Axonal RNA Profiling in CMT for Studying Axon Degeneration

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 Differentiation Somatic cells iPS cells • Phenotype characterization • Cell morphology • Organelle trafficking • •Gene expression • Neuronal-glia interactions • Mechanistic studies • Target identification (pathways, molecules) Drug discovery High throughput screening Neurons Glia Saporta et al, Stem Cell Research and Therapy, 2011

iPSC IN CMT NEFL accumulation Decreased mitochondria displacement NF-L TUJ1 HOESCHT 63x MFN2 iPS Control mitoDsRed eGFP axon mitochondria NFL Clone 2 NFL Clone 1 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 CMT-related 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 2. Motor neuron differentiation 3. Magnetic sorting (L1CAM) 4. Axon isolation 5. Expressing profiling (RNAseq) 6. Gene pathway analysis

ENRICHMENT OF MATURE MOTOR NEURONS L1CAM HB9 positive positive

ISOLATION OF AXONS Merge Hoechst NEFL68 Top Bottom

RNA ISOLATION MFN2 Control

RNA EXPRESSION – TOP 1000 RANKED GENES CONTROL axon somatodendritic SOMATODENDRITIC AXON Control MFN2 Control MFN2 n=1

RNA EXPRESSION – CONTROL VS. MFN2 SOMATODENDRITIC Biological process • ubiquitin-dependent protein catabolic process Control MFN2

RNA EXPRESSION – CONTROL VS. MFN2 AXONS Biological process • protein folding Control MFN2

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 Wisconsin-Madison 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 NEFL control 50 um

MORPHOLOGICAL ANALYSIS – ISOLATED AXONS Axonal beading – CMT2E

Axonal RNA Profiling in CMT for Studying Axon Degeneration

Axonal RNA Profiling in CMT for Studying Axon Degeneration

Presentation Transcript

Gérard FINET . MD PhD

Peter Pronovost, MD, PhD

Philippe Froguel, MD, PhD

Milan Smid, MD, PhD

François Schiele , MD, PhD, Nicolas Meneveau , MD, PhD,

DOMINIQUE CHARRON MD PhD

Serge Resnikoff MD, PhD

Sergio Iavicoli – MD, PhD

Enrico Romagnoli , MD PhD

Paul Kurdyak MD PhD

Laurence de Leval, MD, PhD,* David Waltregny, MD, PhD,w Jacques Boniver, MD, PhD,*

Hasan Bayram, MD, PhD .

Chengpei Xu, MD, PhD

Viroj Tangcharoensathien, MD PhD Phusit Prakongsai, MD Supon Limwattananon, PhD

Szabolcs Péter , MD, PhD Peter Weber, MD, PhD

Gérard Friedlander , MD, PhD

Mario De La Rosa, PhD Director

MICROSURGERY COURSE Prof. Rado Zic, MD, PhD Zlatko Vlajcic, MD, PhD Kresimir Martic, MD, PhD

THE HEADACHE AND SLEEP DIARY Mario F P Peres, MD, PhD