CHMP2B regulates TDP-43 phosphorylation and proteotoxicity via modulating CK1 turnover independent of the autophagy-lysosomal pathway

Abstract

< 250 words) 26Protein inclusions containing phosphorylated TDP-43 are a shared pathology in several 27 neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal 28 dementia (FTD). However, most ALS/FTD patients do not have a mutation in TDP-43 or the 29 enzymes directly regulating its phosphorylation. It is intriguing how TDP-43 becomes 30 hyperphosphorylated in each disease condition. In a genetic screen for novel TDP-43 modifiers, 31we found that knockdown (KD) of CHMP2B, a key component of the endosomal sorting complex 32 required for transport (ESCRT) machinery, suppressed TDP-43-mediated neurodegeneration in 33Drosophila. Further investigation using mammalian cells indicated that CHMP2B KD decreased 34 whereas its overexpression (OE) increased TDP-43 phosphorylation levels. Moreover, a known 35 FTD-causing mutation CHMP2B intron5 promoted hyperphosphorylation, insolubility and 36 cytoplasmic accumulation of TDP-43. Interestingly, CHMP2B did not manifest these effects by its 37 well-known function in the autophagy-lysosomal pathway. Instead, the kinase CK1 tightly 38 regulated TDP-43 phosphorylation level in cells, and CHMP2B OE or CHMP2B Intron5 significantly 39 decreased ubiquitination and the turnover of CK1 via the ubiquitin-proteasome (UPS) pathway. 40Finally, we showed that CHMP2B protein levels increased in the cerebral cortices of aged mice, 41 which might underlie the age-associated TDP-43 pathology and disease onset. Together, our 42 findings reveal a molecular link between the two ALS/FTD-pathogenic proteins CHMP2B and 43 TDP-43, and provide an autophagy-independent mechanism for CHMP2B in pathogenesis. 44 Linking CHMP2B to pTDP-43 via CK1_Sun et al Page 3 / 34 SIGNIFICANCE STATEMENT (< 120 words) 45 TDP-43 and CHMP2B are both ALS/FTD-associated proteins. Protein aggregations containing 46 phosphorylated TDP-43 are a pathological hallmark of ALS/FTD; however, it is unclear how 47 increased phosphorylation of TDP-43 occurs in diseases. The pathogenesis of CHMP2B has 48mainly been considered as a consequence of autophagy-lysosomal dysfunction. Here, we reveal 49 that increase of CHMP2B levels (which occurs in aged mouse brains) or expression of the 50 disease-causing mutation CHMP2B Intron5 promotes TDP-43 hyperphosphorylation, insolubility 51 and cytoplasmic mislocalization. This effect is independent of the autophagy-lysosomal pathway 52 but rather relies on the proteasome-mediated turnover of the kinase CK1 that phosphorylates 53 TDP-43. Together, we provide a new molecular mechanism of CHMP2B pathogenesis by linking 54 it to TDP-43 pathology via CK1. 55

Experimental Disease-Modifying Agents for Frontotemporal Lobar Degeneration

“…137,138 While the disruption of CHMP2B perturbs endo-lysosomal trafficking, vesicle fusion and autophagic degradation, it also promotes TDP-43 hyperphosphorylation and insolubility by the control of the UPS-dependent turnover of CK-1. 139,140 Given that therapeutics have been identified for CHMP2B this could represent a promising therapeutic axis. 139 On the contrary, TDP-43 itself plays an active role in the regulation of these mechanisms, modulating the expression of the phagosome machinery.…”