Abstract
Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.
Highlights
Amyotrophic lateral sclerosis (ALS) is the most common adultonset motor neurone disease, which leads to progressive muscle weakness and eventual respiratory failure
We characterized the biochemical behaviour of the GFP-tagged FL TAR-DNA-binding protein 43 (TDP-43) and the two major fragments derived from its caspase cleavage TDP-35 and TDP-25 in immortalized motor neurone NSC34 cells
By transiently overexpressing the different N-terminal GFP-tagged TDP variants, or GFP itself, we found that, while GFP was distributed in a diffuse manner, GFP-TDP-43 was confined to the nucleus in the vast majority of motor neurones, while both GFP-TDP-35 and GFP-TDP-25 were observed to be mislocalized to the cytoplasm (Figure 1A)
Summary
Amyotrophic lateral sclerosis (ALS) is the most common adultonset motor neurone disease, which leads to progressive muscle weakness and eventual respiratory failure. The two fragments differ in the context that TDP-35 retains both the RRM1 and RRM2 sequences, while only the RRM2 is preserved in TDP-25 It is not clear if this difference can differentially affect TDP-25 and TDP-35 activity, it has been suggested that only TDP-35 is able to deregulate pre-RNA splicing [5,9]. Both TDP-25 and TDP-35 fragments lack the NLS and retain the NES, both mislocalize to the cytoplasm and both are highly aggregation-prone [10]
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