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Axonal RNA profiling of human motor neurons from patients with CMT as a novel approach to study axon degeneration Mario Saporta, MD, PhD ANIMAL MODELS OF CMT: FAILURE TO TRANSLATE TO HUMAN THERAPIES Adebola et al, Hum Mol Genet 2015 Saporta et al, Brain 2012 HUMAN DISEASE MODELING USING INDUCED PLURIPOTENT STEM CELLS (iPSC) Reprogramming iPS cells Phenotype characterization Drug discovery •Cell morphology • Organelle trafficking High throughput screening •Gene expression • Neuronal-glia interactions Mechanistic studies • Target identification (pathways, molecules) Differentiation Somatic cells Neurons Glia Saporta et al, Stem Cell Research and Therapy, 2011 iPSC IN CMT NEFL accumulation Decreased mitochondria displacement mitoDsRed eGFP iPS Control MFN2 NFL Clone 1 NFL Clone 2 mitochondria NF-L TUJ1 HOESCHT 63x axon Saporta et al, Exp. Neurol. 2015 DISEASE MECHANISMS IN CMT RNA? Lupski et al., 2014 DISREGULATION OF RNA METABOLISM IN AXONS Kyota Yasuda and Stavroula Mili, 2016 QUESTIONS: What is the role of axonal RNA transport in CMTrelated axonal degeneration? Can axonal RNA profiling identify common molecular pathways involved in axonal degeneration in distinct types of CMT? RESEARCH STRATEGY OUTLINE 1. iPSC expansion 4. Axon isolation 2. Motor neuron differentiation 5. Expressing profiling (RNAseq) 3. Magnetic sorting (L1CAM) 6. Gene pathway analysis ENRICHMENT OF MATURE MOTOR NEURONS L1CAM HB9 positive positive ISOLATION OF AXONS Hoechst NEFL68 Top Bottom Merge RNA ISOLATION Control MFN2 RNA EXPRESSION – TOP 1000 RANKED GENES CONTROL axon somatodendritic SOMATODENDRITIC Control MFN2 AXON Control MFN2 n=1 RNA EXPRESSION – CONTROL VS. MFN2 SOMATODENDRITIC Biological process • cellular macromolecule localization • ubiquitin-dependent protein • respiratory electron transport chain catabolic process • electron transport chain Control MFN2 RNA EXPRESSION – CONTROL VS. MFN2 AXONS Biological process • cell cycle process • RNA splicing, via transesterification reactions • oxidation reduction • regulation of cellular protein metabolic process • cytoskeleton organization • protein folding • RNA splicing, via transesterification reactions with bulged adenosine as nucleophile • mRNA metabolic process • nuclear mRNA splicing, via spliceosome Control MFN2 • mitotic cell cycle CONCLUSIONS • Human iPSC-derived motor neurons are an useful model to study CMT. • Several optimizations can increase the reliability and relevance of this platform: • Enrichment techniques to generated pure motor neuron cultures • Techniques to isolate axons from the neuronal body • Unbiased gene expression profiling • Preliminary data suggest that several pathways are differently activated in MFN2 axons. • Careful confirmation and detailing of these networks may provide insights into pathomechanisms in Axonal CMT and may help identify new therapeutic targets. Acknowledgement University of Miami Saporta Lab Renata Maciel Züchner’s Lab Adriana Rebelo Dana Bis Cima Saghira Stephan Züchner Michael Benatar University of WisconsinMadison John Svaren Anita Bhattacharyya University of Iowa Michael Shy ENRICHMENT OF MATURE MOTOR NEURONS ISOLATION OF AXONS – ENRICHMENT OF AXONS MORPHOLOGICAL ANALYSIS – ISOLATED AXONS Axonal beading – CMT2E NEFL68 control NEFL 50 um MORPHOLOGICAL ANALYSIS – ISOLATED AXONS Axonal beading – CMT2E