Biochemical Mechanisms, Friedreich's Ataxia - The Children's Hospital of Philadelphia (3 of 5)

>> WELL, THANK YOU FOR THIS NICE INTRODUCTION, AND GOOD MORNING TO EVERYONE. WHAT I'LL TRY TO DO IN THE NEXT 20 MINUTES OR SO IS TO TRY AND REVIEW WHAT ARE THE KNOWN MOLECULAR MECHANISMS THAT ARE INVOLVED IN CAUSING FRIEDREICH'S ATAXIA. AND IT'S IMPORTANT TO SEE A NUMBER OF DIFFERENT STEPS THAT GO FROM THE GENETIC DEFECT TO THE DISEASE ITSELF, BECAUSE EACH OF THESE STEPS CAN BE THE TARGET OF A TREATMENT. AND THEREFORE, IT'S IMPORTANT TO KNOW AND TO CONSIDER IN THE GENERAL PICTURE. SO WITH THAT IN MIND, I WILL TRY VERY BRIEFLY TO REVIEW WHAT'S HAPPENING FROM THE DNA LEVEL TO THE FUNCTIONING OF THE CELL THAT IS NOT ENOUGH FRATAXIN. AND THEN, ROB WILSON WILL TELL YOU HOW WE CAN TARGET IN OTHERS, AND OTHER SPEAKERS AS YOU WILL SEE DURING THE DAY, WE WILL TELL YOU HOW WE CAN TARGET THESE DIFFERENT STEPS WITH POTENTIAL TREATMENTS. SO AS YOU ALL KNOW, THE DISEASE IS A GENETIC DISEASE, IT IS WHAT WE CALL A RECESSIVE DISORDER, WHICH MEANS THAT EACH OF THE TWO COPIES THAT WE HAVE OF OUR GENETIC INFORMATION MUST CARRY THE MUTATION OR THE ABNORMALITY IN ORDER FOR THE DISEASE TO MANIFEST, IT TO APPEAR. AND YOU HAVE LEARNED, YOU HAVE HEARD ALREADY, THAT, LET'S SEE, OKAY, WELL LET ME POINT THIS WAY FOR ONCE. EVERYONE IS, SO YOU HAVE LEARNED, YOU HAVE HEARD ALREADY TODAY AND YOU PROBABLY KNOW, KNEW BEFORE, THAT THE MOST COMMON CAUSE OF THIS DISEASE IS THIS GAA REPEAT EXPANSION, WHICH IS LOCALIZED IN A VERY PECULIAR PART OF THE GENE, IT'S CALLED THE INTRON, A PART OF THE GENE THAT DOES NOT REALLY CODE FOR THE PROTEIN, IT'S THERE FOR UNKNOWN, POSSIBLY REGULATORY PURPOSES. AND, WHAT HAPPENS THAT HAVING TOO MANY OF THESE GAAS IN THIS REGION WILL ALTER THE LEVEL OF EXPRESSION OF THIS GENE AND YOU WILL NOT MAKE ENOUGH OF THE PROTEIN, WHICH IS CALLED FRATAXIN. AS YOU NOTICE IN THIS SLIDE, YOU MAY HAVE OTHER TYPES OF MUTATIONS THAT WE CALL POINT MUTATIONS. THERE ARE ALSO DELETIONS, OTHER TYPES OF MUTATIONS THAT MAY BE INVOLVED IN A MINORITY OF CASES. WE THINK SO FAR THAT MOST OF THESE OTHER TYPES OF MUTATIONS, AND ANYWAY, ASSOCIATED WITH THE EXPANSION, SO THAT YOU HAVE THIS ON ONE CHROMOSOME AND THE EXPANSION ON THE OTHER CHROMOSOME. HOWEVER, THERE MAY BE SOME CASES THAT ARE STARTING TO BE RECOGNIZED VERY RECENTLY, THAT MAY HAVE, ACTUALLY, ONLY THIS KIND OF MUTATION AND NOT THE EXPANSION WITH A SIMILAR RESULT. AND WHAT IS THE RESULT? THE RESULT THAT THIS GENE, LIKE ALL GENES IN OUR GENOME, CONTAINS THE INSTRUCTIONS TO MAKE A PROTEIN, PROTEIN THAT WE CALLED MANY YEARS AGO FRATAXIN, BUT WE DIDN'T KNOW WHAT THIS PROTEIN WAS DOING AND WE NAMED IT AFTER THE DISEASE, IN FACT. AND WHAT WE SEE HERE WITH THIS IN THIS PARTICULAR EXPERIMENT WHICH IS CALLED THE WESTERN BLOT IN WHICH WE USE ANTIBODIES THAT YOU RAISE IN ANIMALS TO DETECT THAT PARTICULAR PROTEIN IN THE TISSUE EXTRACT, IN THESE CASES BRAIN OR CEREBELLUM OF PATIENTS, AND YOU SEE IN THE MIDDLE A DARK SPOT AND THAT'S A NORMAL INDIVIDUAL, AND YOU CAN SEE ON THE SIDES MUCH LIGHTER SPOTS AND THESE ARE PATIENTS WITH FRIEDREICH'S. AND THE FACT THAT THE SPOTS ARE LIGHTER JUST MEANS THAT THEY HAVE MUCH LESS OF THAT PROTEIN. SO THE BASIC PROBLEM IN THIS DISEASE IS THAT THOSE WHO ARE AFFECTED MAKE TOO LITTLE, NOT ENOUGH OF THIS PARTICULAR PROTEIN CALLED FRATAXIN. AND, AGAIN, I COME BACK TO THIS POINT BECAUSE IN THE VAST MAJORITY OF CASES, THIS LONG REPEAT WHICH DOES NOT ALTER THE INFORMATION NEEDED TO MAKE THE PROTEIN, IT JUST CHANGES THE AMOUNT IN WHICH THIS PROTEIN IS MADE. THIS IS VERY IMPORTANT FOR POSSIBLE THERAPEUTIC APPROACHES. SO WHAT DOES THAT REPEAT DO? THE EXPANDED REPEAT CHANGES THE WAY DNA IS PACKED IN THE CELL NUCLEUS, AND WE WILL HEAR FROM DR. JOEL GOTTESFELD LATER TODAY HOW THIS HAPPENS, AND HOW THIS CAN BE TARGETED WITH SPECIFIC TREATMENTS. SO I WOULD NOT GET, WILL NOT SPEND ANY MORE WORDS ON THIS POINT. YOU WILL HEAR ABOUT IT, YOU WILL HEAR SOME DETAILS, AND THIS IS A VERY PROMISING WAY OF POSSIBLY TREATING THE DISEASE. WE WILL SEE WHAT THESE KIND OF TREATMENTS TARGETED THIS PARTICULAR STEP WILL BE AFFECTED, BUT THE PERSPECTIVE LOOKS QUITE PROMISING. SO I SAID YOU DON'T MAKE ENOUGH OF THIS PARTICULAR PROTEIN CALLED FRATAXIN. WHAT DOES THIS PROTEIN DO? WELL, YOU WILL HEAR, AGAIN, FROM ANOTHER SPEAKER, DR. ELAINE POUCHER THIS AFTERNOON, SOME MANY DETAILS ABOUT WHAT THESE PROTEINS DO. SO I WILL NOT ENTER, I WILL NOT SAY ANYTHING ABOUT THE PRECISE FUNCTION OF THE PROTEIN. I WILL JUST TELL YOU SOME GENERAL CONCEPTS ABOUT ITS FUNCTION, AND ESPECIALLY WHAT HAPPENS WHEN YOU DON'T HAVE ENOUGH OF IT. WE KNOW THAT THIS PROTEIN IS ESSENTIAL IN ALL MULTI-CELLULAR ORGANISMS. WHAT DOES THIS MEAN? IT MEANS THAT, FIRST OF ALL, ALMOST EVERY LIVING THING HAS FRATAXIN OR SOMETHING THAT LOOKS LIKE FRATAXIN, INCLUDING BACTERIA. NOT ALL BACTERIA, BUT MANY BACTERIA HAVE SOMETHING THAT LOOKS LIKE FRATAXIN. AND ALSO, OTHER SIMPLE CELLULAR ORGANISMS LIKE YEAST HAVE FRATAXIN. THIS ORGANISM CAN SURVIVE WITHOUT FRATAXIN, ALTHOUGH IT'S IN VERY BAD SHAPE WITHOUT FRATAXIN, BUT CAN LIVE. ANY MULTI-CELLULAR ORGANISM, THAT IS ANY LIVING THING THAT IS COMPOSED BY MANY CELLS, WILL DIE DURING EMBRYONIC DEVELOPMENT WITH NO FRATAXIN AT ALL. THAT HAS BEEN SEEN IN THE MOUSE BY OUR COLLEAGUES IN STRASBOURG WHO DEVELOPED THE MOUSE THAT DOESN'T MAKE ANY FRATAXIN. THIS MOUSE JUST DOESN'T GROW, WILL DIE IN THE UTERUS OF THE MOTHER WHEN JUST A LITTLE CLUMP OF CELLS, BUT IT HAS BEEN SEEN BY OTHER RESEARCHERS IN AS DIFFERENT ORGANISMS AS WORMS, FLIES, EVEN PLANTS. PLANTS WILL NOT DEVELOP WITHOUT FRATAXIN. SO IT'S REALLY NECESSARY FOR THE DEVELOPMENT OF A HUMAN BEING. AND YOU MAY UNDERSTAND THAT THE DISEASE EXISTS BECAUSE THOSE WHO ARE AFFECTED MAKE A LITTLE BIT OF THIS PROTEIN, OTHERWISE THEY WOULD NEVER BE BORN. THEY WOULD DIE IN UTERUS, AND WE WOULDN'T EVEN KNOW ABOUT THIS DISEASE. BUT THE FACT THAT THEY MAKE SOME OF THE PROTEIN WILL ALLOW TO GO THROUGH EMBRYONIC DEVELOPMENT, AND TO BECOME FULLY FORMED INDIVIDUALS, BUT UNFORTUNATELY, WITH NOT ENOUGH OF THIS PROTEIN AND THE CONSEQUENCE WILL SHOW UP, THEN, LATER ON IN LIFE. THIS PROTEIN IS LOCALIZED IN A PARTICULAR PART OF EACH CELL, WHICH IS CALLED THE MITOCHONDRION. HERE IS AN EXPERIMENT THAT SHOWS THAT FRATAXIN LOCALIZES IN THE SAME POSITION WITHIN THE CELL AS A KNOWN PROTEIN, A KNOWN MITOCHONDRION PROTEIN, COX4, AND YOU CAN LABEL THIS PROTEIN WITH TWO DIFFERENT FLUORESCENCES, AND YOU CAN SEE THAT THEIR FLUORESCENCE OVERLAPS SO THEY ARE IN THE SAME, IN THE SAME PORTION OF THE CELL. YOU WILL HEAR ABOUT THE DIFFICULTIES THAT WE HAD FROM THE FACT THAT THE ORGANISM CAN NOT LIVE COMPLETELY WITHOUT FRATAXIN IN TERMS OF DEVELOPING MODELS THAT WE CAN USE IN THE LAB TO WORK ON, TO UNDERSTAND THE PATHOGENESIS OF FRIEDREICH'S. AND YOU WILL HEAR FROM OTHER COLLEAGUES, FROM ELAINE POUCHER AND FROM MIRELLA DOTTORI, WE ARE TRYING TO MAKE ANIMALS AND CELLULAR MODELS THAT CAN HELP US UNDERSTAND THE STUDY OF FRATAXIN. BUT LET'S GO BACK TO MITOCHONDRIA. SO WHAT ARE THESE MITOCHONDRIA THAT CONTAIN FRATAXIN. THE WORD COMES FROM THE GREEK THAT MEANS, "MIT" OR "MITO" MEANS "THREAD" AND "CHONDRION" MEANS "GRANULE" BECAUSE THEY LOOK AT THE MICROSCOPIC LIKE THREADS OR GRANULES INSIDE THE CELL. THIS IS A PECULIAR STRUCTURE SO THAT YOU CAN FIND BETWEEN THE CELLS THERE ARE CONTAINED WITHIN A MEMBRANE THAT WE CALL THE OUTER MEMBRANE, THERE IS A SECOND MEMBRANE INSIDE THAT WE CALL THE INNER MEMBRANE THAT DOESN'T REALLY FOLLOW EXACTLY THE OUTER MEMBRANE BUT MAKES ALL THESE FAULTS THAT ARE CALLED CRISTAE, AND THEN THERE IS A SPACE BETWEEN THESE TWO MEMBRANES WHICH IS CALLED AN INTER MEMBRANE SPACE. AND THERE IS A SPACE INSIDE THE INTERMEMBRANE WHICH IS CALLED A MATRIX, AND ACTUALLY, FRATAXIN IS LOCALIZED IN THE MATRIX OF MITOCHONDRIA, WELL INSIDE THIS ORGANISM. YOU WILL HAVE ALREADY UNDERSTOOD THAT IF YOU DON'T HAVE ENOUGH FRATAXIN, YOU HAVE A PROBLEM WITH YOUR MITOCHONDRIA, OF COURSE, THE PROTEIN IS THERE, IT MUST DO SOMETHING METABOLIC. AND, IN FACT, SUGGESTED TO REMIND YOU, THAT FRIEDREICH'S ATAXIA IS ONE OF MANY DISEASES IN WHICH MITOCHONDRIA DO NOT WORK, AND DO NOT WORK PROPERLY, AND THESE DISEASES HAVE A NUMBER OF COMMON ELEMENTS. THEY OFTEN AFFECT THE NERVOUS SYSTEM. AT THIS POINT IT IS BASICALLY ALMOST THERE. BUT, OKAY. THEY OFTEN INVOLVE THE CENTRAL NERVOUS SYSTEM. THEY OFTEN INVOLVE THE HEART, AS THE CASE OF FRIEDREICH'S ATAXIA. THEY OFTEN INVOLVE THE PANCREAS, AND OTHER TISSUES LIKE THE MUSCLE, AS YOU HAVE HEARD, CAUSING DIABETES OR AT LEAST INCREASING THE RISK OF HAVING DIABETES. SO MANY ASPECTS OF WHAT'S WRONG IN FRIEDREICH'S CAN BE INTERPRETED ON THE BASIS OF MITOCHONDRIA THAT DO NOT WORK PROPERLY. BUT LET'S SEE, FIRST OF ALL, WHAT THESE MITOCHONDRIA ARE DOING NORMALLY. AND MITOCHONDRIA DO A LOT OF THINGS. PEOPLE USUALLY KNOW THAT MITOCHONDRIA ARE KIND OF THE POWER PLANTS OF THE CELL, THE PLACE WHERE ENERGY IS PRODUCED. WHICH IS CORRECT. THE MOST IMPORTANT SOURCE OF ENERGY IN THE CELL IS THE MITOCHONDRIA. THEY'RE THE LAST STEP OF THE UTILIZATION OF FUELS, WHICH MEANS FOOD, COMPONENTS OF THE FOOD THAT WE EAT ARE, SORT OF, METABOLIZED. THEY ARE KIND OF BURNED IN THE MITOCHONDRIA, BOTH OF THE FATTY ACIDS, AND SOME DERIVATIVE AND SOME MOLECULES THAT COME FROM SUGARS ARE OXIDIZED, WE SAY IN MITOCHONDRIA. AND, THE ELECTRONS, I MEAN, THESE ARE KIND OF CHEMICAL REACTIONS THAT TAKE PLACE THERE. THESE METABOLIC PATHWAYS GENERATE ELECTRONS, WE SAY, THAT FLOW THROUGH A COMPLEX, A SERIES OF COMPLEXES OF PROTEINS THAT ARE IN THIS INNER MITOCHONDRIAL MEMBRANE, THAT MEMBRANE THAT IS FOLDED FORMING THE CRISTAE, AND THROUGH, BY FLOWING THERE, THEY GENERATE, THEY END UP GENERATING A SUBSTANCE WHICH IS CALLED ATP, WHICH IS ACTUALLY, IS THE ENERGY THAT IS USED IN THE CELL. SO THIS ATP IS AN ENERGY CHARGED MOLECULE THAT THEN IS UTILIZED BY ALL THE CELLULAR PROCESSES THAT REQUIRE ENERGY. SO THIS IS THE MAIN FUNCTION, BUT IN REALITY, MITOCHONDRIA HAVE A LOT OF ADDITIONAL FUNCTIONS. AND, ACTUALLY ALL MITOCHONDRIA ARE NOT THE SAME, AND THIS IS IMPORTANT TO KNOW THERE IS NOT SUCH A THING AS A GENERIC CELL, OR THERE IS NOT SUCH THING AS A GENERIC MITOCHONDRIA. WE HAVE MANY DIFFERENT CELL TYPES IN OUR BODY, AND MITOCHONDRIA, IT'S IN DIFFERENT CELL TYPES AS YOU CAN SEE HERE, THERE'S A PICTURE OF MITOCHONDRIA IN DIFFERENT CELL TYPES. YOU CAN SEE THEY LOOK SOMEWHAT DIFFERENT, BECAUSE MITOCHONDRIA DO NOT ONLY HAVE THIS FUNCTION OF GENERATING ENERGY, AND THEREFORE, THEY ARE NOT ONLY CONCENTRATED IN CELLS THAT REQUIRE A LOT OF ENERGY, LIKE MUSCLE, HEART, OR BRAIN. BUT, THEY ALSO HAVE A NUMBER OF ADDITIONAL FUNCTIONS THAT ARE MAYBE IMPORTANT FOR CERTAIN CELL TYPES, AND THAT MAY BE THE KEY WHY CERTAIN CELL TYPES ARE MORE AFFECTED THAN OTHERS IN FA, AS YOU HAVE LEARNED ALSO FROM THE FIRST TALK THIS MORNING. AND THERE ARE A NUMBER OF OTHER FUNCTIONAL MITOCHONDRIA THAT ARE SUMMARIZED HERE. ONE IMPORTANT FUNCTION THAT IS RELATED TO FRATAXIN THAT HAS COME OUT TO THE ATTENTION OF RESEARCHERS, PARTICULARLY IN THE RECENT YEARS, IS THE FACT THAT THE MITOCHONDRIA, AMONG OTHER THINGS, ARE THE STRUCTURES IN THE CELL WHERE IRON IS UTILIZED. SO WE NEED A NUMBER, WE NEED TO PUT IRON IN A NUMBER OF MOLECULES SO THEY ARE NECESSARY FOR THE CORRECT FUNCTIONING OF OUR BODY AND OF OUR CELLS. ONE IS HEME, THE MOLECULE IN THE BLOOD THAT CARRIES OXYGEN, WHICH IS MADE IN MITOCHONDRIA. AND THEN, THERE ARE OTHER MOLECULES THAT CONTAIN IRON. THERE ARE MANY MITOCHONDRIA, AND THIS IS A VERY IMPORTANT FUNCTION IN MITOCHONDRIA, BECAUSE BASICALLY, THAT'S WHERE THE IRON CONTAINING MOLECULE OF CELLS ARE MADE. AND, ABOUT NOW FOURTEEN YEARS AGO, WHEN THE GENE MUTATION AND THE LINE FRIEDREICH'S HAD JUST BEEN DISCOVERED, A GROUP OF RESEARCHERS IN FRANCE NOTICED THAT PATIENTS WITH FRIEDREICH'S ATAXIA, IN PARTICULAR WITH HEART DISEASE DUE TO FRIEDREICH'S ATAXIA, HAD A DEFICIENCY OF CERTAIN ENZYMES IN THE HEART THAT CONTAIN, THAT NEEDED TO FUNCTION, SPECIAL MOLECULAR STRUCTURES THAT CONTAIN IRON THAT ARE CALLED IRON-SULFUR CLUSTERS. AND HERE, YOU HAVE A PICTURE OF THESE IRON-SULFUR CLUSTERS, SO YOU DON'T NEED TO BE A CHEMIST, JUST IMAGINE THAT THESE ARE BASICALLY, AS YOU CAN SEE, DIFFERENT TYPES OF COMPOUNDS FORMED BY IRON AND SULFUR ATOMS THAT ARE ASSEMBLED TOGETHER IN A NUMBER OF GEOMETRIC SHAPES, AND THAT MANY PROTEINS THAT ARE NEEDED TO FUNCTION, THEY WOULD NOT FUNCTION WITHOUT IRON-SULFUR CLUSTERS. AND GUESS WHAT? IRON-SULFUR CLUSTERS ARE MADE IN MITOCHONDRIA. THESE PROTEINS THAT NEED IRON-SULFUR CLUSTERS TO FUNCTION ARE AWARE THAT NOT ONLY MITOCHONDRIA PROTEIN, AND IN THE MITOCHONDRIA THEY INCLUDE MANY OF THESE PROTEINS THAT ARE NEEDED TO MAKE ENERGY TO BURN COMPLETELY PRODUCTS COMING FROM FOOD AND COMPONENTS OF THIS ELECTRON TRANSPORT CHAIN THAT WILL GENERATE THIS ENERGY-RICH MOLECULE ATP, BUT IRON-SULFUR CLUSTERS ARE NEEDED BY PROTEINS IN ALL THE CELLS, INCLUDING IN THE NUCLEUS, THE PROTEINS THAT NOT NEEDED FOR THE INTEGRITY OF DNA, AND IN THE CELLS FOR A NUMBER OF OTHER MOLECULAR PROCESSES. WHAT HAPPENS, AND DR. POUCHER WILL EXPLAIN TO YOU, WILL GIVE TO YOU THE DETAILS THIS AFTERNOON IS THAT IF YOU DON'T HAVE ENOUGH FRATAXIN, IRON-SULFUR CLUSTERS ARE MADE VERY INEFFICIENTLY. AND I TOLD YOU THESE ARE MADE IN MITOCHONDRIA. SO WE HAVE, FIRST OF ALL, A NUMBER OF CONSEQUENCES THAT ARE SUMMARIZED IN THIS SLIDE. YOU DON'T MAKE ENOUGH OF THESE IRON-SULFUR CLUSTERS, YOU, OF COURSE, WILL HAVE DEFICIENCY IN ALL THESE PROTEINS, IN THE FUNCTION OF ALL THESE PROTEINS THAT NEED IRON-SULFUR CLUSTERS. SO YOU WILL HAVE DEFICIENCY IN ENERGY PRODUCTION BECAUSE IN THE MITOCHONDRIA, SOME OF THESE PROTEINS ARE INVOLVED IN ENERGY PRODUCTION, BUT YOU WILL HAVE ALSO DEFICIENCIES IN OTHER PROCESSES INCLUDING, FOR INSTANCE, THE CONTROL OF DNA INTEGRITY THAT TAKE PLACE IN OTHER PARTS OF THE CELL. YOU WILL ALSO HAVE SOME IMPORTANT CONSEQUENCES AT THE LEVEL OF IRON METABOLISM. AND, WELL, YOU WILL HAVE WHAT IS CALLED OXIDATIVE STRESS, WHICH MEANS THAT THESE ENERGY PRODUCING MOLECULES, THESE ENERGY PRODUCING COMPLEXES THAT EXIST IN THE MITOCHONDRIA, IN FACT, USE OXYGEN, THEY TRANSFORM OXYGEN INTO WATER AT THE END OF THESE REACTIONS THAT PRODUCE ENERGY. AND, IF THESE COMPLEXES DON'T WORK PROPERLY BECAUSE THEY DO NOT, BECAUSE THEY CONTAIN IRON-SULFUR CLUSTERS THAT ARE NOT AS SUFFICIENTLY AVAILABLE, SOME OF THIS OXYGEN, INSTEAD OF BEING REDUCED IN A CONTROLLED WAY, WILL REACT WITH OTHER, WITH OTHER COMPOUNDS IN THE CELL AND WILL FORM TOXIC SUBSTANCES THAT ARE CALLED FREE RADICALS, OR REACTIVE OXYGEN SPECIES, THAT WILL MAKE DAMAGE IN THE MITOCHONDRION, BUT ALSO WILL GET OUT OF THE MITOCHONDRION AND MAKE DAMAGE IN THE REST OF THE CELL. BUT ALSO, ANOTHER IMPORTANT POINT THAT THE CONTROL OF IRON METABOLISM WILL BE ALTERED. THIS CELL SOMEWHAT SENSES THE FACT THAT THESE IRON-SULFUR CLUSTERS ARE NOT MADE, AND HOW THE CELL WOULD INTERPRET THIS THING, I MEAN, THE CELL IS NOT A BEING WITH A BRAIN, BUT THERE ARE MECHANISMS, CONTROL MECHANISMS, AND BASICALLY, THE LACK OF IRON-SULFUR CLUSTERS IS FELT BY THE CELL AS A LACK OF IRON. BECAUSE NORMALLY, IF YOU DON'T MAKE ENOUGH SULFUR CLUSTERS IT'S BECAUSE YOU DON'T HAVE ENOUGH IRON, SO WHAT THE CELL WILL DO WILL TRY TO GET MORE IRON, AND WILL ACTIVATE MECHANISMS THAT PICK UP IRON FROM THE ENVIRONMENT, FROM THE BLOOD, FROM THE EXCESS CELLULAR FLUID, AND BRING IT INTO THE CELL. AND WHERE WILL THIS IRON GO? IT WILL GO WHERE IT'S NEEDED TO MAKE IRON-SULFUR CLUSTERS IN MITOCHONDRIA. SO THE CELL WILL, THERE ARE SENSOR SYSTEMS, SENSORS IN THE CELL THAT WILL FEEL LIKE THERE IS NOT ENOUGH IRON, WILL MAKE IRON GET INTO THE CELL AND ALL GO INTO THE MITOCHONDRIA. WHAT ARE THESE SENSORS? ONE OF THESE SENSORS IS A PROTEIN THAT ACTUALLY CONTAINS AN IRON-SULFUR CLUSTER, WHICH IS CALLED IRP1, IRON RESPONSIVE PROTEIN 1. AND IN FACT, IF THIS PROTEIN THAT DOES NOT, IS WITHOUT THE IRON-SULFUR CLUSTER, IT WILL ACTIVATE THIS MECHANISM. SO ACTUALLY, THE FACT THAT YOU CANNOT MAKE ENOUGH IRON-SULFUR CLUSTERS WILL MAKE THIS IRP1 FEEL LIKE THERE IS NOT ENOUGH IRON. AND AS I SAID, THIS IRON WILL END UP IN MITOCHONDRIA, BUT IN MITOCHONDRIA, THE SYSTEM THAT MAKES IRON-SULFUR CLUSTER IS NOT WORKING BECAUSE YOU DON'T GET ENOUGH FRATAXIN. SO ALL THIS IRON WILL GET INTO THE MITOCHONDRIA, AND WILL NOT BE UTILIZED AND WILL MAKE DAMAGE. AND THAT'S ANOTHER THING THAT HAPPENS, IRON CAN BE ACCUMULATED IN MITOCHONDRIA, AND THIS IS TAKEN FROM A PATIENT, FROM THE HEART OF A PATIENT, AND WHICH IS BEING STAINED WITH A PARTICULAR STAIN THAT WILL RECOGNIZE IRON, AND THESE DARK SPOTS ARE BASICALLY DEPOSITS OF IRON THAT ARE LOCALIZED WITHIN MITOCHONDRIA. WE CAN SEE THAT BY ELECTRON MICROSCOPY. AND THIS IRON WILL ALSO AGGRAVATE THE PROBLEM OF OXIDATIVE STRESS BECAUSE THIS IRON WILL REACT WITH THOSE OXYGEN DERIVATIVES THAT ARE FORMED BECAUSE THE RESPIRATORY CHAIN IS NOT REALLY EFFICIENT, AND WILL CREATE EVEN MORE OF THESE FREE RADICALS THAT WILL BE DAMAGING FOR THIS CELL. AND ALL THE STEPS, BY THE WAY, CAN BE TARGETED BY TREATMENT. THERE IS ONE FINAL POINT THAT I WILL LIKE TO ATTRACT TO YOUR ATTENTION BEFORE LEAVING THE PODIUM TO THE NEXT, AND MY PARTNER SPEAKER FOR THIS PART, FOR THIS PART OF THE PRESENTATION. MITOCHONDRIA THAT, OF COURSE, DON'T WORK PROPERLY, THEY DON'T MAKE ENOUGH ENERGY, THEY FILL UP WITH IRON, THEY MAKE THESE FREE RADICALS, THEY ARE SUFFERING, SOMEWHAT HAVE TO SIGNAL TO THE REST OF THE CELL THAT THERE IS SOMETHING WRONG WITH THEM, AND USUALLY THE CELL REACTS TO THE FACT THAT THERE IS SOMETHING WRONG WITH THE MITOCHONDRIA BY MAKING MORE MITOCHONDRIA. SO THERE IS A WHOLE COMPLEX SIGNALING MECHANISM THAT IS SUMMARIZED HERE BUT, AGAIN, AS DR. LYNCH SAID, I WILL NOT GO AROUND WITH A QUESTIONNAIRE ABOUT THE CONTENT OF THIS SLIDE. THERE IS THIS KIND, AND IN FRIEDREICH'S, THIS SIGNALING IS ALSO NOT FUNCTIONING PROPERLY. THERE IS, WE KNOW, AND THAT'S A VERY COMPLEX SLIDE, BUT WE KNOW THAT IN MANY TISSUES, NOT IN ALL TISSUES, FOR INSTANCE, IN THE HEART SOMEHOW THE SIGNALING WORKS AND THE HEART MAKES MORE MITOCHONDRIA TO TRY TO COMPENSATE. BUT OTHER TISSUES DO NOT, LIKE THE MUSCLE OR THE NERVE CELLS, BECAUSE THERE IS SOMETHING ABNORMAL IN THIS COMMUNICATION. SO IF YOU DON'T HAVE ENOUGH FRATAXIN, ALSO THIS RESPONSE OF MAKING MORE MITOCHONDRIA IS NON-FUNCTIONAL, AND WE THINK THAT THE REASON IS THIS ABNORMALITY IN IRON. THIS IS ALSO A VERY COMPLEX EXPERIMENT THAT BASICALLY SHOWS THAT IF YOU TAKE AWAY IRON FROM THE CELL, YOU TEND TO (UNINTELLIGIBLE) IT IN THE FORMATION OF NEW MITOCHONDRIA. AND PROBABLY SINCE THE CELL SENSES, FEELS LIKE IT DOES NOT HAVE ENOUGH IRON, KEEPS THE FORMATION OF MITOCHONDRIA LOW, AND ACTUALLY, THAT'S ANOTHER POSSIBLE TARGET FOR TREATMENT, BECAUSE IN A NUMBER OF MITOCHONDRIAL DISEASES, IT HAS BEEN SHOWN THAT IF YOU INCREASE THE FORMATION OF NEW MITOCHONDRIA, YOU CAN IMPROVE THE CLINICAL PICTURE. AND THERE HAS BEEN EVEN A VERY RECENT STUDY FROM A FRIEND OF MINE FROM MILAN, MASSIMO ZEVIANI, IN OTHER MITOCHONDRIAL DISEASES WERE SHOWN VERY ELEGANTLY BY USING ANIMAL MODELS, THAT YOU CAN IMPROVE, ACTUALLY, THE CLINICAL PICTURE IN A NUMBER OF MITOCHONDRIAL DISEASES BY DOING, BY ACTING ON THIS PATHWAY. I WILL THANK YOU NOW FOR YOUR ATTENTION, AND I WILL LEAVE THE PODIUM TO DR. WILSON, WHO WILL, I SUPPOSE, TRY TO USE, TO RE-EVALUATE SOME OF THIS INFORMATION TO TALK ABOUT POTENTIAL TREATMENTS. AND, AS I SAID, YOU WILL HEAR OTHER SPEAKERS TODAY GIVING YOU MORE DETAILS ABOUT THE FUNCTION OF FRATAXIN, AND ABOUT POSSIBLE THERAPEUTIC APPROACHES PARTICULARLY THIS AFTERNOON. THANK YOU FOR YOUR ATTENTION. [APPLAUSE] >> I'M COGNIZANT OF THE FACT THAT I STAND BETWEEN YOU AND THE BREAK. I CAN NEVER SPEAK AS QUICKLY AS DAVE, BUT I SHALL TRY TO BE EFFICIENT. I'M CHARGED TODAY WITH TELLING YOU ABOUT THERAPEUTIC PATHWAYS IN FRIEDREICH'S ATAXIA, AND IT WAS WISE OF THE ORGANIZERS TO PAIR MASSIMO AND ME IN THIS WAY, BECAUSE THE THERAPEUTIC PATHWAYS AND THE UNDERSTANDING OF THE BIOCHEMICAL MECHANISMS OF THE DISORDER ARE TOTALLY ENTWINED. THE MORE WE LEARN ABOUT THE UNDERLYING BIOCHEMICAL DEFECTS IN FRIEDREICH'S ATAXIA, THE MORE THERAPEUTIC POSSIBILITIES SUGGEST THEMSELVES. SO, LET ME SEE, HOW DO I ADVANCE THE SLIDE? >> PUSH THE GREEN BUTTON. I PUSH THE--, THIS THING, THE GREEN BUTTON. ALL RIGHT. THANKS. I ALWAYS RELY ON MASSIMO. HE SAVES ME. SO THE BIOCHEMICAL DEFECTS THAT GUIDE MOST, NOT ALL, BUT MOST OF THE THERAPEUTIC DEVELOPMENT FOR FRIEDREICH'S ATAXIA ARE THREE: THE DECREASED EXPRESSION OF THE FRATAXIN GENE, WHICH MASSIMO TALKED ABOUT; THE MITOCHONDRIAL IRON ACCUMULATION, WHICH MASSIMO TALKED ABOUT; AND OXIDATIVE STRESS, WHICH MASSIMO TALKED ABOUT. YOU SEE WHY WE'RE TOGETHER. SO LET'S START WITH THE FIRST ONE, DECREASED EXPRESSION OF THE FRATAXIN GENE. TO REVIEW BRIEFLY, THERE ARE THOSE GAA REPEAT EXPANSIONS IN THE FIRST INTRON OF THE GENE. AS MASSIMO MENTIONED, THE INTRON OF THE GENE DOES NOT ENCODE FOR PROTEIN. THAT'S ACTUALLY VERY IMPORTANT. IN A LOT OF GENETIC DISORDERS, THE PROBLEM IS IN THE EXON, THE PART THAT ACTUALLY ENCODES THE FUNCTIONAL PROTEIN. IN MOST DISEASE GENES IN FRIEDREICH'S ATAXIA, OR DISEASE ALLELES, A VERSION OF A GENE, THE EXPANSIONS, THE EXTRA BASES, GAA GUANINE-ADENINE-ADENINE, ARE IN THE FIRST INTRON, SO THE PROTEIN ENCODING PARTS OF THE GENE ARE STILL INTACT. WHICH MEANS THAT IF YOU COULD RE-EXPRESS THE GENE, YOU COULD GET FUNCTIONAL PROTEIN. ALL RIGHT? SO THAT'S VERY IMPORTANT. NORMAL, 7 TO 40 REPEATS. DISEASE LENGTHS ARE 7 TO 1800, MAYBE EVEN MORE NOW. AND THIS IS ACTUALLY A SLIDE FROM MASSIMO'S REVIEW, EXCELLENT REVIEW, IN THE ARCHIVES OF "NEUROLOGY" 2008, AND YOU SEE AT THE TOP THE NORMAL GAA REPEATS IN THE FIRST INTRON. OKAY? NOT IN THE EXONS. THE EXONS ENCODE THE PROTEIN, RIGHT? AND YOU SEE THE NORMAL AMOUNT OF FRATAXIN BEING MADE THERE IN THE UPPER RIGHT, AND THEN THERE'S AN EXPANSION, THE GAA 900, EXTRA GAAS, AND THAT LEADS TO DECREASED EXPRESSION, SO YOU SEE MUCH LESS FRATAXIN, JUST THAT ONE CIRCLE UNDERNEATH, SO MUCH LESS PROTEIN GETS MADE AS MASSIMO SAID. WHY THAT OCCURS IS AN INTERESTING BASIC SCIENCE QUESTION, AND HYPOTHESES ARE SHOWN ON THE THIRD LINE THERE, AND ON THE THIRD LINE ON THE RIGHT, TO THE RIGHT OF THE QUESTION MARK YOU SEE CHROMATIN CONDENSATION. SO DNA IS WRAPPED UP AROUND PROTEINS CALLED HISTONES, AND IT'S COMPACTED AND THERE'S LESS EXPRESSION PERHAPS DUE TO THAT. AND SO, WHAT'S THE APPROACH? WHAT'S THE THERAPEUTIC PATHWAY BASED ON THIS? WELL, OBVIOUSLY, TO MAYBE RE-EXPRESS THE FRATAXIN GENE. HDAC INHIBITORS, HISTONE DEACETYLASE INHIBITORS ARE BEING PIONEERED BY JOEL GOTTESFELD WHO WILL TALK LATER, SO I WON'T SPEND A LOT OF TIME ON IT, BUT WHAT THEY DO IS THEY UNPACK THAT PACKAGING OF DNA, AND HOPEFULLY RE-EXPRESS THE GENE. AND IN FACT, JOEL WILL TELL YOU ABOUT COMPOUNDS THAT HE STUDIED NOW WITH REPLIGEN THAT DO INCREASE FRATAXIN EXPRESSION. WHAT REPLIGEN IS TRYING TO DO IS TRYING TO FIND COMPOUNDS THAT RE-EXPRESS FRATAXIN WITHOUT TOO MANY SIDE EFFECTS, PROBABLY FROM OVEREXPRESSING OTHER GENES, AND JOEL WILL TELL YOU ABOUT THAT. ANOTHER DRUG WHICH COULD INCREASE FRATAXIN EXPRESSION IS ERYTHROPOIETIN, OR EPO. EPO IS A HORMONE SECRETED BY YOUR KIDNEYS, ACTUALLY, THAT TELL THE BONE MARROW TO MAKE MORE RED BLOOD CELLS. NOW RED BLOOD CELLS, AS MASSIMO MENTIONED, MAKE A LOT OF HEMOGLOBIN, AND DO A LOT OF IRON METABOLISM. AND IT SEEMS THAT EPO HAS THE PROPERTY OF TURNING ON A LOT OF GENES ASSOCIATED WITH IRON METABOLISM, INCLUDING FRATAXIN. THE SIDE EFFECT OF EPO THEY INCREASE THE NUMBER OF RED BLOOD CELLS IN YOUR BLOODSTREAM. THIS IS GOOD, UP TO A POINT, PARTICULARLY IF YOU ARE A TOUR DE FRANCE RIDER, BUT YOU TAKE TOO MUCH EPO, YOU CAN HAVE TOO MANY RED BLOOD CELLS IN YOUR BLOOD, AND THAT CAN ACTUALLY MAKE YOUR BLOOD FLOW VERY SLUGGISH AND IS PARTICULARLY BAD IF YOU HAVE HEART DISEASE. SO YOU WANT TO BE VERY CAUTIONS ABOUT TAKING EPO, AND THAT'S THE SIDE EFFECT OF EPO, IF YOU WILL, FOR INCREASING FRATAXIN EXPRESSION. SO PEOPLE ARE STUDYING NOW EPO MIMETICS. EPO-LIKE COMPOUNDS THAT HOPEFULLY WILL INCREASE FRATAXIN EXPRESSION WITHOUT THE SIDE EFFECT OF INCREASING OR MAKING TOO MANY RED BLOOD CELLS IN THE BLOOD. A THIRD APPROACH TO DECREASED EXPRESSION OF FRATAXIN IS TO REPLACE IT WITH TAT-FRATAXIN. SO PROTEINS DON'T NORMALLY CROSS VERY WELL INTO CELLS. YOU CAN'T EAT A PROTEIN AND EXPECT THAT IT'LL GET INTO YOUR BLOODSTREAM AND THEN GET INTO CELLS. IT'S BROKEN DOWN IN YOUR GUT, AND EVEN IF YOU INJECT IT INTO YOUR BLOOD, IT NEVER GETS INTO CELLS. BUT IF YOU TAKE A SMALL PEPTIDE, A SMALL PIECE OF A PROTEIN CALLED TAT, AND CONNECT IT TO FRATAXIN, IT CAN GET INTO CELLS TO A CERTAIN DEGREE, AND MARK PAYNE HAS STARTED STUDYING THIS IN MOUSE MODELS, AND THE FRATAXIN CAN ACTUALLY GET INTO THE CELLS. PROTEIN BASED THERAPEUTICS IS A VERY NEW AREA, HOWEVER, AND I THINK IT FACES A LOT OF REGULATORY HURDLES, BUT I THINK IT'S DEFINITELY SOMETHING THAT SHOULD BE PURSUED, AND A STORY THAT YOU SHOULD FOLLOW. HIGH-THROUGHPUT SCREENING IS SORT OF A MORE UNBIASED APPROACH TO DISCOVERING DRUGS FOR FRIEDREICH'S ATAXIA, AND MAREK NAPIERALA WILL SPEAK THIS AFTERNOON ABOUT HIS HIGH-THROUGHPUT SCREEN TO LOOK FOR COMPOUNDS THAT INCREASE FRATAXIN EXPRESSION. IN HIGH-THROUGHPUT SCREENING, BASICALLY YOU LOOK AT HUNDREDS OF THOUSANDS OF DIFFERENT CHEMICAL COMPOUNDS. YOU BASICALLY SAY, "I DON'T KNOW WHAT'S GOING TO WORK BEST. I'M GOING TO LET THE CELLS TELL ME WHAT WORKS BEST." AND YOU TEST EACH OF THOSE COMPOUNDS INDIVIDUALLY IN A ROBOTICS FACILITY, AND LOOK FOR COMPOUNDS THAT DO WHAT YOU WANT. AND I'LL LET MARK TELL YOU ABOUT THAT. SO THE SECOND BIOCHEMICAL DEFECT I MENTIONED WAS MITOCHONDRIAL IRON ACCUMULATION, ALSO MENTIONED BY MASSIMO. AS MASSIMO SAID, FRATAXIN HELPS FORM IRON-SULFUR CLUSTERS, THOSE LITTLE PROSTHETIC GROUPS THAT HELP ENZYMES DO THEIR THING IN THE CELL. AND DECREASED FRATAXIN CAUSES IRON-SULFUR CLUSTER DEFECTS, AS MASSIMO SAID, AND THAT'S ASSOCIATED WITH MITOCHONDRIAL IRON ACCUMULATION. SO HERE'S MY SUPER-SIMPLIFIED VERSION: HERE'S FRATAXIN, TAKES IRON, MAKES AN IRON-SULFUR CLUSTER. OKAY? SO IRON-SULFUR CLUSTERS ARE IN THAT ELECTRON TRANSPORT CHAIN THAT MASSIMO TALKED ABOUT. THE ELECTRONS MOVE THROUGH THIS SERIES OF PROTEINS, IT'S LIKE A WIRE ALMOST, AND USE THAT TO GENERATE ENERGY FOR THE CELL. SO HERE'S AN ELECTRON GOING THROUGH TO GENERATE ENERGY. SO THIS IS MY SUPER-SIMPLIFIED VERSION. SO IF YOU MAKE TOO LITTLE FRATAXIN, WHICH I INDICATE BY A MUCH SMALLER FONT SIZE, YOU DON'T MAKE AS MANY IRON-SULFUR CLUSTERS AS WELL, SO THE ELECTRONS DON'T GO THROUGH AND YOU DON'T GENERATE QUITE AS MUCH ENERGY FROM YOUR MITOCHONDRIA. AS MASSIMO SAID, IT'S MORE COMPLICATED THAN THAT. THERE ARE MANY ENZYMES THAT USE IRON-SULFUR CLUSTERS, BUT THIS MAY BE PART OF THE SOURCE OF THE DEFECT IN MITOCHONDRIAL ENERGY PRODUCTION. SO THERE'S AN ACCUMULATION OF IRON IN THE MITOCHONDRIA, AND THAT IRON CAN BE TOXIC. SO AN APPROACH, A THERAPEUTIC PATHWAY, IF YOU WILL, IS TO REVERSE THE MITOCHONDRIAL IRON ACCUMULATION USING WHAT ARE KNOWN AS LIPID-SOLUBLE CHELATORS. SO LET ME UNPACK THAT FOR YOU. A CHELATOR IS A COMPOUND THAT GRABS ON TO SOMETHING LIKE A HEAVY METAL. OKAY? THE CHELATORS THAT WE WANT TO USE GRAB ON TO IRON, BECAUSE WE WANT TO GET THAT IRON OUT OF THE MITOCHONDRIA THAT'S ACCUMULATING THERE AND CAUSING TOXICITY. LIPID SOLUBLE MEANS THAT IT CAN CROSS MEMBRANES. AS MASSIMO SHOWED YOU, THE MITOCHONDRIA IS SURROUNDED BY TWO MEMBRANES, THE CELL IS SURROUNDED BY ONE MEMBRANE. SO A CHELATOR HAS TO GO THROUGH THREE MEMBRANES TO GET ALL THE WAY INTO THE CENTER PART OF THE MITOCHONDRIA, THE MITOCHONDRIAL MATRIX, TO TAKE THE IRON OUT. THAT'S WHY WE'RE LOOKING AT LIPID-SOLUBLE CHELATORS TO TRY AND DECREASE THAT MITOCHONDRIAL IRON ACCUMULATION, AND THE LEADER THERE IS PROBABLY THE COMPOUND DEFERIPRONE, WHICH IS BEING STUDIED BY A COMPANY CALLED APOPHARMA, AND IT'S STILL IN THE PROCESS OF PHASE II, PHASE III TRIALS. MY OPINION OF IT RIGHT NOW IS THAT SOME PATIENTS SEEM TO RESPOND WELL, OTHERS LESS SO, BUT IT DEFINITELY NEEDS TO BE STUDIED FURTHER. AND, THE FOLKS WHO STUDY IT ALWAYS EMPHASIZE THAT WHEN I TALK TO YOU, I SHOULD TELL YOU THAT, AND THIS IS TRUE, IT CAN BE VERY, VERY TOXIC. THIS IS NOT SOMETHING THAT YOU SHOULD BUY OFF THE INTERNET AND TAKE. YOU MUST TAKE THIS UNDER SUPERVISION, BECAUSE YOU NEED TO GET A BLOOD COUNT ONCE A WEEK. AND THE REASON IS, A SUBSET OF PATIENTS WHO TAKE THIS DRUG, THEIR WHITE CELL COUNTS PLUMMET, AND THEY CAN GET INFECTED AND DIE. SO THIS IS A VERY, VERY DANGEROUS DRUG. YOU SHOULD NOT TAKE THIS ON YOUR OWN. AND I HOPE YOU ALL HEAR ME THERE. OKAY? THAT'S VERY IMPORTANT. THERE ARE OTHER CHELATORS IN PRE-CLINICAL DEVELOPMENT THAT I COULD TELL YOU ABOUT, BUT THERE ARE A WHOLE BUNCH OF THEM. THE BASIC IDEA, THOUGH, IS THAT HERE'S THIS BIOCHEMICAL DEFECT, MITOCHONDRIAL IRON ACCUMULATION, AND IT SUGGESTS A THERAPEUTIC PATHWAY. AND THAT'S, I THINK, WHAT I WANT. THE TAKE HOME MESSAGE HERE IS THAT THE MORE YOU UNDERSTAND, AS I SAID BEFORE ABOUT THE BIOCHEMICAL DEFECTS IN A DISORDER, THE MORE THERAPEUTIC POSSIBILITIES SUGGEST THEMSELVES. THIRD DEFECT IS, OF COURSE, OXIDATIVE STRESS, WHICH YOU'VE ALL HEARD ABOUT. THIS IRON-SULFUR CLUSTER ASSEMBLY DEFECT LEADS TO INCREASED OXIDANT FORMATION DIRECTLY AND THROUGH INCREASED IRON. SO LET'S LOOK AT THAT. SO HERE'S MY LITTLE CARTOON AGAIN. TOO LITTLE FRATAXIN, IRON-SULFUR CLUSTERS ARE NOT MADE PROPERLY, SOME OF THOSE ELECTRONS DON'T GET THROUGH, YOU MAKE LESS ENERGY. THERE'S ALSO THIS FORMATION OF OXIDANTS FROM THESE EXCESS ELECTRONS. THE ACCUMULATION OF IRON ALSO LEADS TO THE FORMATION OF OXIDANTS. NOW, WHAT ARE THESE OXIDANTS? YOU'LL HEAR DIFFERENT TERMS. YOU'LL HEAR OXIDANTS, YOU'LL HEAR REACTIVE OXYGEN SPECIES, AND YOU'LL HEAR FREE RADICALS. OKAY? THOSE ARE THREE THINGS YOU'LL HEAR. A FREE RADICAL IS A CHEMICAL COMPOUND THAT HAS AN UNPAIRED ELECTRON. OKAY? SO ELECTRONS LIKE TO GO IN PAIRS, AND IF A MOLECULE HAS AN UNPAIRED ELECTRON, IT SEARCHES AROUND FOR ANOTHER ELECTRON TO GET. AND WHAT IT CAN DO IS IF IT BUMPS INTO A BIOMOLECULE IN YOUR CELL, IT TRIES TO EXTRACT AN ELECTRON SO IT CAN HAVE A PAIRED ELECTRON. WHEN IT TAKES AN ELECTRON AWAY FROM A BIOMOLECULE, IT IS OXIDIZING THAT MOLECULE. THAT'S OXIDATIVE STRESS. OKAY? SO FREE RADICALS PULL ELECTRONS OFF OF BIOMOLECULES AND DAMAGE THEM BY TAKING AN ELECTRON AWAY. THAT'S OXIDATION. ONE FORM OF FREE RADICAL IS REACTIVE OXYGEN SPECIES. THESE ARE FREE RADICALS THAT HAVE OXYGEN IN THEM, AS MASSIMO SHOWED ON ONE OF HIS SLIDES. OKAY. SO OXIDATIVE STRESS DAMAGES BIOMOLECULES IN THE CELLS. SO, OBVIOUSLY, THE THERAPEUTIC PATHWAY, THE APPROACH, WOULD BE ANTIOXIDANTS. OKAY? SO WHAT ARE SOME ANTIOXIDANTS? WELL, YOU HEARD FROM DAVE THIS MORNING ABOUT IDEBENONE. IT'S AN ANTIOXIDANT. IT'S SOMETHING CALLED A PARABENZONEQUINONE, MEANING IT HAS A CHEMICAL STRUCTURE SIMILAR TO COENZYME Q10, WHICH OCCURS NATURALLY IN OUR BODY AND IS PART OF THAT ELECTRON TRANSPORT CHAIN, ACTUALLY. IDEBENONE HAS THE SAME REACTIVE GROUP AS COQ10, BUT IT'S ACTUALLY A SMALLER MOLECULE SO IT'S ABSORBED BETTER INTO THE BLOODSTREAM, SO IT'S MORE DRUG-LIKE. BUT IT IS METABOLIZED A LOT, SO IT'S NOT AS DRUG-LIKE AS ONE MIGHT WANT, AND DAVE TOLD YOU ABOUT THE PRELIMINARY RESULTS WITH IDEBENONE, WHICH ARE SORT OF MIXED. A0001 AND EPI-743 ARE SORT OF SECOND-GENERATION IDEBENONES. OKAY? IT'S A SIMILAR IDEA TO IDEBENONE IN TERMS OF HOW IT WORKS. BUT A0001 LOOKS MORE LIKE A DRUG. IT'S ABSORBED BETTER, IT'S MORE STABLE IN THE BLOOD, IT HAS MORE DRUG-LIKE PROPERTIES, AND SO AS DAVE SAID, THAT ONE'S GOING INTO PHASE II IMMINENTLY, AND WE'LL SEE. PIOGLITAZONE, I'M SO GLAD MASSIMO MENTIONED, PUT UP THAT LAST SLIDE THAT HE PUT UP WITH THE COMPLICATED BIOCHEMICAL PATHWAY. PIOGLITAZONE ACTS THROUGH THOSE PATHWAYS THAT MASSIMO WAS PUTTING UP TO INCREASE THE EXPRESSION OF GENES THAT ENCODE PROTEINS THAT ARE IMPORTANT FOR REPAIRING AND BUILDING NEW MITOCHONDRIA. OKAY. SO PIOGLITAZONE ISN'T ITSELF AN ANTIOXIDANT, BUT SOME OF THE GENES THAT IT ACTIVATES, WHEN IT ACTIVATES GENES THAT MAKE NEW MITOCHONDRIA, ARE ANTIOXIDANT ENZYMES. SO IN EFFECT, WHAT PIOGLITAZONE MIGHT BE DOING IS ACTIVATING THE ANTIOXIDANT CAPACITY OF THE CELL ITSELF IN ADDITION TO BUILDING NEW MITOCHONDRIA. AND IT'S IN CLINICAL TRIALS IN FRANCE RIGHT NOW, AND IT'S USED IN DIABETES. OKAY? REMEMBER STEVE WILLI'S TALK THAT THESE INTERESTING LINKS BETWEEN DIABETES AND FRIEDREICH'S ATAXIA, AND SO THERE'S, THERE MIGHT BE SOMETHING THERE. BUT, THE BOTTOM LINE, AS DAVE SAID THIS MORNING, IS YOU'VE GOT TO TEST IT AND YOU HAVE TO DO IT IN A RIGOROUS WAY AND GET GOOD DATA TO REALLY KNOW FOR SURE. EGB 761 IS A FREE RADICAL SCAVENGER. OKAY? MEANING IT SEES FREE RADICALS, COMPOUNDS WITH THAT EXTRA ELECTRON, AND IT LOOKS FOR THOSE, AND IT EITHER GRABS THE ELECTRON OR PAIRS UP THE ELECTRON AND DETOXIFIES IT IN SOME WAY. THAT'S ENTERING INTO CLINICAL TRIALS. RESVERATROL IS THE FAMOUS COMPOUND THAT'S IN RED WINE, AN ANTI-AGING COMPOUND, AND IT HAS SOME ANTIOXIDANT PROPERTIES. AND THERE ARE MANY RESVERATROL DERIVATIVES NOW THAT ARE BEING TESTED FOR VARIOUS CONDITIONS, INCLUDING DIABETES. OX1 IS A NEW MOLECULE, AT LEAST IT CAME ONTO MY RADAR SCREEN FAIRLY RECENTLY, FIRST STUDIED BY INTELLECT NEUROSCIENCES, AND IT'S A SMALL MOLECULE, HAS GOOD DRUG-LIKE PROPERTIES, AND IS A POTENT ANTIOXIDANT. AND IT'S BEING TESTED IN VARIOUS FRIEDREICH'S MODELS NOW WITH THE HOPE OF GOING INTO CLINICAL TRIALS AS SOON AS POSSIBLE. HECHT WIZARDRY, WHAT DOES THAT REFER TO? THAT REFERS TO MY COLLEAGUE, SID HECHT. SID HECHT IS A BRILLIANT, HENCE WIZARDRY, MEDICINAL CHEMIST AT ARIZONA STATE UNIVERSITY. HE'S THE GUY BEHIND THE DRUG BLEOMYCIN, FOR THOSE OF YOU WHO KNOW ABOUT, THOSE OF YOU IN MEDICINE. HE TURNED HIS ATTENTION TO MITOCHONDRIA IN THE LAST FEW YEARS AND IS TRYING TO RATIONALLY DESIGN MOLECULES SPECIFICALLY FOR THE DEFECTS IN FRIEDREICH'S ATAXIA. AND I'VE TESTED SOME OF THESE COMPOUNDS IN MY LAB IN CELL CULTURE MODELS AND THEY WORK EXTREMELY WELL. THAT DOESN'T MEAN THEY'LL GO ON TO BE DRUGS, BUT IT'S A VERY NICE FIRST STEP. SO THAT'S SOMETHING TO STAY TUNED FOR. SO THERE ARE SOME WHO THINK THAT THESE DEFECTS THAT I'M TALKING ABOUT ACTUALLY FORM A VICIOUS CYCLE, THAT WHEN YOU HAVE DECREASED ASSEMBLY OF IRON-SULFUR CLUSTERS, THIS LEADS TO THE MITOCHONDRIAL IRON ACCUMULATION, WHICH IN TURN, INCREASES REACTIVE OXYGEN SPECIES, FREE RADICALS, OXIDANTS. OKAY? AND THAT IRON-SULFUR CLUSTERS ARE, IN TURN, DISRUPTED BY REACTIVE OXYGEN SPECIES. NOW, THIS SORT OF VICIOUS CYCLE VIEW OF THE FRIEDREICH'S UNIVERSE HAS BEEN CHALLENGED. THERE WAS A POSTER IN STRASBOURG FROM PIERRE RUSTIN'S GROUP, WHICH I DIDN'T READ SUPER CAREFULLY, SO MAYBE YOU GUYS COULD COMMENT THIS AFTERNOON ON WHETHER YOU BUY WHETHER THIS VICIOUS CYCLE IS TRUE OR NOT. IF THIS VICIOUS CYCLE IS TRUE, YOU WOULD IMAGINE THAT HITTING IT AT ANY POINT MIGHT HELP THE OTHER TWO POINTS. THERE MAY BE SOME SYNERGY THERE. ALSO, AS DAVE MENTIONED, WE MIGHT BE ABLE TO COMBINE DRUGS FROM THESE DIFFERENT GROUPS THAT I'VE TALKED ABOUT. SO THESE ARE THE DRUGS THAT I'VE TALKED ABOUT TODAY. AND, EVERY DRUG HAS SIDE EFFECTS. ALL RIGHT. SO, BUT WHAT IF, YOU KNOW, THE SIDE EFFECTS FOR THREE DIFFERENT DRUGS, ONE HITTING EACH CORNER OF THIS TRIANGLE, LET'S SAY, HAD SIDE EFFECTS IN DIFFERENT ORGAN SYSTEMS? YOU COULD PERHAPS LOWER THE DOSE OF ALL THREE OF THEM TO REDUCE THE SIDE EFFECTS, BUT THEY WOULD OVERLAP SYNERGISTICALLY IN THEIR BENEFICIAL EFFECT FOR FRIEDREICH'S ATAXIA. SO I IMAGINE THE FUTURE MIGHT HOLD A COMBINATION DRUG APPROACH TO FRIEDREICH'S ATAXIA. YOU'VE SEEN THIS SLIDE. OKAY? I TOO, LIKE DAVE, STOLE IT FROM THE FARA WEBSITE. IT'S, I NOW KNOW, BECAUSE I SAT IN THE BACK FOR DAVE'S TALK, THAT YOU CAN'T REALLY SEE IT FROM ANYWHERE EXCEPT THE VERY FRONT. SO APOLOGIES FOR THAT. HOWEVER, I WANT YOU TO-- THE REASON I PUT IT UP THERE IS THE SAME REASON DAVE DID. IT'S TO EMPHASIZE, YOU KNOW, PEOPLE ALWAYS ASK ME, THEY SAY, "ROB, YOU KNOW, WHICH IS THE ONE THAT GOING TO BE THE WINNER?" AND THE CORRECT ANSWER TO THAT QUESTION IS, "I HAVE NO IDEA." OKAY? AND IF ANYONE TELLS YOU THEY THINK THEY KNOW WHICH ONE THE WINNER IS, TAKE IT WITH A MAJOR GRAIN OF SALT. THEY PROBABLY HAVE A CONFLICT OF INTEREST. OKAY. SO YOU REALLY HAVE TO TAKE OPTIMISM FROM THE FACT THAT WE HAVE SO MANY SHOTS ON GOAL. DRUG DEVELOPMENT IS FRAUGHT WITH PERIL. A LOT OF THESE DRUGS WILL FAIL. OKAY? I CAN TELL YOU THAT RIGHT NOW. I DON'T KNOW WHICH ONES. ALL RIGHT? THESE ARE THE ONES THAT REALLY MERIT INVESTIGATION, AND THAT'S WHY THE BARS ARE EXTENDING UP INTO THE PHASE II AND PHASE III TRIALS. OKAY? AND, THAT'S WHY I'M OPTIMISTIC ABOUT GETTING GOOD TREATMENTS FOR FRIEDREICH'S ATAXIA, BECAUSE WE HAVE SO MANY SHOTS ON GOAL. SOME OF THEM ARE PROBABLY GOING TO MAKE IT THROUGH. SOME OF THEM WILL FAIL, BUT THAT'S WHERE MY OPTIMISM LIES. I ALSO WANT TO, THIS REMINDS ME TO TELL YOU THAT IF YOU GO TO THE FARA WEBSITE, CUREFA.ORG, YOU CAN FIND THIS, WHICH JEN FARMER UPDATES FROM TIME TO TIME, AND YOU CAN ALSO FIND CAPSULE SUMMARIES OF MOST OF THE DRUGS ON THIS PIPELINE. JUST CLICK ON THE RESEARCH LINK, AND THEN ON THE PIPELINE. AND, YOU CAN READ ABOUT EACH OF THESE DRUGS, YOU KNOW. I PUT SOME NOTES ABOUT THE ONES I'VE TALKED ABOUT IN MY, IN YOUR HANDOUT, BUT, IT'S ALWAYS THERE ON THE FARA WEBSITE, AND THEY UPDATE IT REGULARLY. IT'S ACTUALLY A FANTASTIC WEBSITE AS A RESOURCE FOR THE PATIENT COMMUNITY. SO TAKE ADVANTAGE OF THAT. I ALWAYS ACKNOWLEDGE MY FRIEND, DAVE LYNCH, WHO I WORK WITH HAND IN HAND AT THE UNIVERSITY OF PENNSYLVANIA. RON BARTEK, JENNIFER FARMER, AND FELICIA DEROSA ARE THE FULL-TIMERS AT THE FRIEDREICH'S ATAXIA RESEARCH ALLIANCE. THERE ARE A LOT OF OTHER FARA PEOPLE HERE, TOO. I SEE KYLE BRYANT, HOLLY HEDRICK, PAUL AVERY IS HERE, SANDY LANE. I CAN'T EMPHASIZE HOW MUCH FARA DOES FOR YOU. ALL RIGHT? THAT SOME YOU KNOW ABOUT AND SOME YOU DON'T. FARA IS THE ENVY OF OTHER RARE DISEASE ORGANIZATIONS, IN PART BECAUSE OF THAT LAST SLIDE I SHOWED YOU. A LOT OF OTHER RARE DISEASE ORGANIZATIONS SAY, "LET ME GET THIS STRAIGHT. THE GENE WAS DISCOVERED IN THE LATE 90S, AND YOU ALREADY HAVE THAT MANY DRUGS IN CLINICAL TRIALS? THAT'S IMPOSSIBLE." ALL RIGHT, WELL, IT'S BECAUSE OF THOSE PEOPLE THAT I JUST MENTIONED, AND MANY OTHERS THAT IT'S POSSIBLE. SO, THANK THEM IF YOU SEE THEM. I ALSO ALWAYS MAKE A POINT OF MENTIONING WILLIAM HARTNETT, MARIANNE WILCOX, AND MARTIN OHMAN, AND THE REST OF THE EDS TEAM. THESE ARE THE FOLKS WHO DONATED THEIR TIME TO BUILD OUR WEB-BASED DATA ENTRY SYSTEM FOR MULTI-CENTER TRIALS. AND THEY WORK TOTALLY BEHIND THE SCENES, YOU NEVER SEE THEM, YOU NEVER HEAR ABOUT THEM. THAT'S WHY I'M SAYING THEIR NAMES NOW, BECAUSE IT REALLY HAS FACILITATED DEVELOPMENT OF THE ATAXIA SCALES THAT DAVE TALKED ABOUT, AND HAS GREASED THE SKIDS TO GET THESE DRUGS INTO CLINICAL TRIALS AS QUICKLY AS POSSIBLE. AND, OF COURSE, PATIENTS AND THEIR FAMILIES, AND I WILL STOP THERE. [APPLAUSE]