| Abstract No.: | B-C2111 |
| Country: | Canada |
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| Title: | HEAT SHOCK PROTEINS REDUCE NEUROFILAMENT AND MITOCHONDRIAL ABNORMALITIES IN CULTURE MODELS OF CHARCOT MARIE TOOTH TYPE 2E (CMT2E) |
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| Authors/Affiliations: | 2 Benoit Gentil*; 2 Miranda Tradewell; 1 Walter Mushynski; 2 Heather Durham ;
1 McGill University, Montreal, QC, Canada ; 2 MNI McGill University, Montreal, QC, Canada
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| Content: | Objective: CMT2E is caused by dominantly inherited mutations in the nefl gene encoding the neurofilament light subunit (NFL). Mutations disrupt the assembly and transport of neurofilaments in motor and sensory axons. The goal of the present study was to investigate the effect of heat shock proteins (HSPs) with protein chaperoning activity on neurofilament and mitochondrial abnormalities induced by NFL mutants.
Material and methods: A primary culture model of CMT2E was established by microinjecting plasmids encoding NFLP8R or NFLQ333P into nuclei of motor neurons in long term dissociated cultures of murine embryonic spinal cord (with NFLwt serving as control). Multiple or individual HSPs were expressed by co-injection of plasmid encoding a constitutively active Hsf1 (the major heat shock transcription factor), Hsp27 or Hsp70. The influence of NFM or NFH on neurofilament abnormalities induced by CMT-related mutant NFL and the sensitivity to intervention by HSPs was investigated in SW13- cells, which lack endogenous intermediate filaments. Mitochondrial morphology was assessed in the primary culture model by microfluorometric imaging of motor neurons co-expressing pOCT-GFP and NFL constructs, mitochondrial length serving as the quantitative measure.
Results: Mutant NFL initially integrated into endogenous neurofilaments in motor neurons, but subsequently disrupted the network. After 7 days, about 50% of motor neurons expressing either NFLP8R or NFLQ333P had aggregated neurofilament proteins, bundled/fragmented neurofilaments, or axonal swellings (significant cell death was not observed with injection of this plasmid concentration). Co-expression of HSPs significantly reduced these abnormalities, but in a mutant-specific manner: Hsp27 was more effective in preventing cytoskeletal abnormalities induced by NFLP8R, whereas Hsp70 was more effective against NFLQ333P. In the SW13- model, HSPs were more effective in preventing abnormalities of neurofilaments made of NFL and NFM compared to NFL and NFH. A novel finding in the motor neuron culture model was a profound effect of mutant NFL on mitochondrial morphology and distribution long before abnormalities of the neurofilament network were detected. Overexpression of HSPs significantly reduced mitochondrial rounding and maintained a more even axonal distribution with the selectivity described above for preventing cytoskeletal abnormalities.
Conclusions: In a primary culture model of CMT2E, the predominant phenotype is mitochondrial and the abnormalities are similar to those that occur with mutant mitofusin2 responsible for CMT2A. That mitochondrial abnormalities precede neurofilament disruption suggests an important role in pathogenesis. That both mitochondrial and cytoskeletal abnormalities are reduced by HSPs supports the development of chaperone-based therapies for CMT. The results also support a normal physiological role for HSPs as chaperones assisting in neurofilament assembly.
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