Download Axonal RNA Profiling of Human Motor Neurons from Patients with

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