTINUE THIS IN THE RECEPTION. HE WILL BE AVAILABLE RIGHT AFTERWARDS IN THE LIBRARY. THANK YOU AGAIN. [ APPLAUSE ]

2 Comments

  • Reply Adelgaza20 con Ingrid Macher October 11, 2013 at 4:43 pm

    very informative lecture.

  • Reply REDES GYMA September 3, 2014 at 2:44 pm

    Pathetic Introduction,

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