SO WELCOME, EVERYBODY
TO TODAY'S WALS SEMINAR.
FOR THOSE OF YOU ONLINE, I
SHOULD MENTION OR FOR ANYONE
WHO'S GETTING CME CREDITS FOR
THIS, REMEMBER THAT THE CODE IS
57681.
SO THAT'S THE CODE YOU WILL HAVE
TO ENTER TO GET CREDIT.
FOR THOSE OF YOU WHO DON'T KNOW
ME, I'M BEN WHITE, I'M A MEMBER
OF THE DROSOPHILA NEUROSCIENCE
INTEREST GROUP AT NIH AND OUR
GROUP IS COSPONSORED TOGETHER
WITH THE WOMEN'S SCIENTISTS
ADVISORS COMMITTEE OF TODAY'S
WALS SPEAKER DR. DANIELA
ZARNESCU, OF THE COLLEGE OF PENN
STATE MEDICINE.
OT DROSOPHILA SIDE I SHOULD SAY
THAT DR. ZARNESCU, WAS AN
APPROPRIATE PICK FOR THIS
SEMINAR SERIES AND HER WORK
SHOWS THE POWER OF WORK OF
STUDYING FLIES TO BRIDGE THE GAP
IN UNDERLYING HUMAN PROCESSES
AND HUMAN DISEASE.
AND MOST OF US WHO WORK IN FLIES
KNOW THAT OUR WORK ALTHOUGH IT'S
AIMED AT BIOMEDICAL ADVANCES,
OFTEN WE WORK PRETTY FAR FROM
THE CLINIC AND YOU WILL SEE THAT
DR. ZARNESCU'S RESEARCH IS
DIFFERENT IN A SENSE THAT IT'S
VERY MUCH DIRECTED AT
UNDERSTANDING THE CLINICAL
NEURAL PATHOLOGIES BASED ON WORK
THAT DONE'S IN THE FLY.
SO HER LAB DEVELOPED FLY MODELS
FOR ALS AND FRONTAL LOBE
DEMENTIA AND THEN SHE USED THOSE
MODELS TO GAIN INSIGHT INTOS
MOLECULAR MECHANISMS THAT THEN
COULD BE TESTED IN HUMAN TISSUES
AND PATIENT DERIVED IPSC
NEURONS.
SO USING THAT STRATEGY HER LAB
HAS DISCOVERED NOVEL PATHOGENIC
ROLES FOR A VARIETY OF MUTANT
PROEN TOOS WHICH I AM SURE YOU
WILL HEAR PLENTY MORE ABOUT IN
HER TALK TODAY.
IT'S WORTH POINTING OUT HOWEVER,
THAT IN ADDITION TO USING THE
FLY TO ELUCIDATE THE KEY
MOLECULES INVOLVED IN PROCESSES,
UNDERLYING NEUROPATHOGENESIS
THAT DR. ZARNESCU IS ALSO
LEVERAGING THE FLY MODEL AS A
FIRST PASS TOOL FOR DRUG
DISCOVERY, KREENING FOR SMALL
MOLECULES THAT MAY PROVIDE
THERAPEUTIC BENEFITS AND HUMANS,
SO JUST AS A BIT OF BACKGROUND
DR. ZARNESCU STUDIED PHYSICS IN
ROMANIA BEFORE COMING TO THE
U.S. BEFORE GETTING HER DEGREE
IN MOLECULAR BIOLOGY AT PENN
STATE.
SHE WENT ON TO DO POST DOCTORAL
WORK AT EMORY WITH KEVIN MOSES
WITH THE FRAGILE X PROTEIN OF
DROSOPHILA AND THEN JOINED THE
FACULTY AT UNIVERSITY OF ARIZONA
IN 2006.
AND IT'S INTERESTING AT LEAST I
WAS INTERESTED IN LOOKING AT HER
CEV, TO SEE THAT THE GROWING
CLINICAL RELEVANCE OF HER WORK
COULD ALMOST BE FOLLOWED IN HER
TRAJECTORY FROM ASSISTANT
PROFESSOR TO FULL PROFESSOR AT
THE UNIVERSITY OF ARIZONA IN THE
JOINT APPOINTMENTS THAT SHE ADO
YOU MEANULATED.
SO BY THE TIME SHE LEFT THE
UNIVERSITY OF ARIZONA IN 2022,
TO GO BACK TO PENN STATE, SHE
HAD APPOINTMENTS NOT ONLY IN HER
PRIMARY DEPARTMENT OF MOLECULAR
AND CELLULAR BIOLOGY, BUT ALSO
IN NEUROLOGY, CELLULAR AND
MOLECULAR MEDICINE, NEUROSCIENCE
AND GENETICS.
SO I THINK THAT SPEAKS TO THE
BROAD IMPACT OF HER WORK.
SO FINALLY AS I MENTIONED,
DR. ZARNESCU, IS A -- IS ALSO
SPONSORED TODAY BY THE WOMEN
SCIENTISTS ADVISOR'S COMMITTEE
AND THAT REFLECTS HER LONG
STANDING COMMITMENT TO MENTORING
EDUCATION AND TRAINING, FOR
WHICH SHE HAS WON NUMEROUS
AWARDS.
SHE'S CURRENTLY ASSOCIATE DEAN
FOR GRADUATE EDUCATION AND POST
DOCTORAL TRAINING AT PENN STATE
COLLEGE OF MEDICINE AND I WANT
TO MENTION THAT SHE KINDLY
OFFERED TO ALSO LEAD A SPECIAL
CAREER WORKSHOP TOMORROW FOR
FELLOWS, THAT'S ENTITLED STAYING
THE COURSE, LESSONS IN BUILDING
RESILIENCE.
SO FOR FELLOWS WHO WOULD LIKE TO
ATTEND THE WORKSHOP, IT'S
TOMORROW AT 2:00 P.M. IN
BUILDING 40.
SPOTLIGHT TODAY OF COURSE IS ON
HER SCIENCE, HER EXCITING
SCIENCE AND SO, WITHOUT FURTHER
ADO, I WANT TO TURN THE PODIUM
OVER TO DR. ZARNESCU, WHO'S TALK
TITLE PERHAPS WITH APOLOGIES TO
ROBERT BERNE AND JOHN STEIN BECK
IS OF FLIES AND MEN-HARNESSING
THE POWER OF DROSOPHILA TO
DECIPHER THE MOLECULAR
MECHANISMS OF NEURO
DEGENERATEDDATION GENERATEDDATI-
DEGENERATION.
SO PLEASE HELP ME WELCOME HER.
>> CAN YOU HEAR ME OKAY, IT
FEELS SPECIAL TO BE HERE SO
THANK YOU MUCH FOR HAVING ME,
EVERYONE AT MY INSTITUTION ASKS
ME ARE YOU SURE THE SEMINAR IS
STILL ON, AND I SAID AS FAR AS I
KNOW YES, SO, I WAS REALLY
IMPRESSED TO SEE THIS, I CAN
TAKE THESE BACK AND PROVE THAT I
WAS HERE AND THE SEMINAR TOOK
PLACE IN CASE THEY'RE WONDERING
WHAT'S GOING ON.
SO THANK YOU VERY MUCH AGAIN, I
PUT TOGETHER PERHAPS TOO MANY
SLIDES SO A MAY NOT GO THROUGH
ALL OF THEM BUT I DO WANT TO SAY
THAT I LOVE FLIES AND I WAS
ATTRACTED BY THE POWER OF FLY
GENETICS WHEN I STUDIED GRADUATE
SCHOOL AND THEN I DISCOVERED
WHEN I WAS A POST DOC THAT THE
FLY CAN BE A WONDERFUL SYSTEM TO
UNDERSTAND BASIC MECHANISMS OF
DISEASE AND I WAS WORKING ON
FRAGILE X SYNDROME AT THE TIME
AND IN MY LAB WE MADE THE SWITCH
TO AGE DEPENDENT
NEURODEGENERATIVE DISEASES
BECAUSE AS YOU CAN TELL AGING
AND INEVITABLE AND THAT BECAME
RELEVANT AND INTERESTING TO ME
AT THE TIME.
SO I'M GOING TO TRY TO DO A BIT
OF A TOUR DEFORCE THROUGH SOME
OF THE WORK WE'VE DONE SINCE WE
DEVELOPED FLY MODELS OF LATERAL
SCLEROSIS AND I WOULD LIKE TO
SHARE NEW AND UNPUBLISHED DATA
AND I LOOK FORWARD TO GETTING
FEEDBACK FROM ALL OF YOU AND
ALSO PLEASE FEEL FREE TO
INTERRUPT AND ASK QUESTIONS AT
ANY TIME AND THAT LIGHT IS A BIT
BLINDING SO IF I SQUINT, IT'S
BECAUSE OF THAT.
BUT -- SO NEURODEGENERATIVE
DISORDERS ARE A GROUP OF
HETEROGENEOUS DISEASES AND I WAS
REALLY GLAD TO SEE THIS REVIEW
THAT REALLY PUTS TOGETHER AWL
HARKERS NEURODEGENERATIVE
DISEASES VERY MUCH LIKE THE
CANCER FIELD HAS DONE A LONG
TIME AGO AND I LIKE TO
PERSPECTIVE BECAUSE IT'S REALLY
HIGHLIGHTS THE FACT THAT
NEURODEGENERATIVE DISORDERS
SHARE ALTERED COMMON PATHWAYS
THAT INCLUDE ABNORMALITIES
BROIGATION OF PROTEINS, INSIGNE
APTIC DYSFUNCTION, DEFECTING
PROTEIN COMPLEXIO STASIS,
CYTOSKELETAL ABNORMALITIES
AFFECTING METABOLISM AND ENERGY
HOMEOSTASIS, DEFECT IN DNA AND
RNA, AS WELL AS INFLAMMATION
THAT ULTIMATELY LEADS TO
NEURONAL DETH -- DEAG.
AND I THINK THIS SCHEME PROVIDES
A SCHEME TO THINK MORE
WHOLISTICALLY OF THE
NEURODEGENERATIVE DISORDIERS
LIKE ALS, OR PARKINSONS THAT I
WORK ON, BECAUSE ULTIMATE GOAL
MITTLY THEY SHARE ALL OF THIS
AND IT'S BECOME MORE AND MORE
CLEAR IN RECENT YEARS THAT AS WE
UNDERSTAND THE PATHOMECHANISMS
IN 1 DISEASE THEY BECOME
APPLICABLE OR PROVIDE INSIGHT
INTOS WHAT WE CALL RELATED
NEURODEGENERATIVE DISORDERS.
IS THAT A LASER POINTER OR
SHOULD I -- I WILL USE THE
CURSOR, OKAY.
SO IN NIGH LAB VERMEN INFECTED
BEEN FOCUSING ON MI LATERAL
SCLEROSIS SINCE 2008.
IT'S A PROGRESSIVE
NEURODEGENERATIVE DISORDER WITH
A PREVALENCE OF 43 OR HIGHER
DEPENDING ON WHAT PAPER YOU
READ, A IN A HUNDRED THOUSAND
PEOPLE.
AGE IS THE BIGGEST RISK FACTOR,
AND I'VE HAD REALLY WONDERFUL
TALKS IN MORNING ABOUT AGING AND
WHAT DOES THAT REALLY MEAN.
THE AVERAGE LIFE SPAN IS ABOUT 2
AND HALF YEARS FOR PATIENTS POST
DIAGNOSIS, AND THERE ARE ONLY 4
FDA APPROVED DRUGS THAT HAVE
VARIOUS DEGREES OF IMPACT ON THE
PROGRESSION OF THE DISEASE.
WHAT IS REALLY REMARKABLE ABOUT
ALS AND BRINGS AGAIN THE HILIGHT
BACK ON THESE HALLMARKS OF
NEURODEGENERATION IS THIS RNA
BYPASSING PROTEIN CALLED TDP43.
WHICH WE THINK OF AS A COMMON
DENOMINATOR IN ALS AS WELL AS
RELATED NEURODEGENERATIVE
DISORDERS.
IT'S AN AUTONOMIC BINDING
PROTEIN WHEN WITH 2 DIFFERENT
ROM DOMAINS AND A C-TERMINUS
STRUCTURE.
AND I'M HIGHLIGHTING THE FACT
THAT PATIENTS IN TDP43, THAT
ONLY REPRESENT 2-4% OF THE ALS
POPULATION, THIS MUTATION'S
CLUOF THENER THE C-TERMINUS
DOMAIN OR UNSTRUCTURED DOMAIN
AND THERE'S EVIDENCE THAT THESE
MUTATIONS MAKE TDP 43, WHICH IS
IN ITS WILD-TYPE FORM PRONE TO
ABNORMALITIES BROIGATION, EVEN
MORE BROWN TO ABNORMALITIES
BROIGATION, SO THESE MUTATIONS
ACCELERATE THE FORMATION OF
AGGREGATES WHICH WAS 1 OF THE
HALLMARKS OF NEURODEGENERATION
THAT I HIGHLIGHTED EARLIER.
BUT WHAT IS EVEN MORE REMARKABLE
ABOUT THIS RNA BINDING PROTEIN,
IS THAT 97% OF ALS PATIENTS HAVE
WILD-TYPE TDP 43 AGGREGATES.
SO WHILE NORMALLY THIS PROTEIN
IS DOICATE INDEED THE NUCLEOS --
LOCATED IN THE NUCLEUS, 97% OF
PATIENTS EXHIBIT TPD43 IN THE
CYTOPLASM THAT EXHIBIT THREEZ
VARIOUS SHAPES HERE.
SO BASED ON THIS PATHOLOGY, WE
AND OTHERS THINK OF THIS
PATHOLOGY BEING CAUSED BY A
COMBINATION OF LOSS OF NUCLEAR
TDP 43 FUNCTION AS WELL AS GAIN
OF TOXIC CYTOPLASMIC FUNCTION.
AND DEPENDING ON WHO YOU TALK
TO, THERE WILL BE SOME THAT
THINK IT'S THE LOSS OF NUCLEAR
FUNCTION THAT MATTERS THAT WILL
BE SOME THAT THINKS IT'S THE
GAIN OF CYTOPLASMIC TOXIC
FUNCTION THAT MATTERS AND AS
TRUE IN LIFE, THE TRUTH IS
SOMEWHERE IN BETWEEN.
THE OTHER REMARKABLE THING ABOUT
TDP43 IS THAT WHILE 97% OF ALS
PATIENTS HAVE WILD-TYPE TDP 43
AGGREGATES, 40% OF THE FRONTAL
TEMPORAL PATIENTS ALSO HAVE
WILD-TYPE TDP43 AGGREGATES.
IN ADDITION ABOUT 50% OF
ALZHEIMER'S PATIENTS EXHIBIT
WILD-TYPE TDP43 AGGREGATE,
INDIVIDUALS THAT SUFFERED
TRAUMATIC BRAIN INJURY HAVE
WILD-TYPE TDP 43 AGGREGATE,
FOOTBALL PLAYERS WHO GET HIT A
LOT WHO IS DIAGNOSE WIDE CET,
HAVE WILD-TYPE AGGREGATES, SO IF
YOU SEE IF WE STUDY THE BIOLOGY
OF TDP43, HOW MIGHT THIS
AGGREGATES COME ABOUT IN WHAT
ARE THE PHENOTYPIC CONSEQUENCES
OF THESE AGGREGATES?
YOU MIGHT BE ABLE TO UNDERSTAND
A LOT ABOUT MANY OF THESE
DISORDERS THAT HAVE AS A COMMON
DENATIONAL LIBRARY OF MEDICINE
THEINATOR TDP43.
SO AS TRAINED AS A FLIGHT
GENETICIST DURING GRADUATE
SCHOOL AND WORKING WITH FRAGILE
X SYNDROME MODELS AS A POST DOC,
I THOUGHT WHAT A GREAT
OPPORTUNITY TO MODEL THIS IN
FLIES AND I PROBABLY DON'T NEED
TO SAY THIS TO MANY OF YOU IN
THE AUDIENCE BUT I WILL SAY IT
ANYWAY, FLIES ARE AWESOME,
THERE'S A HIGH DEGREE OF
CONSERVATION THAT ALLOWS US TO
STUDY GENE FUNCTION AND IDENTIFY
RELEVANT PATHWAYS TO HUMAN
BIOLOGY AND HUMAN DISEASE IN THE
FLY, THERE ARE MANY HUMAN
DISEASE GENES THAT HAVE
ORTHOLOGS IN THE FLY AND
ALTHOUGH THERE ARE NOT IDENTICAL
SYSTEMS, RIGHT?
FLIES DON'T HAVE A LIVER PER SE
BUT THEY HAVE A FAT BODY THAT
PERFORMS MANY OF THE FUNCTIONS
THE LIVER DOES, SO WHEN IT COMES
TO THE NERVOUS SYSTEM, WE HAVE A
PRETTY NICE SIMILARITY OR
CORRELATION BETWEEN THE 2
SYSTEMS, BOTH AT THE LEVEL OF
CENTRAL NERVOUS SYSTEM AND
PERIPHERAL NERVOUS SYSTEM,
AGAIN, NOT IDENTICAL, BUT
SUFFICIENT SIMILARITIES AND
WONDERFUL GENETIC TOOLS THAT,A
ALLOW US TO UNDERSTAND BASIC
BIOLOGICAL QUESTIONS AND I DON'T
NEED TO TELL YOU THAT THEY HAVE
OTHER ADVANTAGES SUCH AS LOWER
COST AND SHORT GENERATION TIME
WHICH STUDENTS LIKE TO HEAR
ABOUT BECAUSE THAT MEANS THEY
WILL FINISH THEIR Ph.D. A
LITTLE FASTER, RIGHT?
THAT'S A GREAT SELLING POINT
ANDIME THE ONLY FLIGHT
GENETICIST IN MY COLLEGE, SO
THAT'S BEEN A SUCCESSFUL
RECRUITING TOOL.
SO IN 2008, TDP 43 MUTATIONS
WERE IDENTIFIED IN ALS AND
THAT'S WHEN I DECIDED TO APPLY
OUR SKILLS AND KNOWLEDGE OF
FLIGHT IMENETTICS AND DISEASE
MODELS AND MAKE FLY MODELS OF
ALS, AND WE DECIDED TO DO THIS
BY OVEREXPRESSING EITHER HUMAN
TDP43 OR DO SOFILLA HOMOLOGUE OF
TDP43 AND WE CAN DO IN THIS IN
MOTOR NEURONS OR GLIAL CELLS OR
MUSCLE CELLS, THOSE ARE ALL THE
CELLS SKTAD IN THIS DISEASE.
SO WE OVEREXPRESSED WILD-TYPE
TDP43 AS A PROXY FOR THOSE 97%
OF CASES WITH THE WILD-TYPE TDP
PATHOLOGY, AND WE ALSO
OVEREXPRESS VARIOUS MUTANT FORMS
OF TDP43 AS A PROXY FOR TOAZ
2-4% OF PATIENTS THAT EXHIBIT
MUTATIONS IN TDP43.
AND I'M SHOWING YOU HEAR,
EXAMPLES OF PHENOTYPES THAT WE
THINK ARE RELEVANT TO ALS, AND
ALLOW US TO DO ALL KINDS OF
PHENOTYPIC SCREENS INCLUDING
DRUG SCREENS AND GENETIC SCREENS
TO FIGURE OUT THE MECHANISTIC
UNDERPINNINGS OF TDP43 PROTEIN
OPEN MEETINGATHYS.
SO OVEREXPRESS OF TDP43 MOTOR
NEURONS CAUSES MOTOR NEURON
DEATH, YOU CAN SEE FEWER
MOTONEURONS IN THE THORACIC
GANGLIA WHEN WE OVEREXPRESS
HUMAN TDP 43, THERE ARE SYNAPTIC
ABNORMALITIES OF THE
NEUROMUSCULAR JUNCTION AND
WITHOUT GOING INTO TOO MUCH
DETAIL, I CAN TELL YOU THOSE
SYNAPSES ARE ABNORMAL, THEY'RE
SMALLER, SO THE SYNAPTIC
DPLIEWRONS ARE THERE AND ALSO
THERE ARE FUNCTIONAL DEFECTS IN
SYNAPTIC VESICLE CYCLING, THE
LIFE SPAN IS ALSO USED COMPARED
TO WILD-TYPE FLY, WILD-TYPE
TDP43 OVEREXPRESS CAUSES A
REDUCTION IN LIFE SPAN AND
MUTANT TDP43 OVEREXPRESS CAUSES
AN EVEN MORE DRAMATIC AREY DUKS
AND FOR AN ALS MODEL WE WANTED
TO SEE WHETHER WE CAN HAVE OR
DEVELOP ASSAYS THAT EXHIBIT
OTHER HIGHLIGHT LOCOMOTOR
DEFECTS CAUSED BY TDP43, AND
THERE ARE MANY WAYS TO DO THIS
IN THE FLY, MY FAVORITE ASSAY IS
THE LARVA TURNING ASSAY BECAUSE
IT'S QUICK, QUANTITATIVE, IT'S
VISUALLY VERY SATISFYING AND
COULD ALSO BE DONE BY
UNDERGRADUATE STUDENTS WHICH
WERE THE ENGINEER OF MY LAB WHEN
I WAS AT THE UNIVERSITY OF
ARIZONA.
SO JUST TO GIVE YOU AN EXAMPLE,
OVEREXPRESS OF WILD-TYPE TDP43
NEURONS CAUSES A LARVA TURNING
PHENOTYPE.
SO WE FLIP THE LARVA AND THEN WE
TAKE A TIMER AND COUNT LOW LONG
IT TAKES FOR THIS ANIMAL TO TURN
BACK AND START THE FIRST FORWARD
MOVEMENT AND AFTER THIS GUY
STOPS, I HOPE YOU CAN SEE THAT
THIS CAUSES THE ANIMAL TO DRAG
IT'S POSTERIOR END.
SO I WILL START THIS AGAIN, AND
I'M GOING TO ALSO START THE 1 ON
THE RATE, THAT ATE WHAT WE CALL
A PRODUCTIVE DRUG, SO I HOPE YOU
CAN SEE THAT THE 1 ON THE RIGHT
HAS ALREADY TURNED, WHILE THE 1
ON THE LEFT THAT HASN'T EATEN,
THE PROTECTIVE DRUG IS STILL
STRUGGLING.
SO WE USE THIS MODEL AND TRY TO
UNDERSTAND ALS FOR MULTIPLE
PERSECTIONAL ANALYSISSIVE USING
GENETIC SCREENS EITHER UNBIASED
OR CANDIDATE GENETIC SCREENS
USING DRUG SCREENS, USING THE
ALTERATIONS IN THIS ANIMAL, AND
WHEN WE'RE THINKING ABOUT TDP 43
AND GENERAL THINKING IN THE
FIELD, AND I TOLD THAT YOU TDP43
IS LOCATED IN THE NUCLEUS,
HOWEVER THE CONTEXT OF DISEASE,
IT LEAVES THE NUCLEUS, GOES INTO
THE CYTOPLASM AND ASSOCIATES
WITH VARIOUS COMPLEXES MOST OF
WHICH CONDANE VARIOUS RNA
BINDING PROTEINS AND AS
AQUENCEQUENCE OF THAT, THERE ARE
ALTERATIONS IN VARIOUS ASPECTS
OF RNA METABOLISM, INCLUDING RNA
TRANSLATION AND TRANSPORT.
AND WHILE MANY LABS ARE FOCUSING
ON THE NUCLEAR LOSS OF FUNCTION
CONSEQUENCES OF TDP 43, 1 OF
WHICH IS SPLICING DEFICITS, WE
ARE FOCUSING MORE ON THE
CYTOPLASMIC TOXIC ASPECTS OF TDP
43.
AND 1 OF THE HYPOTHESIS THAT HAS
BEEN PUT FORTH IN THE FIELD BY
COLLEAGUES MANY YEARS AGO, IS
THAT ONCE IN THE CYTOPLASM, AND
THAT CAN BE CAUSED BY STRESS
WHETHER IT'S INJURY OR MUTATION
OR AGING, TDP 43 CAN ASSOCIATE
WITH OTHER RNA BINDING PROTEINS
AND IN THE PROCESS CAN SEQUESTER
THESE PROTEINS AND SEQUESTER
COGNATE RNAs AND AS A
CONSEQUENCE THOSE RNAs ARE NOT
AVAILABLE TO THE TRANSLATIONAL
MACHINERY FOR PROTEIN SYNTHESIS
THIS WAS COINEDLET RIBOSTASIS
HYPOTHESIS, AND IT MAKES A LOT
OF SENSE, WE AND OTHERS HAVE
BEEN TESTING THIS HYPOTHESIS AND
I WILL SHOW YOU DATA THAT WE GOT
FROM SPRPTS TESTING THESE --
EXPERIMENTS TESTING THESE
HYPOTHESIS, BUT I WILL ALSO SAY
THAT WHILE THIS ALL MAY SEEM
REALLY INDIRECT, TDP43 MAY PLAY
A MORE DIRECT ROLE IN
TRANSLATION AND THAT IS
SUGGESTED BY EVIDENCE THAT TDP43
BINDING TO RAC 1, WHICH ALSO
BINDS TO THE RIBOSOME, AND
TDP43, WHICH ASSOCIATES WITH
PROTEINS SO THERE MAY BE MORE TO
TDP43'S ROLE IN TRANSLATION THAN
THESE SORT OF SEQUESTRATION THAT
CAN OCCUR IN THE CONTEXT OF
DISEASE.
TO TO ADDRESS THIS POSSIBILITY
MANY YEARS AGO, FORMER GRADUATE
STUDENT IN MY LAB, PERFORMED
POLYSOME FRACTIONATIONS FROM
FLIES OVER EXPRESSING WILD-TYPE
OR MUTANT TDP 43OT RIGHT BUT
ALSO FROM CONTROL FLIES AND ON
THE LEFT IT CAN LOOK AT
ENDOGENOUS TDP 43 AND YOU CAN
SEE THAT IT BASICALLY
DISTRIBUTES THROUGHOUT THE
GRADIENT SUGGESTING THIS PROTEIN
CAN HAVE A ROLE AS A NEGATIVE
REGULATOR AS WELL AS POSITIVE
REGULATOR OF TRANSLATION, AND
THE OVEREXPRESSION OF HUMAN
TDP43 EXHIBITED A SIMILAR
PATTERN WITHIN THE POLYSOMES.
TO FURTHER EXPLORE THE POTENTIAL
RELATIONSHIP BETWEEN TDP43 AND
TRANSLATION, 2 FORMER STUDENTS
IN MY LAB, WHILE AT THE
UNIVERSITY OF ARIZONA USE
GENETIC INTERACTIONS TO MODULATE
TRANSLATION, EITHER BY KNOCKING
DOWN 40 DP WHICH IS AN INHIBITOR
OF INITIATION OR OVEREXPRESSING
THE CONSTITTATIVELY ACTIVE F6
KINASE OF THE LEVEL OF
QUANTIFICATION.
SO WHAT YOU'RE LOOKING AT IS THE
LEVEL OF ASSAY, I SHOWED YOU
EARLIER, THE TAKE IS LONGER THAN
CONTROLS AND THIS IS RESCUED BY
KNOCKING DOWN FOUR BT OR
CONSTITTATIVELY ASSESSING
KINASE, CONSISTENT ALTOGETHER
WITH THE FACT THAT THE NOTION
THAT IF YOU PROMOTE TRANSLATION,
YOU CAN MITIGATE TDP43
ASSOCIATED LOCOMOTOR DEFECTS.
OTHER WAYS IN WHICH WE'RE
STUDYING THE ROLE, OR THE
CONNECTION, INVOLVEMENT OF TD43
IN TRANSLATION, IS USING
GENETICALLY ENCODED APPROACH
CALLED NONCANNONICLE TAGGING
DEVELOPED IN SHYOUMAN'S LAB
WHERE WE BASICALLY OVEREXPRESS A
MUTANT FORM OF TRNA SYNTHETASE
AND THIS MUTANT FORM OF
METHIONINE SYNTHETASE CANNOT
CHARGE THE IPADDA 9 ON TO NEW
PEPTIDES BUT CAN DO SO WITH
AN ANALOGUE WHICH IS CALLED ANL,
AND IF WE FEED ANL IN THE FEED
THEN WE OVEREXPRESS IT IN THE
RNA SYNTHETASE IN MOTOR NEURONS,
ALL THE NEWLY SIPGHT SIZED
PROTEINS NEURONS WILL NOW HAVE
NEWEL MADE PROTEINS STARTING
WITH A, IN L AND THE NICE THING
ABOUT ANL IS THAT IT CAN BE
LABELED USING CLICK CHEMISTRY
OTHERS EITHER WITH FLUORESCENT
MARKERS AND WE CALL THAT
APPROACH FUN CAT OR CAN BE
LABELED WITH BIOTIN AND CALL IT
BBONCAT, AND TURNS OUT WE ARE
CAT LOVERS SO WE USE FUNCAT AND
BONCAT, TO STUDY THESE ON NEWLY
SYNTHITIZED PROTEINS.
AND I HOPE THAT WHAT YOU CAN SEE
HERE, IS THAT AT THE NEURAL
MUSCULAR JUNCTION, THE
FLUORESCENT LABEL OF ANL PROTEIN
SYSTEM REDUCED COMPARED TO
CONTROLS.
WITH THE BONCAT APPROACH, WE CAN
PULL DOWN THE BIOATTENUATE
ANL PROTEIN AND DO MASS
SPECTROMETRY AND IERK DENTIFY
SPECIFIC CHANGES IN DOSE
PROTEINS THAT ARE NOT
SYNTHESIZED APPROPRIATELY IN THE
CONTEXT OF TDP 43.
THEN IN MOTOR NEURONS, THE
EXPERIMENTS ARE IN PROGRESS AND
THERE ARE A LITTLE BIT MORE
CHALLENGING BECAUSE, THERE ARE
FEW MOTOR NEURONS AND THE SIGNAL
TO NOISE RATIO IS CHALLENGING
BUT WE'RE WORKING ON IT.
AND I WILL SHOW YOU A LITTLE BIT
LATER DATA USING THIS,A PROACH
IN MUSCLE, WHERE -- THIS
APPROACH IN MUSCLE WHERE THE
DEFECT IS VERY CLEAR.
SO, THINKING ABOUT TRANSLATION,
THINKING ABOUT RNAs THAT
ASSOCIATE WITH TDP 43, AND ARE
DISREGULATED IN THE CONTEXT OF
TDP 43 PROTEIN OP A THY, WE OVER
THE YEARS WE IDENTIFY A NUMBER
OF RNAs THAT ARE ASSOCIATE
WIDE TDP43 AND DISREGULATED IN
MOTOR NEURONS IN THE CONTEXT OF
TDP43, OUR VERY FIRST TARGET WE
IDENTIFIED WAS THE CYTOSKELETAL
COMPONENT CALLED FORTUNE FLIES,
IS THE ORTHOLOG OF MAP 1 B IN
HUMANS WHICH IS A MICROTUBEULAR
ASSOCIATED PROTEIN THAT
STABILIZES PROTEINS AND AS YOU
CAN SEE USING THIS NEUROMUSCULAR
SYSTEM IN THE FLY, WE WERE ABLE
TO STUDY PROTEIN GENE EXPRESSION
IN THE VENTRAL CORD WITH ALL THE
MOTOR NEURONS THAT ARE
ALLOCATED, WE CAN LOOK AT THE
AXON, WE CAN LOOK AT THE
SYNAPTIC TERMINAL SO USING THE
SYSTEM, WE FOUND THAT PROTEIN,
IS REDUCED AT THE SYNAPTIC
TERMINAL BUT IS INCREASED IN THE
MOTOR NEURON CELL BODY AND IN
THE VENTRAL QUARTER AND THIS IS
QUANTIFIED HERE AND THIS IS
IMMUNO FLUORESCENCE OF FOOT
PROTEIN IN THE TDP43 EXPRESSING
ANIMAL, AND THAT WAS THE VERY
FIRST TIME WHEN WHICH WAS
BROUGHT TO MY ATTENTION THAT
THIS IS ALL NICE AND INTERESTING
BUT WHAT DOES THIS MEAN FOR THE
HUMAN DISEASE AND THE CLINICIAN
TOLD ME THAT.
I SAID OKAY, THATIA A GOOD POINT
SO THAT'S WHEN WE STARTED
COLLABORATIONS WITH COLLEAGUES
IN THE FIELD THAT WE'RE WORKING
WITH PATIENT TISSUES AND WE
STARTED LOOKING AT PATIENT
TISSUES OURSELVES, TO VALIDATE
THE TARGETS WITH WHETHER THE
TARGETS WERE IEE, AUDIENCE
DENTIFIED IN THE FLY OR ALSO
ALTERED IN THE CONTEXT OF HUMAN
DEC TO ULTIMATELY TEST THE
RELEVANCE OF OUR WORK TO ALS.
AND I HOPE YOU CAN SEE HERE,
THAT IN PATIENTS SPINAL CORDS,
MAP 1 B, IS ALSO UPREGULATED IN
MOTOR NEURON CELL BODIES AS WE
IDENTIFIED IT IN THE FLY.
AND THIS WAS INTERESTING AT THE
TIME BECAUSE SINGLE CELL RNA SEQ
WASN'T AVAILABLE, RIGHT, LIKE
THE IDENTIFICATION OF TARGETS IN
HUMAN TISSUES WAS MUCH HARDER SO
HAVING THE ABILITY TO DO THIS IN
THE FLY WAS REALLY HELPFUL IN
IDENTIFYING MARKERS OF PATHOLOGY
IN PATIENT TISSUES.
SOON AFTERRER WE FOUND A VESICLE
CALLED HSP8, AND WE FOUND IN
FLIES AS WELL AS PATIENT DERIVED
NEURONS THE EXPRESSION OF THE
SYNAPTIC TERMINAL WAS REDUCED
OVER EXPRESS OF IT IN THE FLY
RESCUED ALL THE PHENOTYPES
CAUSED BY TDP43, AND THIS IS
WHEN WE STARTED EXPANDING FROM
LOOKING AT TDP43 PROTEIN OP A
THY TO OTHER TYPES OF ALS, THAT
EXHIBIT THE PATHOLOGY.
SO WE'VE BEGUN TO EXPAND OUR
ABILITY TO SWIR ANSWER QUESTIONS
AND UNDERSTAND OTHER TYPES OF
ALS BY USING THESE OTHER TYPES
OF MODELS AND INTERESTINGLY
ENOUGH, CHAPERON WAS ALSO
ALTERED IN THE FLY MODELS AND
THIS IS AN EXPERIMENT WHERE
LOOKING AT THE UPTAKE OF TDP43,
AND YOU CAN SEE THIS IS REDUCED
IN THE CONTEXT OF THE MODEL BUT
OVER EXPRESS OF HSC7-4 RESCUED
THE VESICLE CYCLE.
SO THIS WAS A TYPE OF APPROACH
THAT WE USE LIKE SORT OF GOING 1
TARGET AT A TIME, IDENTIFYING
WHAT IS TDP43, REGULATING AND
WHAT DO THESE TARGETS -- HOW DO
THEY MEDIATE THE TOXICITY OF
TDP43, DO THEY AFFECT MOTOR
FUNCTION, DO THEY AFFECT
DEGENERATION, DO THEY AFFECT
SYNAPTIC FUNCTION, SO THE NEXT
TARGET WE WORKED ON IS THIS
DALLY LIKE PROTEIN, IT PLAYS AN
IMPORTANT PROTEIN IN WIN
SIGNALING AND IN HUMANS 1 OF THE
ORTHOLOGS WAS GPC 6, AND WHAT WE
FOUND IS THAT AT THE SYNAPTIC
TERMINAL THIS PROTEIN IS REDUCED
IN EXPRESSION.
THIS TIME WE ALSO STARTED
LOOKING AT TDP43 LOSS OF
FUNCTION TO REALLY COMPARE THE
OVEREXPRESSION MODEL WITH THE
LOSS OF FUNCTION MODELS, AND I
HOPE YOU CAN SEE THAT THE
SYNAPTIC TERMINAL WE HAVE A
SIMILAR EFFECT, A SLIGHT BUT
STATISTICALLY SIGNIFICANT
REDUCTION IN DLP EXPRESSION, BUT
TO OUR SURPRISE, THERE WAS A
COMPARTMENT SPECIFIC PHENOTYPE,
WHEN WE LOOKED IN THE VENTRAL
CORD WHERE THE MOTOR NEURONS ARE
LOCATED WE FOUND THAT DLP WAS
FORMING THESE ACCUMULATIONS THAT
WERE SPECIFIC TO THE TDP43
OVEREXPRESSION, THEY ARE NOT
PRESENT IN THE CONTROL, AND
THEY'RE SORT OF MIDDLE OF THE
ROAD IN THE LOSS OF FUNCTION.
AND SO, AGAIN WOORKSY WANTED TO
KNOW WHETHER GPC 6 IS ALTERED IN
PATIENT TISSUES, AND LOOKING AT
SPINAL CORDS FROM ALS CASES OF
TDP43 PATHOLOGY, WE FOUND THERE
IS ACCUMULATION OF GPC 6 TARGET
THAT IS QUANTIFIED OVER HERE IN
PATIENT TISSUE, ONCE AGAIN
HIGHLIGHTING THE POWER, THE
PREDICTIVE POWER OF THE FLY, OUR
ABILITY TO IDENTIFY MRNA TARGETS
THAT ARE ALTERED IN FLIES BUT
ALSO IN PATIENT TISSUES.
AND WE'RE CURRENTLY TRYING TO
FIGURE OUT WHAT IS THIS
COMPARTMENT SPECIFIC
DISREGULATION OF DLP MEAN, SO
FOR EXAMPLE, WE SEE THESE
ACCUMULATION IN THE CELL BODY,
DOING MEMBRANE FRACTIONATIONS,
WE FOUND THAT THE ENDOPLASMIC
RETIC LUMIS FRAGMENTED IN THE
CONTEXT, THERE ARE INTRACELLULAR
TRANSPORT DEFICITS THAT CAUSE
FRAGMENTATION IN THE ENDOPLAZ
MIDSIC RETIC LUMAND SURELY
ENOUGH WHILE DLPs ARE
ASSOCIATE WIDE THE CONTROL, YOU
CAN SEE IT'S KIND OF ENRICHED IN
THE FRAGMENTED ENDOPLASMIC RETIC
LUMTHAT WE FIND IN THE CONTEXT
OF TDP43 MUTANT MODEL AND THE
SYNAPTIC TERMINAL, THIS IS SUPER
INTERESTING BECAUSE DLP IN
RESPONSE TO CCLSTIMMULATION GOES
TO THE MEMBRANE AND THAT'S WHAT
IT SIGNALS TO WINLESS RECEPTORS
AND POTENTIALLY TDMP RECEPTORS
AND WE FIND THAT WHILE DLP CAN
DO THAT IN THE CONTEXT OF THE
MUTANT, THERE IS MUCH LESS OF IT
BECAUSE OF THE EFFECT OF TDP43
ON THE OVERALL EXPRESS OF THE
SYNAPTIC TERMINAL, SO WE
HYPOTHESIZED THIS HAS AN EFFECT
ON THE WINLESS SIGNALING AT THE
NEURAL MUSCULAR JUNCTION IS THIS
IS SOMETHING WE'RE CURRENTLY
WORKING ON.
SO DURING THIS 1 TARGET OF TIME
IS INTERESTING BUT WE ALSO
WANTED TO HAVE A BETTER
UNDERSTANDING OF ALL THE TARGETS
THAT TDP43 MAY REGULATE IN MOTOR
NEURONS AND SO 2 OF MY STUDENTS
DECIDED TO USE RIBOTAGGING TO
IDENTIFY ALL THE MRNAs THAT DO
NOT ASSOCIATE WITH RIBOSOMES
NORMAL IN THE CONTEXT OF TDP43,
SO THESE ARE JUST VISUALIZATION
OF DATA WE GOT THROUGH
RIBOTAGGING SHOWING YOU THAT THE
RNATHAT IS HAVE DIFFERENT LEVELS
OF ASSOCIATION WITH RIBOSOMES IN
METEOR NEURONS SPECIFICALLY WHEN
WE OVEREXPRESS THE TDP43,
FALLING TO SPLICING IF YOU DO A
TERM ANALYSIS, MANY ARE
ASSOCIATE WIDE SPLICING BUT
WHAT'S DIFFICULT TO FIGURE OUT
HERE IS YOU SEE MORE ARE MORE
ASSOCIATED WITH RIBOSOMES AND
SOME ARE LESS ASSOCIATE WIDE
HYPOSTUDIES OF MULTIPLE
ENDOCRINES SO IT'S DIFFICULT TO
MAKE SENSE OF WHAT'S GOING ON IN
REGARD TO THIS PATHWAY.
WHEN WE LOOK THEA ELECTRON
TRANSPORT, YOU CAN SEE THAT
COMPLEX 1 COMPONENT TRANSCRIPT,
ACTUALLY MORE ASSOCIATE WIDE
RIBOSOME IN THE CONTEXT OF
TDP43.
AND WE THINK THAT THAT'S
ACTUALLY A COMPENSATORY
MECHANISM BECAUSE OF DEFICITS IN
MITOCHONDRIA, THERE IS THE
COMPENSATORY MECHANISM, TRYING
TO MAKE MORE OF COMPLEX 1
PROTEIN AND TRY TO RESTORE
MITOCHONDRIA FUNCTION IN THIS
WAY, BUT WHAT WAS A LITTLE BIT
MORE CLEAR, AND EASY TO APPROACH
WAS A DOWN REGULATION OF THE
TRANSCRIPTS INVOLVED IN
TRANSLATION INITIATION.
SO YOU CAN SEE THAT MANY OF
THESE ARE LESS ASSOCIATE WIDE
RIEB SOSOMES IN THE CONTEXT OF
TDP43 CONSISTENT WITH TDP43
OVEREXPRESSION HAVING AN EFFECT
ON TRANSLATION AND INITIATION IN
GENERAL.
AND 1 OF THE OTHER INTERESTING
PATHWAYS THAT SHOWED A VERY
CLEAR EFFECT FROM THE
RIBOTAGGING EXPERIMENT, IS THE
PHOSPHATE PATHWAY.
THIS IS SORT OF AN ARMADILLOS OF
GLYCOLYSIS, THAT NEURONS USE AND
CELLS USE, WHEN THEY ARE
UNDERSTRESS, AND IT WAS REALLY
INTERESTING TO SEE THAT THE
COMPONENTS OF THE PENTINE
REGIMEN OS PHOSPHATE PATHWAY
WERE LESS PREDICTIVE.
THEY WERE LESS RIBOSOME
INCLUDING ZEST WHITE WHICH IS IN
FLIES THE RATE LIMITING ENZYMES
ALSO KNOWN AS GSPDIN THE
PATHWAY.
AND INTERESTINGLY ENOUGH JUST A
FEW YEARS BEFORE WE DID THIS
WORK, WE HAVE DONE A WHOLE
METABOLOMIC STUDY OF OUR FLY
MODEL AND FOUND THAT BOTH TBLI
COLSIS AND PHOSPHATE RELATED
METABOLITES WERE ALTERED WITH
PENTHOSPHOSPHATE WERE REDUCED
CONSISTENT WITH THE FACT THAT WE
MIGHT HAVE A DEFECT IN THE
PATHWAY POTENTIALLY BECAUSE THE
RATE LIMITING ENZYME GP D IS NOT
TRANSLATED SUFFICIENTLY AS TDP43
AS INDICATED BY RIBOTAGGING.
AND THE METABOLOMICS DATA WERE
CONFIRMED USING QPC R OF VARIOUS
-- I'M JUST GOING TO SHOW YOU
FROM HERE, IT'S HARD FOR ME TO
SEE HERE BUT FROM HERE THIS IS
G6 PC D IN CONTRAST TO THE
MUTANT SYSTEM HIGHER IN CONTROLS
AND IN PATIENT TISSUES, AS WELL
AS IN PATIENT DERIVED NEURONS,
THE TRANSCRIPT OF G6 PD IS
INCREASED AND YOU CAN --
PROBABLY LOOKING HERE AT
COMPENSATORY MECHANISM, WHERE
YOU KNOW WE HYPOTHESIZED THAT
THE TRANSLATION OF G6 PD'S
REDUCED THERE IS PERHAPS A
COMPENSATORY MECHANISM AT THE
LEVEL OF TRANSCRIPTION TRYING TO
MAKE MORE OF THIS TRANSCRIPT TO
ULRICH MATILY MAKE MORE OF THIS
PROTEIN.
AND WE ADDRESSED THIS ISSUE
USING -- WE ADDRESS THIS
QUESTION USING GENETIC
INTERACTION WHEREBY WE OVER
EXPRESS GSPD AND USING THAT
ASSAY I SHOWED YOU, YOU CAN SEE
THAT TDPOVEREXPRESSION CAUSES A
DEFECT, THERE WAS A HIGHER
TURNING TIME, BUT THIS TIME, IS
REDUCED FOR MUTANT TDP43 WHEN WE
OVEREXPRESS G6 PD, SO THIS
TURNING TIME IS SMALLER,
COMPARED TO TDP43 ALONE.
SUGGESTING THAT IF WE
OVEREXPRESS G6 PD, POTENTIALLY
RESTORE THE PENTOS-PHOSPHATE
PATHWAY WE SEE THIS IN THE
MODEL.
IT WAS NICE THAT CAME TOGETHER
WITH THE RIBOTAGGING APPROACH OR
THE METABOLOMICS APPROACH.
SO, THIS IS THE WORK WE'VE DONE
TO MODEL ALS WHICH IS
ESSENTIALLY TDP43 PROTEIN OP A
THY IN MOTOR NEURONS BUT I
HIGHLIGHTED TO YOU EARLIER THAT
ALS AND FTD ARE A SPECTRUM
DISORDER WHERE TDP43
CHARACTERIZEs A LARGE,
PROTEINS OP A THY CHARACTERIZES
A LARGE FRACTION OF PATIENTS AND
WE WANTED TO SEE WHETHER WE CAN
FOCUS ON THE MUSHROOM BODY
CIRCUIT IN THE FLY BRAIN, TO
MODEL DEMENTIA AND A FORMER VERY
TALENTED POST DOC IN MY LAB,
OVEREXPRESSED TDP43 IN MUSHROOM
NEURONS AND I HOPE YOU CAN SEE
HERE THAT EARLY ON IN THE LARVA,
MOST OF THE PROTEIN TAG WIDE YFP
IS IN THE NUCLEUS, BUT IN ADULT
FLIES, THE PROTEIN COMES OUT OF
THE NUCLEUS AND YOU CAN SEE THIS
HERE WITH THE DOUBLE ARROWS AND
THE ARROW, CAN YOU SEE THESE
MISLOCALLIZATION FROM THE
NUCLEUS TO THE CYTOPLASM THAT
IT'S OBSERVED IN PATIENTS.
SHE THEN DID AN ANALYSIS OF
NUCLEAR TO CYTOPLASMIC RATIO OF
TDP43, SO WITHOUT GOING THROUGH
ALL THE DETAILS OF THIS FIGURE,
I WANT TO DRAW YOUR ATTENTION TO
THE GRAPH.
IN LARVA MOST OF THE PROTEIN IS
IN THE NUCLEUS, IN YOUNG ADULTS
ABOUT DAY 1-3, A LOT OF THE
PROTEIN COMES OUT OF THE
NUCLEUS, SO YOU SEE THIS REDUCED
NUCLEUS TO CYTOPLASMIC RATIO,
BUT TO OUR SURPRISES, ALL FLIES
AROUND 50 OR SO DAYS OF AGE,
THERE ARE MORE NEURONS WITH MORE
TDP43 IN THE NUCLEUS THAN THE
CYTOPLASM WHICH SEEMED COUNTER
INTUITIVE.
ON HOWEVER WHEN WE PAID
ATTENTION AND STARTED COUNTING
THE REMAINING NEURONS IN THE FLY
BRAIN AT DAY 50, WE FOUND THAT
YOU CAN SEE THIS IN THIS GRAPH,
THERE WERE FEWER NEURONS ALIVE,
IN THE OLD BRAIN, SUGGESTING
THAT THE NEURONS THAT SURVIVED
ARE ABLE TO RETAIN TDP 43 IN THE
NUCLEUS, AND THEN WE CONFIRM
THIS WITH KASP A R PACE
STAININGS, THE CELLS THAT ARE
LOSING TDP43 IN THE NUCLEUS, AND
TDP43, MISLOCALLIZES TO THE
CYTOPLASM, THOSE CELLS ARE LOST
DURING LIFE SPAN AND HENCE THE
REDUCTION IN OVERALL NEURONS
WITH AGE.
WITH THEN LOOKED AT A NUMBER OF
PHENOTYPES INCLUDING AXONAL
DEGENERATION, SO IF YOU LOOK IN
TDP43 OVEREXPRESSING FLIES, YOU
CAN SEE A THINNING OF THE AXONS,
THIS IS THE CARTOON OF THE
MUSHROOM BODY CIRCUIT,
ALPHABETTA, GAMMA, NEURONS, AND
YOU CAN SEE THEY ARE THINNING
WITH AGE AND YOU CAN SEE THIS
QUANTIFIED OVER HERE AND WE ALSO
SEE AN INCREASE IN THE PUNTA
THAT THE TDP43 FORMS AND YOU CAN
SEE THIS QUANTIFIED OVER HERE,
SO DURING LIFE SPAN THESE AXONS
ARE DEGENERATING THE LOBES ARE
THINNER AND THE TDP43 PANTA ARE
ACCUMULATED AND INCREASED WITH
SIZE IN AGE.
WE THEN WANTED TO SEE IF WE CAN
MEASURE A BEHAVIORIAL ASSAY
THAT'S REALLY VANT TO DEMENTIA.
AND WE COLLABORATED WITH A CLOAG
AT ARIZONA WHO MADE THIS FOR
FLIES.
YOU MAY BE FAMILIAR WITH THIS
ASSAY FROM RODENTS, THEY'RE USED
TO QUANT TIE SPATIAL MEMORY AND
WHAT WE FOUND IS HERE'S A LITTLE
MOVIE.
WE PUT INDIVIDUAL FLIES IN THESE
LITTLE Y-MAZE AND WE IMAGE THEM
FOR 10 MINUTES THEN WE EVALUATE
THE MOVEMENT AND THE ABILITY TO
SHIFT ARMS IN IN PERIOD OF TIME.
AND WE FOUND THAT WHEN WE
MEASURED DISTANCE, AND THE
ALTASDINATION MEANING ABILITY TO
CHANGE ARMS CONSECUTIVELY, WE
FIND THAT ALTHOUGH THE OVERALL
MOVEMENT EXPRESSION IN MUSHROOM
BODY WAS HIGHER, THE
ALTASDINATION WAS REDUCED
CONSIST EPT WITH THE DEFECT IN
SPATIAL WORKING MEMORY IN THESE
FLIES.
AND THAT WAS THROUGH BOTH FOR
MALES AND FEMALES.
WE THEN LOOKED AT WHETHER THERE
ARE DEFECT IN SLEEP, AND JUST TO
FOCUS ON THE KEY POINT HERE IS
THAT TO OUR SURPRISE, WE FOUND
INCREASED SLEEP CAUSED BY TDP43.
WHICH IS KIND OF UNEXPECTED BUT
THEN AS I READ MORE ABOUT
PATIENT PHENOTYPES, IT TURNS OUT
PATIENTS WITH DEMENTIA DO SLEEP
MORE DURING THE DAY BUT THEY DO
EXHIBIT SLEEP FRAGMENTATION
DURING THE NIGHT.
AND THE ONLY TIME OR THE ONLY
PLACE WHERE WE IDENTIFIED SLEEP
FRAGMENTATION OF TDP43
OVEREXPRESSION IN BODIES IS IN
YOUNG MALES BUT YOU CAN SEE THAT
AT NIGHT THERE ARE MORE SLEEP
BOUTS BUT THERE ARE SHORTER.
SO THE SLEEP IS MORE FRAGMENTED
AT NIGHT.
LATTER ON THE PHENOTYPES BECOME
MORE COMPLEX, BUT AT LEAST THIS
1 POINT IN TIME, AT LEAST FOR
MAN'S EXPRESSING TDP43 AND
KENYAN CELLS WE COULD IDENTIFY
SLEEP FRAGMENTATION, SIMILAR TO
WHAT'S IN PATIENTS.
THERE IS ALSO A SLIGHT BUT
SIGNIFICANT REDUCTION IN LIFE
SPAN, WHICH WAS KIND OF
UNEXPECTED BUT ALSO INTERESTING
BECAUSE WE'RE ONLY MANIPULATING
KENYAN CELLS HERE, NOT ALL THE
NEURONS IN THE BRAIN.
AND WE ALSO USED A NONCANNONICLE
ACID APPROACH TO SEE WHEN TDP43
IN THE KENYAN CELLS CAUSES A
REDUCTION IN THE NEW PROTEIN
SYNTHESIS AND THIS IS THE FUN
CAT VERSION OF THIS EXPERIMENT
AND YOU CAN SEE THAT THAT THERE
IS A REDUCTION IN SIGNAL IN THE
CONTEXT OF TDP43, SO WE'RE
CURRENTLY USING THE BONCAT
APPROACH TO IDENTIFY WHAT ARE
NOT DONE CORRECTLY IN THIS
CIRCUIT.
SO THE REASON -- 1 OF THE
REASONS WE WANTED TO MANIPULATE
THE CIRC YOUS TO DEVELOP ALS
RELEVANT MODEL BECAUSE OF
DEMENTIA RELEVANT MODEL IS
BECAUSE WE WANT TO LOOK AT THE
OVERLAP AS WELL AS UNIQUE
FEATURES THAT MAY BE RELEVANT TO
ALS OR FTD SPECIFICALLY AND WE
DID AN OVERLAP OF THE MRNAs
THAT ARE ASSOCIATED WITH TDP43,
WANT TO DO THIS EXPERIMENT IN
MOTOR NEURONS VERSUS KENYAN
CELLS, IN THE DEMENTIA MODEL,
AND THE REASON I SHOWED YOU DLP
BEFORE IS BECAUSE WE ALSO FOUND
DLP AS A POTENTIAL TARGET OF
TDP43 IN THE MUSHROOM BODIES.
SO THIS MAY BE 1 OF THE THOSE
MRNAs THAT IS REGULATED BY
TDP43, BOTH IN ALS, AS WELL AS
IN THE CONTEXT OF FTD, WHILE
OTHER MRNARKs ARE SPECIFICALLY
REGULATED BY TDP43 MOTOR NEURONS
OR SPECIFICALLY IN THE MUSHROOM
BODY CIRCUIT, AND WE HOPE THAT
THIS APPROACH IS GIVING US THE
ABILITY TO IDENTIFY THE
COMMONALITIES BETWEEN THESE 2
FORMS OF THE SPECTRUM DISORDER,
AS WELL AS SPECIFIC
MANIFESTATIONS OF THESE 2.
SO, THIS IS DLP EXPRESSION IN
YOUNG VERSUS OLD FLIES AND IT'S
A LITTLE BIT DIFFICULT BECAUSE
OF AGE DLP KIND OF EXPRESSES
EVERYWHERE BUT WE CAN HIGHLIGHT
THE MUSHROOM BODY SPECIFICALLY
CAN DIFFERENT MARKIERS AND WHAT
WE FOUND IS THAT ALTHOUGH DURING
AGING DLP IN A NORMAL CONDITION
IS REDUCED IN EXPRESSION IS
REDUCED EVEN FURTHER BY TDP 43
OVEREXPRESSION, AND JUST LIKE IN
MOTOR NEURONS IN THE MUSHROOM
BODY CIRCUIT, DLP IS THOUGHT TO
PLAY A REEL KEEPSAKES IN
SIGNALING AS WELL AS SYNAPTIC
PLASTICITY.
AND THEN WE WANTED TO KNOW -- WE
TOOK ADVANTAGE OF POWER OF FLY
GENETICS AND WE WANTED TO KNOW
WHETHER OVEREXPRESSION OF DLP
CAN RESCUE TDP 43 DEPENDENT
PHENOTYPE, SO USING THAT ASSAY I
SHOWED YOU, YOU CAN SEE THAT DLP
OVEREXPRESSION RESCUES THE
MOVEMENT DEFICIT AND IT ALSO
RESCUES THE ALTASDINATION
DEFICIT.
AND THEN WE WERE ASKING
OURSELVES, OKAY, THIS WORKS
GREAT IN FLIES BUT HOW ABOUT IN
PATIENTS AND WE TRIED OUR HANDS
AT STAINING FOR THE ORTHOLOG
GP16 FTD PATIENT TISSUES BUT THE
STAINING IS VERY COMPLEX AND
HETEROGENEOUS ORY GENIUS, SO
THEN WE TOOK ADVABTAGE OF A DATA
SET THAT WAS GENERATED BY OUR
COLLABORATORS WHERE THEY DID
SINGLE NUCLEUS RNA SEQ, FROM FTD
PATIENT BRAINS AND WHAT THEY DID
IS THEY LOOKED WITHIN THE FTD
PATIENT BRAIN POPULATION AT
NEURONS THAT STILL HAVE TDP43 IN
THE NUCLEUS, VERSUS NEURONS IN
THAT PATIENT POPULATION THAT
LOST TDP43 IN THE NUCLEUS,
MEANING THEY HAVE THE PATHOLOGY
AND THIS CAN BE DETERMINED VIA
THE PRESENCE OF CRYPTICAXONS,
THAT ARE ASSOCIATED WITH LOSS OF
TDP43 FROM THE NUCLEUS, SO WHEN
DOING THIS KIND OF ANALYSIS,
DIFFERENTIAL GENE EXPRESSION
WITHIN THE FTD POPULATION,
BETWEEN NEURONS THAT HAVE NORMAL
TDP 43 IN THE NUCLEUS, VERSUS
TDP 43 PATHOLOGY, GUESS WHICH
WAS 1 OF THE BEST-WHICH WAS 1 OF
THE TARGETS, 1 OF THE MOST
DIFFERENTIAL EXPRESSED GENES WAS
GPC 6, WHICH WAS OUR DLP
ORTHOLOG.
SO ESSENTIALLY THIS IS TELLING
US THAT ALTHOUGH IT'S NOT
NECESSARILY THE SAME EXPERIMENT,
NOT THE SAME DIRECTION THAT IT
HAPPENS IN THE FLY, WE CAN LINK
DLP/GPC 6 TO TDP43 PATHOLOGY
FROM FLY ALL THE WAY TO PATIENT
TISSUES.
SO TO SUMMARIZE THIS FESTER PART
OF MY TALK, WHICH TALK TOOK MORE
THAN A THOUGHT, I WANTED TO
HIGHLIGHT TO YOU THE POWER OF
MODELING TDPPROTEIN OPERATING
GLOBALLYATHY IN THE CIRCUITS 1
THAT'S RELEVANT TO ALS AND 1
THAT'S RELEVANT TO FTD AND THE
DISCOVERIES WE MADE IN THE FLY
THAT MORE THAN 90% EVEN TO MY
SURPRISE AS A FLY GENETICIST
VALIDATED PATIENT TISSUES ALMOST
EVERY SINGLE TIME, WERE TARGETS
THAT HAVE TO DO WITH
CYTOSKELETON FUNCTION, MICROTUBE
Y'ALL ABILITY, SYNAPTIC VESICLE
CYCLE, METABOLISM AND WIN
SIGNALING, WITH -- WE'RE PROUD
OF THIS FTD MODEL BECAUSE IT
ALLOWS US TO DO ADDITIONAL
GENETIC SCREENS THAT ARE
SPECIFIC TO THE CIRCUIT AS WELL
AS DRUG SCREENS, AND DLPs, THE
FIRST TARGET WE FOCUSED ON,
BECAUSE IT ALLOWED US TO TIE
INTO THE 2 MODELS BUT THERE ARE
MANY, MANY MORE INTERESTING
TARGETS THAT ARE ALS SPECIFIC
VERSUS FTD SPECIFIC THATY WOO
WOULD LIKE -- THAT WE WOULD LIO
WORK ON.
WE SO THIS MISLOCALLIZATION IN
THE CELL DEATH OF THE FTD MODEL
AND WE WERE ABLE TO TIE IN THE
TARGETS THAT ARE DISREGULATED IN
THE FLY MODEL TO DATA FROM
PATIENT TISSUES WHERE THE SAME
TARGETS ARE ALSO ALTERED IN
NEURONS OF TDP43 PATHOLOGY AND
WHILE THESE MODELS OF
OVEREXPRESSION HAVE SIGNIFICANT
CAVEATS BECAUSE WE RECOGNIZE
THAT EVERY TIME YOU OVEREXPRESS
AN RNA BINDING PROTEIN THERE
WILL BE SOMEART FACTS, ON 1 HAND
IT WAS SATISFYING THAT WE WERE
ABLE TO VALIDATE IN-PATIENT
TISSUES, THE TARGETS WE FOUND IN
THE FLY, BUT AT THE SIEM TIME
WE'RE COGNIZANT THAT WE NEED TO
ALSO TAKE DIFFERENT APPROACHES
SO WE'VE BEEN USING A C RISPR
KNOCK-IN MODEL THAT REPLACES
TDPIN THE FLY WITH HUMAN TDP43,
WHETHER IT'S WILD-TYPE OR MUTANT
AND A VERY TALENTED STUDENT IN
MY LAB WANTS ON DO SINGLE CELL
RNA SEQ AND LOOKA THE EFFECTS OF
TDP43 PROTEIN OP A THY, DURING
AGING.
AND SOME OF YOU IN THIS ROOM
HELPED US DO THESE EXPERIMENTS,
SO THANK YOU VERY MUCH FOR THAT.
AND THIS IS THE LITERALLY DATA
AS OF MONDAY IT WAS REALLY
EXCITING TO SEE THAT OUR VERY
FIRST ATTEMPT WITH A LOT OF HELP
FROM ELLA'S LAB, ROSARIO IN
PARTICULAR, WE WERE ABLE TO
GENERATE DATA THAT LOOKED LIKE
IT REALLY ISOLATED BEAUTIFUL
CLUSTERS IN THE FLY BRAIN AND
FOR THIS 1 WE WANT ON DO A QUICK
DIFFERENTIAL GENE EXPRESSION IN
THE KENYAN CELLS WHICH REGULATE
SPATIAL MEMORY, SLEEP AND OTHER
BEHAVIORS THAT ARE RELEVANT TO
DISEASE, AND INTERESTINGLY
ENOUGH, WE FOUND SOME
DISREGULATED GENES SOME OF WHICH
ARE LINKED TO LEARNING AND
MEMORY SUCH AS DANCE SHOWN HERE.
SO THIS IS VERY NEW, WE'RE JUST
BEGINNING TO UNDERSTAND THAT,
BUT JUST TO GIVE YOU AN IDEA
THAT WHILE THE OVEREXPRESSION
MODELS HAVE TAUGHT US A LOT,
THAT VALIDATED IN PATIENT
TISSUES, WE ARE ALSO USING OTHER
APPROACHES SUCH AS THIS CRISPR
KNOCK INS TO UNDERSTAND TDP43
MECHANISMS, AND SO SINCE I ONLY
HAVE 13 MINUTES I HAVE 2 OTHER
STORIES I WANTED TO TALK ABOUT,
1 IS IN MUSCLES AND THE OTHER IS
METABOLISM SO I DON'T IF I
SHOULD TAKE A VOTE?
GO FOR IT.
>> METABOLISM BUT THAT'S THE
SECOND 1.
SO.
HOW MANY PEEP WANT TO SEE
METRICS TAB -- HOW MANY PEOPLE
WANT TO DO METABOLISM.
I WILL DO THIS, THIS IS SO COOL,
THE MUSCLE BON, NCAT DATA IS
AMAZING, SO LOOK AT THIS, YOU
OVEREXPRESS TDP43 IN THE
MUSCLES, THIS IS THE NEWEL
SYNTHESIZED PROTEIN IN THE
NORMAL MUSCLE, THIS IS REDUCTION
IN SIGNATURES WILL NAING CONTEXT
OF TDP43, AND THIS IS THE
VOLCANO PLOT OF THE BIOATTENUATE
PROTEINS SO THERE'S A LOT OF
THINGS THAT ARE ALTERED AND MANY
OF THEM, ARE LISTENED TO
METABOLISM, WHETHER IT'S TCA
CYCLE OR LIPID METABOLISM,
OROXIDATIVE STRESS, AND THIS IS
SOMETHING THAT WE'RE JUST NOW
BEGINNING TO LOOK AT AND
VALIDATE.
THERE ARE A NUMBER OF PHENOTYPES
THAT WE SEE IN THE MUSCLE.
AND 1 OF THE THINGS THAT WE SEE
IS A REDUCTION IN FRAGILE X
MENTAL RETARDATION PROTEIN, I
WAS TELLING SOMEONE THAT I
STARTED MY CAREER IN FRAGILE X
SYNDROME AND I MOVED TO AGE
DEPENDENT NEUROLOGICAL DISORDERS
ONLY TO FIND A CONNECTION
BETWEEN THE RNA BINDING PROTEINS
THAT PLAY A ROLE IN THE AGE OF
NEURODEGENERATION RESPECTIVELY
SO WE FOUND THAT FMRP IS
OVEREXPRESSED IN DDP43, SO AND
IF WE OVEREXPRESS THEM, WE SEE
THIS, AND RESCUE THE LEATHALLITY
IN TDP43 MUSCLE.
WE SEE STRUCTURAL DEFECTS AND IN
COLLABORATION WITH DR. CRUZ'S
LAB, WE FOUND IN THE MASS MODEL
OF TDP43 PROTEIN OP A THY, THE
ORTHOLOG, CALLED FXR1 IS REDUCED
IN THE MOUSE, SO THERE'S THE
FLY, THE MOUSE AND ALSO THE
HUMAN SO LOOKING AT MUSCLE,
POSTMORTEM SAMPLES FROM ALS
PATIENTS, WE CAN SEE WITH THIS
EXCEPTION HERE, A SPORADIC CASES
THAT HAVE TDP43 PROTEIN
OPERATING GLOBALLYATHY, THE
MUSCLE ALSO OF FMRP, IS REDUCED
IN PATIENT SAMPLES, HIGHLIGHTING
THE FLY TOA IDENTIFY TARGETS
THAT ARE REFINED WITH THE
PATIENT TISSUES AND USING
GENETIC APPROACHES WE'VE SHOWN
THAT RESTORING FMRP FUNCTION WE
CAN RESCUE THE TDP 43 ASSOCIATED
PHENOTYPES SO NOWIME READY TO GO
TO METABOLISM, LET'S SEE HOW
FAST CAN I SPEAK?
REALLY FAST.
SO THIS PROJECT CAME ABOUT BY
ACCIDENT.
ON 1 HAND, MY YOUNGER DAUGHTER
WHO WAS A TEENAGER AT THE TIME
WAS OBSESS WIDE METABOLISM AND
WHAT YOU SHOULD EAT TO MAINTAIN
YOUR FIGURE AND THEN I HAD A
GRADUATE STUDENT IN THE LAB WHO
WAS INTERESTED IN THE EEIVETS OF
FOOD AND DIFFERENT COMPONENTS OF
THE FOOD ON TDP43 PHENOTYPES SO
THIS PROMPTED STUDYING THE
METRICS TAB O LOAM OF ANIMALS
WITH TDP43 PROTEIN OP A THY, BUT
IT WAS MOTE VIETED BY CLINICAL
OBSERVATIONS THAT SHOWED THAT
PATIENTS HAVE DEFECTS IN
METABOLISM ARE HYPOMETABOLIC
SPECIFICALLY AND HONESTLY I
DIDN'T REALLY UNDERSTAND WHAT
THAT MEANT BECAUSE THEY WERE
REFERRING TO THIS AS THE
ORGANISMAL LIMP BUT NOBODY COULD
TELL ME WHAT HAPPENED AT THE
LEVEL.
SO THE FLY GAVE US AN
OPPORTUNITY TO LOOK AT METABOLIC
CHANGES AT THE CELLULAR LEVEL.
SO A FORMER VERY TALENTED
STUDENT IN MY LAB, PURSUED THIS
METABOLOMIC APPROACH AND I
SHOWED YOU THIS GRAPH THAT ARE
CHANGES IN THE GLYCOLYSIS, AND
IN PYRUVATE SPECIFICALLY
SUGGESTING THIS THE HIGHER GLIEK
LYTIC OUTPUT AND THIS WAS ALSO
IDENTIFIED IN TISSUE CULTURE
MODELS AS WELL AS IN SOME MOUSE
MODELS.
I SHOWED YOU THIS FIGURE BEFORE,
WE VALIDATED USING QPC R, RANGES
IN THE LEVELS OR THE EXPRESSION
OF RATE LIMITING ENZYMES BOTH IN
GLYCOLYSIS, AS IN FLIES AS WELL
AS IN PATIENT TISSUES AND IMN,
SO CONSISTENTLY ACROSS MODELS I
FOUND AN UPREGULATION OF PFK
TRANSPORRATE APPROXIMATE
TENTIALLY WITH ENHANCED
GLYCOLYSIS.
HOWEVER, TO OUR SURPRISE WHEN WE
USE THE LARVA RETURNING ASSAY TO
MODULATE GLYCOL SIT EAR
EXPRESSING OR KNOCKING IT DOWN
WE FOUND THAT IN THE CONTEXT OF
PDFTREY APPROXIMATE, WE PFK
RESCUED THE LOCOMOTOR DEFECT,
EVEN IF THE TRANSCRIPT IS
HIGHER, IT OVEREXPRESSING PFK
CAN RESCUE, SUGGESTING TO US
THAT PFK UPREGULATION AT THE
TRANSCRIPT LEVEL MUST BE A
COMPENSATORY MECHANISM,
POTENTIALLY TO MAKE UP FOR ATP
PRODUCTION DEFICITS CAUSED BY
MITOCHONDRIA DEFECTS.
SO THEN WE WANTED TO KNOW -- OH
I FORGOT TO MENTION THAT WE,
PROACH THIS PROBLEM, ALSO WITH I
DIETARY APPROACH SO IF YOU FEED
THE HIGH SUGAR DIET TO A NORMAL
FLY, THAT'S NOT VERY GOOD.
THAT FLY WILL HAVE AN EFFECT ON
LOCOMOTOR FUNCTION AS WELL AS
LIFE SPAN.
NOT IN A GOOD WAY, BUT IF YOU
FEED A HIGH SUGAR DIET TO AN ALS
FLY, THERE WILL BE AN
IMPROVEMENT IN LOCOMOTOR
FUNCTION AND INCREASING LIFE
SPAN AND ACHIEVE THAT AS WELL AS
GENETICALLY, BY OVEREXPRESSING
HUMAN GLUCOSE TRANSPORTER JUST
IN METEOR NEURONS, SO OVERALL IT
LOOKS LIKE, ENHANCING GLYCOLYSIS
IN MOTOR NEURON, ALTHOUGH IT MAY
BE A DESPERATE MEASURE, AND MAY
NOT BE, PER SE, IT IS HELPING
SURVIVE AND FUNCTION BETTER.
SO THEN WE WANTED TO KNOW HOW
DOES THIS HAPPEN?
PFK IS AN ENZYME THAT HAS AN ATP
BINDING DOMAIN THAT CAN BE
REGULATORY AND ANOTHER 1 THAT'S
CATALYTIC.
SO THE FIRST THING WE DID IS WE
MADE NEW LINES AND WE
OVEREXPRESSED PFK AND WE FOUND
THAT THAT CAUSED AN IMPROVEMENT
IN LOCOMOTOR FUNCTION USING THE
LARVA TURNING ASSAY BOTH FOR
WILD-TYPE AS WELL AS MUTANT
TDP43.
WE ALSO FOWBD THE SURVIVAL OF
THE FLY WAS INCREASED BY
WILD-TYPE PFK OVEREXPRESSION
BOTH FOR WILD-TYPE AS WELL AWE
MUSEUM TABT TDP43.
AND THEN WE WANTED TO MAKE A
TRANSGENE WHERE THE CATALYTIC
ATP BINDING SITE IS ALTERED, SO
THIS FORMER STUDENT IN MY LAB,
DID SOME MODELING AND WE
GENERATED TRANSGENIC FLIES THAT
OVEREXPRESSED THIS MUTANT FORM
OF PFK THAT IS CATALYTICALLY
INACTIVE, SO YOU CAN SEE THAT
WHEN WE OVEREXPRESS THIS FORM OF
PFK WE DO NOT RESCUE THE
LOCOMOTOR PHENOTYPE AND WE DO
NOT RESCUE THE SURVIVAL
PHENOTYPE.
CONSISTENT WITH THE IDEA THAT
THE ATP BINDING SITE OR THE
CATALYTIC ACTIVITY OF PFK IS
REQUIRED FOR IMPROVING TDP43
ASSOCIATED PHENOTYPES.
AND THEN WE WANTED TO KNOW A
LITTLE BIT MORE ABOUT HOW MIGHT
THIS HAPPEN AND WORK IN DANIEL
RAMS ON LAB AT YALE HAS SHOWN
MANYIERS AGO THROUGH AN
INTERESTING ACCIDENT THAT PFK
FORMS PUNTA UNDER THEOXIDATIVE
STRESS.
IT TELLS A NICE STORY ABOUT A
STUDENT WHO S&P DOING A SCREEN
AND THE STUDENT LEFT THE COVER
SLIP ON THE WORM FOR A LITTLE
LONGER AND THE WORM WAS
SUGGESTED TOOXIDATIVE STRESS AND
THAT CAUSED PFK INTO THE
SYNAPTIC TERMINAL AND IT TURNS
OUT THIS CAN BE SMOOTH, IN YEAST
AS WELL, WHERE ANY
IMMUNOPRECIPITATION HAVE SHOWN
THAT A NUMBER OF OTHER DPLIEK CO
LYTIC ENZYMES AND CHAPERONS AND
EVEN TRANSLATION FACTORS CAN
ASSOCIATE WITH PFK PUNTA INIEST
UNDER STRESS SO THEN WE WANTED
TO KNOW WHETHER THIS MIGHT BE
HAPPENING IN OUR MODEL, SO THE
FIRST QUESTION WAS, DOES PFK
FORM PUNTA, AT THE SYNAPTIC
TERMINAL UNDER NORMAL CONDITION
AND THE NEXT QUESTION IS DO
THOSE PUNTA, ARE THOSE PUNTA
DIFFERENT IN IS THE ASSUM
BLEOMYCIN OF PUNTA DIFFERENT IN
THE CONTEXT OF TDP43 PERHAPS TO
COMPENSATE FOR ENERGY ENERGY D.
SO A NEW STUDENT IN MY LAB WITH
THE NEUROSCIENCE PROGRAM WHO'S
VERY TALENTED USED A STED HIGH
RESOLUTION MICROSCOPE TO LOOK AT
PFK, THIS IS CRISPR KNOCK IN
WHERE WE GIVE THE PFK IN THE
ENDOGENOUS CONTEXT SO WE CAN SEE
PFK IN THE MUSCLE, SO YOU SEE
THAT GREEN PUNTA THAT'S THE
MUSCLE, AND THEN THE RED
STAINING HIGHLIGHTS THE SYNAPTIC
TERMINAL AND YOU CAN SEE THAT
WITHIN THE SYNAPTIC TERMINAL,
THERE ARE PFK PUNTA AND THERE
UNDER NORMAL CONDITIONS AND WE
WERE EXCITED ABOUT THAT, AND
THEN WE STARTED LOOKING AT
TDP43, EXPRESSING FLIES AND WE
FIND THAT THERE ARE PUNCTA, SO I
DON'T HAVE AN ANSWER YOU EXCEPT
TO SAY THEY LOOK A LITTLE
DEFINITE AND WE'RE TRYING TO
FIGURE OUT WHAT MEANS.
AND I WOULD SAY, IT LOOKS LIKE
TDP43 ALTERS THE PFK PUNCTA, AND
THEN WE WENT TO USE AMERIS TO
LOOK AT THIS AND BEGIN TO
QUANTIFY THEM.
SO HERE IS PFK IN A NORMAL
SYNAPTIC TERMINAL SO ACTUALLY
THIS IS A QUESTION I HAD FOR
YOU, IF YOU SEE A DIFFERENCE
BECAUSE I SEE A DIFFERENCE BUT I
WOULD LIKE TO KNOW WHAT YOU SEE.
SO HERE'S A CONTROL, AND HERE'S
A TDP43 FREE RADICALS,
EXPRESSING ANIMAL.
QUANTIFICATION IS IN PROGRESS
AND IT'S SO NEW THAT I'M NOT
QUITE SURE WE'RE READY BUT
VISUALLY, I'LL BE CURIOUS IF YOU
ALL SEE SOMETHING SO WE CAN TALK
ABOUT IT BECAUSE I DO SEE
SOMETHING.
AND THEN THE LAST SLIDE I HAVE
IS HOW MIGHT THIS WORK?
SO THE WHEEL IDEA THAT PFK IS IN
THE TERMINAL IT MAY BE FORMING
GLYCOLYTIC PERIODS IN RESPONSE
TO STRESS.
WE TESTED THAT IDEA BY USING THE
FM143 DISCIPLINARY OPTIC ASSAY
WHICH IS A MEASURE OF SYNAPTIC
ASSAY IN THE SIGNIFYITOSEIS AND
YOU CAN SEE IN THE CONTEXT OF
TDP43, THE SIGNAL IS REDUCED
BECAUSE THERE ARE REDUCES IN THE
VESICLE IN THE SIGNIFYITOSEIS.
OVEREXPRESSION OF PFK RESTORES
THE SIGNAL IN THOSE MUTANTS
SUGGESTING THAT PFK
OVEREXPRESSION RESTORES THE
ENDOCYTOSIS, AND THIS IS MY LAST
SLIDE.
JUST TO SORT OF PUT IT
ALTOGETHER, WE ARE FOCUSING ON
THE -- THESE GLYCOLYTIC BODIES.
WE ARE ISOLATING SYNAPTICSOMES,
WE ARE DOING PROTEOMICS ON THESE
TO FIGURE OUT WHAT ARE THE PFK
ASSEMBLIES, HOW MIGHT THEY BE
DIFFERENT AND COLLABORATING WITH
THE COLLEAGUE WHO DOES RYE O EM
TO LOOK AT CRYO STRUCTURES IN
THE CHANGES OF THESE MOLECULES
AT THE SYNAPSE BUT WE DO -- WE
HAVE OBSERVED AN UPREGULATION
THAT'S COMPENSATORY.
THAT IS PROTECTIVE AND THE
CATALUTTIC ACTIVITY OF PFK IS
REQUIRED FOR THIS
NEUROPROTECTIVE ACTIVITY AND IT
DOES SO BY MITIGATING SYNAPTIC
VESICLE DEFICITS.
AGAIN, THE QUESTION STILL
REMAINS, WHAT DO THESE PFK ASSEM
MYELINS DO, WHAT ARE THEY, HOW
DO THEY CHANGE IN THE CONTEXT OF
TDP 43 AND THIS IS WORK THAT
WE'RE DURANNENTLY DOING AND HOPE
TO APPROACH THIS WITH BIOCHEMIST
RADIOY AS WELL AS CRYOEM
APPROACHES AND WITH THAT I WANT
TO THANK YOU FOR YOUR ATTENTION
AND APOLOGIES I WENT TO THE FULL
HOUR.
BUT I DO WANT TO SAY THANKS TO
THE NEW PEOPLE THAT I RECRUITED
IN MY LAB AT PENN STATE, VERY
WONDERFUL COLLABORATORS ACROSS
THE COUNTRY, ALL THE WAY FROM
ARIZONA TO PENN STATE AND
BELGIUM, FORMER STUDENTS IN THE
LAB WHO CONTRIBUTED TO THIS
WORK.
AND THE FUNDING AGENCIES AND,
PATIENTS AND THEIR FAMILIES AND
SPECIAL THANKS TO THE NIH WHO
NOT ONLY FUNDED THIS WORK BUT
ALSO FUNDED THE TRAINING OF THE
PEOPLE THAT DID THIS WORK.
SO THEY WILL CARRY IT FORWARD.
SO THANK YOU.
[ APPLAUSE ]
>> --I DON'T WANT TO CURTAIL ANY
TALKS WITH THESE BUT QUESTIONS?
>> SO WONDERFUL TALK, ROSS
OROXIDATIVE STRESS HAS BEEN
IMPLICATED IN ALS QUITE A BIT.
IF YOU TREAT ANTIOXIDANT LIKE
NAC, HOW MUCH OF THE PATHOLOGY
CAN YOU REVERSE?
>> WE TRY TO FEED AD4 WHICH I
GUESS IS THE SAME THING, THERE
ARE -- THERE IS A PHENOTYPIC
RESCUE, HOWEVER, WE WERE NOT
ABLE TO IDENTIFY ROSS, IN A
CONSISTENT WAY.
SO, EVERY TIME WE USED VARIOUS
APPROACHES TO MEASURE FREE
RADICALS IN OUR PREPARATIONS,
THE RESULTS WERE TOO VARIABLE TO
TELL, AND IN SPEAKS TO EXPERTS,
IT SEEMS LIKE IT MAY BE
TRANSIENT AND VERY DIFFICULT,
TOO, SO I THINK IT'S HAPPENING
BUT WE HAVEN'T BEEN QUITE ABLE
TO CAPTURE IT, AND SIMPLY
FEEDING AN ANTIOXIDANT IS
PROTECTIVE, BUT I AM NOT SURE
HOW THAT HAPPENS.
NTHANK YOU.
>> THANKS.
>> SO SINCE YOU ASKED OUR
OPINION ON THE LAST EXPERIMENT,
THE QUESTION --
>> DO YOU SEE A DIFFERENCE?
>> WELL, LET ME ASK YOU A
QUESTION FIRST BEFORE I GIVE AN
ANSWER.
HAVE YOU TRIED DOING THAT LIVE
-- IS THIS -- I'M ASSUME
THANKSGIVING IS FIXED TISSUE?
>> YEAH.
>> HAVE YOU TRIED DOING IT LIVE
BECAUSE PARTICULARLY IF YOU
THINK THAT THESE ARE YOU KNOW
CONDEN SITES AND SO FORTH YOU
OFTEN GET LARGER EFFECTS ON THE
DYNAMICS THAN YOU GET ON STEADY
STATE SO SOMETHING LIKE EITHER
DOING IT IN LIVE IMAGING OR
DOING SOMETHING LIKE FRAPP OR
PHOTO ACTIVATION MAY GIVE YOU A
MUCH CLEANER ANSWER, MAYBE THE
DYNAMICS OF THINGS GOING IN AND
OUT THAT MATTERS MORE THAN
WHETHER YOU SEE THE AGGREGATES.
>> WE DID THINK THIS EXPERIMENT.
>> IT'S VERY HARD I REALIZE.
>> IT'S VERY HARD, SO AT PENN
STATE, AT ARIZONA, I USED TO
HAVE AN UPRIGHT MICROSCOPE WITH
IMMERSION LENS.
PENN STATE DOESN'T HAVE THIS, I
HAVE TO GO TO ANOTHER CAMPUS BUT
WE WOULD DO THAT.
MY STUDENT SARAH AND I TALKED
ABOUT THIS AND SAID WE WOULD
LIKE TO DO THAT.
WE WOULD ALSO LIKE TO DO TISSUE
AND MACROPHAGESSOGRAPHY AND LOOK
AT THESE STRUCTURES BUT THE
THING I WE WEREN'T QUITE SURE
THAT FPK IS THERE BECAUSE WHEN
YOU USE THE REGULAR CONFOCAL,
IT'S THERE, BUT WE WANTED A
HIGHER, WE WANTED TO KNOW USING
THE HIGH RESOLUTION WHETHER WE
REALLY DO SEE TRUE FFK PUNCTA,
THAT'S WHY WE WENT THERE FIRST
BUT I AGREE WITH YOU THE LIVE
DYNAMICS OF THIS IS REALLY WHAT
IT'S GOING TO BE INTERESTING.
SO, YES, THANKS.
BUT DO YOU SEE A DIFFERENCE OR
NO?
>> I MEAN, I -- RIGHT.
IN LOOKING AT 1 PICTURE, RIGHT
WHAT CAN YOU SAY, BUT IF YOU --
YOU COULD IMAGINE LOOKING THE
AMOUNT OF CLUSTERING AT VARIOUS
LEVELS, THERE'S WAYS TO QUANTIFY
THAT THAN JUST LOOKING AT
PUNCTA, FOR LOOKING FOR IMMUNO
GENERATED AITY AND THINGS LIKE
THAT.
>> SO I WILL GIVE YOU WHAT I
THINK, THIS AREA LOOKS LIKE IT
HAS FEWER PUNCTA, THERE ARE
AREAS IN THE SYNAPTIC TERMINAL
WHERE PFK'S NOT AS MUCH
LOCALIZED AT THE MEMBRANE AS IT
IS IN THE CONTROL, YOU KNOW
QUANTIFICATION IS IN PROGRESS.
BUT WHEN I SAW THIS, I WAS SUPER
EXCITED AND WE DID THE BLIND
CEST OF EVERYBODY IN MY LAB, AND
SAID DO YOU SEE A DIFFERENCE AND
EVERYBODY DID WITHOUT TELLING
THEM WHAT IT WAS, SO, I'M
HOPEFUL.
THERE WILL BE A DIFFERENCE WHEN
WE DO QUANTIFICATION.
>> THANK YOU.
FOR THE TALK.
I WAS WONDERING ABOUT THE
MECHANISM OF THIS AGGREGATE LIKE
FORMATION STHERE ANY POST
TRANSLATIONAL MODICTION IN THE
PROTEIN THAT IS LIKE TRIGGERED
BY THE OTHER AGING OR BY STRESS
OR SOMETHING ELSE?
WHICH KIND OF SWITCHES THIS FROM
NORMAL --
>> THERE ARE SO POST
TRANSLATIONAL MODIIVESES AND NEW
1S ARE COMING ONLINE EVERY DAY
SO PHOSPHORYLATION WAS THE FIRST
1 THAT WAS DETECTED AND THEN
THERE IS ACETYLATION AND THEN
THERE IS SIMULATION AND THERE IS
ANOTHER 1 THAT I JUST LEARNED
ABOUT, CITRONNULATION OR
SOMETHING LIKE THAT, THERE IS
MULTIPLE POST TRANSLATIONAL
MODIFICATION
MODIFICATIONS THAT PLAY A ROLE
AND I WILL LET OTHER PEOPLE WHO
WORK ON STRUCTURES KNOW MORE
ABOUT THAT.
>> THANK YOU.
>> THANKS.
>> HI, SO VERY INTERESTING TALK,
I HAVE LIKE COUPLE OF QUESTIONS.
SO, THE FIRST QUESTION IS,
REGARDING YOUR FUNCAT
EXPERIMENT.
I WAS JUST WONDERING, HOW WAS
THE EXPERIMENT DONE, WAS LIKE
THESE IMMUNO ASSAYS SKIPPED IN
THE MEDIA FOR A PERIOD OF TIME
OR WAS IT LIKE A POSITIVE --
>> WE TRIED DIFFERENT
APPROACHES, RIGHT BECAUSE YOU
CAN DO A TIME COURSE, WE REALIZE
THAT IN ORDER TO REALLY
UNDERSTAND WHAT'S GOING ON AND
OPTIMIZE THIS EXPERIMENT, IT'S
BETTER TO START WITH A CHRONIC
TREATMENT, SO THE EXPERIMENTS I
SHOWED YOU ARE THROUGHOUT LARVA
TIME, SO ABOUT 3, 3 AND HALF
DAYS OF FEEDING THE ANL, IF YOU
FEED IT LESS, THE SIGNAL TO
NOISE RATIO IS NOT AS GOOD, SO
WHY WOULD OPTIMIZING THIS
CHRONIC EXPOSURE, WE HOPE TO GO
BACK TO THOSE MORE ACUTE OR TIME
COURSE EXPERIMENTS.
>> YES, SO FOLLOWING THAT UP, SO
WHAT YOU ARE SIGNAL IS SHOWING
THAT IN THESE MUTANT MODELS, YOU
HAVE LESS OF NEW PROTEINS BEING
MADE?
>> YEAH.
>> AND ALSO YOU ARE SHOWING SOME
KIND OF ENERGY DEFICIT SO YOU'RE
BASICALLY SAYING THERE'S A
MITOCHONDRIA DEFECT BUT HAVE YOU
LIKE CHECKED IT DIRECTLY?
>> YOU MEAN LIKE FUNCTIONALLY
WITH THE MITOCHONDRIA
FUNCTIONAL?
>> FOR COMPLEX 1 FUNCTION OR --
>> I WE DON'T KNOW HOW TO DO
THAT, WE WOULD HAVE TO WORK WITH
-- WE DID THINK ABOUT THAT TO
LOOK AT COMPLEX 1 FUNCTION AND
THE EXPECTATION WOULD BE THAT
IT'S ALTERED JUST BECAUSE ALTER
INDEED THE NEURODEGENERATIVE
DISEASES BECAUSE OF THE COMPLEX
1 COMPONENT.
WE HAVE DONE TMRE LOOKING AT
MEMBRANE POTENTIAL OF
MITOCHONDRIA AND THAT'S ALTERED
SO WE KNOW THERE'S ALTERATIONS
ALSO THE STRUCTURE AT DATA, I
KIND OF BRUSHED OVER THAT BUT
ULTRA STRUCTURALLY THE
MITOCHONDRIA ARE REALLY WEIRD,
LIKE IN THE MUSCLE WE ACTUALLY
SEE SOME MITOCHONDRIA THAT ARE
LOOK LIKE THEY'RE ENGULFING
RIBOSOMES SO THEY'RE JUST VERY
WEIRD BUT THE EXPECTATION WILL
BE THAT THE FUNCTIONAL ASSAY
WOULD DEVELOP A DEFECT.
>> OKAY, SO SORRY 1 LAST
QUESTION, --
>> SO ACTUALLY IN THE INTEREST
OF TIME, THERE WILL BE
OPPORTUNITIES FOR FURTHER
DISCUSSION OUTSIDE FOR THE
RECEPTION.
I WANT JUST TAKE 1 LAST QUESTION
AND THEN WE RESILIENCE TIRE TO
THE RECEPTION FOR FURTHER
QUESTIONS AND DISCUSSION.
>> OKAY, THANKS.
>> DON'T LIVE LEAVE, I CAN
ANSWER YOUR QUESTION LATER.
>> THIS IS AICH SIMPLE 1 I
THINK, YOU SHOWED US A NUMBER OF
CASE WHERE IS YOU HAVE REDUCED
PROTEIN SYNTHESIS, USING FUN CAT
SYSTEM OR JUST LOOKINGA THE
STEADY STATE LEVELS, AT THE
SYNAPSES, MY QUESTION IS, DO YOU
REALLY KNOW THAT THAT THIS IS
REDUCED TRANSLATION, ARE THE
RNAs REDUCED AT THOSE PLACES?
COULD IT BE A TRANSPORTER OR
EVEN A VERY INDIRECT
TRANSCRIPTION DEFECT RATHER THAN
A TRANSLATION PROBLEM IN.
>> WE DON'T THINK IT'S A
TRANSCRIPTION DEFECT BECAUSE
WE'VE DONE TRANSCRIPTOMICS IN
PARALLEL, THERE DEFINITELY A
TRANSPORTATION DEFICIT.
THOSE 2 MIGHT BE COUPLED YOU ARE
ABSOLUTELY RIGHT.
THERE ARE OTHERS IN THE FIELD
WHO HAVE USED MICROFLUIDICS AND
LOOK AT LOCAL TRANSLATION
DEFECTS CAUSED BY TDP43 AND THEY
SHOWED THAT IN THAT SYSTEM WHERE
YOU CAN LOOK LOCALLY, SO IN OUR
SYSTEM, I CANNOT DISCONNECT THE
TRANSPORT FROM TRANSLATION BUT I
DO KNOW THAT TRANSCRIPTION IS
NOT ALTERED FOR THIS.
>> THANKS.
>> SO THANK YOU VERY MUCH AND
JUST AS A REMINDER, WELL IS A
RECEPTION OWZ, A SMALL RECEPTION
SO IF YOU HAVE FURTHER QUESTIONS
OR WANT TO DISCUSS OTHER THINGS
PLEASE COME TO THAT AND HELP ME
THANK DANIELA FOR THE WONDERFUL
TALK TODAY.
>> THANK YOU.
[ APPLAUSE ]