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

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