![]() ![]() Mean ± SEM, n = ∼10 eyes/group (a–f), n = ∼10 vials/group with ∼20 flies per vial (g) the number of flies tested in each group is as indicated (h) and n = 3 (i, k, and l). The protein levels are normalized to tubulin or total hTDP-43 and shown as percentages to that of the RNAi-Ctrl group. hTDP-43, human TDP-43 p-hTDP-43, phosphorylated hTDP-43. (j–l) Western blot analysis (j) and quantifications of phosphorylation (p409/410 k) and total protein levels (l) of hTDP-43 in the fly heads. The mRNA levels are normalized to actin and shown as the average percentage compared to that of the control flies (UAS- lacZ). (i) qPCR analysis of the mRNA levels of CHMP2B in the heads of TDP-43 flies. (g and h) Adult-onset, neuronal ( elavGS) down-regulation of CHMP2B suppresses hTDP-43–induced climbing deficits (g) and extends the shortened lifespan (h). The degeneration score (mean ± SEM) and the statistical significance compared with the UAS- lacZ (UAS-Ctrl) flies are indicated. (a–f) KD of CHMP2B (#28531 GMR-Gal4) suppresses hTDP-43–induced eye degeneration in flies. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.ĬHMP2B modifies the neurotoxicity and affects the phosphorylation levels of hTDP-43 in an in vivo Drosophila model. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. ![]() In this study, we identify CHMP2B as a modifier of TDP-43-mediated neurodegeneration in a Drosophila screen. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy-endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. ![]()
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