No abstract

The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. 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. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43–mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. 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. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.

TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, we uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). We also detected a robust immune overactivation in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, we showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, our findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases.

< 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

RNA-binding proteins (RBPs) and RNAs can form dynamic, liquid droplet-like cytoplasmic condensates, known as stress granules (SGs), in response to a variety of cellular stresses. This process is driven by liquid–liquid phase separation, mediated by multivalent interactions between RBPs and RNAs. The formation of SGs allows a temporary suspension of certain cellular activities such as translation of unnecessary proteins. Meanwhile, non-translating mRNAs may also be sequestered and stalled. Upon stress removal, SGs are disassembled to resume the suspended biological processes and restore the normal cell functions. Prolonged stress and disease-causal mutations in SG-associated RBPs can cause the formation of aberrant SGs and/or impair SG disassembly, consequently raising the risk of pathological protein aggregation. The machinery maintaining protein homeostasis (proteostasis) includes molecular chaperones and co-chaperones, the ubiquitin-proteasome system, autophagy, and other components, and participates in the regulation of SG metabolism. Recently, proteostasis has been identified as a major regulator of SG turnover. Here, we summarize new findings on the specific functions of the proteostasis machinery in regulating SG disassembly and clearance, discuss the pathological and clinical implications of SG turnover in neurodegenerative disorders, and point to the unresolved issues that warrant future exploration.

TAR DNA binding protein 43 kDa (TDP-43) undergoes liquid-liquid phase separation (LLPS) and forms reversible, cytoprotective nuclear bodies (NBs) in response to stress in cells. Abnormal liquid-to-solid phase transition condenses TDP-43 into irreversible pathological fibrils, which is associated with neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). However, the mechanisms how cells maintain the dynamics of TDP-43 NBs in stressed conditions are not well understood. Here, we show that the molecular chaperon heat shock protein 70 (Hsp70) is recruited into TDP-43 NBs in stressed cells. It co-phase separates with TDP-43 and delays the maturation of TDP-43 droplets in vitro. In cells, downregulation of Hsp70 not only diminishes the formation but also reduces the dynamics of TDP-43 NBs especially during prolonged stress, which potentiates the cytotoxicity of TDP-43. Using NMR, we reveal that Hsp70 binds to the highly aggregation-prone, transient α-helix of TDP-43 via its nucleotide-binding domain, which keeps TDP-43 in the highly dynamic, liquid-like phase and prevents pathological aggregation of TDP-43 both in vitro and in cells. Collectively, our findings demonstrate a crucial role of Hsp70 in chaperoning TDP-43 in the liquid-like phase, which provides a novel layer of the molecular mechanism how chaperons help proteins to remain functional and protect cells from stressed and/or diseased conditions.