The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity

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

OVEREXPRESSION OF HSPB8 PROTECTS AGAINST TDP43-MEDIATED TOXICITY IN DROSOPHILA

Several neurodegenerative diseases are characterized by the accumulation of aggregates. Boosting aggregate clearance by proteasome and autophagy, with the assistance of chaperones, exerts protective functions in these diseases. We focus on the HSPB8-BAG3-Hsp70 chaperone complex. In cells HSPB8 reduces the aggregation of a truncated form of TDP43 associated with ALS (Crippa 2010). First, we tested whether overexpression of HSP67Bc (Drosophila HSPB8) decreases TDP43-mediated toxicity in vivo, using Drosophila expressing mutated or truncated TDP43. HSP67Bc decreases the eye degeneration caused by NLS-TDP43, which by accumulating in the cytosol causes toxicity (Ritson 2010). This correlates with a reduction of NLS-TDP43 protein levels mediated by HSP67Bc. Inversely, silencing HSP67Bc increases both NLS-TDP43 and ubiquitinated proteins levels, suggesting that HSP67Bc participates to proteostasis. Next, we used flies expressing a truncated form of TDP43 (TDP35), which causes pupae lethality (similarly to truncated TDP25; Gregory 2012). Co-expression of HSP67Bc with TDP35 rescued pupae lethality, further supporting its protective role in vivo. We then asked how mechanistically HSPB8 protects against TDP43-mediated toxicity. Recent studies revealed the presence of RNA-binding proteins component of stress granules (SG) in the proteinaceous inclusions. SGs are mRNA-protein aggregates induced by stress, which serve prosurvival functions. Interestingly, autophagy and specific chaperones indirectly assist SG dynamics (Buchan 2013; Seguin 2014). We investigated whether the HSPB8-BAG3-Hsp70 complex, may indirectly modulate SG dynamics. Intriguingly, upon stress HSPB8 is recruited into SG whereas BAG3 colocalizes with ubiquitinated defective ribosomal products, which are excluded from, but adjacent to SG. Our data open the possibility that HSPB8 and BAG3 may contribute to regulate both protein clearance and SG dynamics, thereby assisting restoration of protein and RNA homeostasis