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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 ***.
SO THIS IS THE *** HEAD, THE
TAIL, AND YOU CAN SEE THE PIECE
OF THE *** 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 ]