Modulation of synaptic plasticity, motor unit physiology, and TDP-43 pathology by CHCHD10.

Mutations in CHCHD10, a gene coding for a mitochondrial intermembrane space protein, are associated with Frontotemporal dementia (FTD)-Amyotrophic lateral sclerosis (ALS) spectrum disorders, which are pathologically characterized by cytoplasmic inclusions containing TDP-43. FTD/ALS-linked CHCHD10 mutations and TDP-43 inclusions similarly induce mitochondrial defects in respiration, fusion/fission, mtDNA stability, and cristae structure, while sizeable amounts of cytoplasmic TDP-43 aggregates are found in mitochondria. However, the mechanistic link between CHCHD10 and TDP-43 pathogenesis remains unclear. In this study, we present immunohistochemical and biochemical evidence demonstrating that insoluble CHCHD10 aggregates accumulate and colocalize with phospho-TDP-43 inclusions in brains of FTLD-TDP and AD patients, and that insoluble CHCHD10 levels tightly correlate with insoluble TDP-43 levels in control and FTLD-TDP brains. In an experimental exploration of this pathological phenotype, transgenic mice neuronally expressing FTD/ALS-linked CHCHD10R15L or CHCHDS59L mutations but not CHCHD10WT transgenic mice exhibit significantly increased CHCHD10 aggregation and phospho-TDP-43 pathology, which often colocalize within the same inclusions. Such pathologies are reflected in poor functional outcomes in long-term synaptic plasticity, motor unit physiology, and behavior in CHCHD10R15L and CHCHDS59L transgenic mice. In contrast, expression of CHCHD10WT in hTDP-43 transgenic mice (TAR4;CHCHD10WT) significantly mitigates phospho-TDP-43 pathology and rescues TDP-43-induced impairments in synaptic integrity and long-term synaptic plasticity. In isolated mitochondria, the S59L mutation induces the aggregation of resident CHCHD10S59L protein as well as the aggregation and slower turnover of recombinant TDP-43 imported into mitochondria. Likewise, in an in vitro cell-free system, the S59L mutation induces the aggregation of CHCHD10S59L protein while simultaneously enhancing the aggregation of recombinant TDP-43, as evidenced by filter trap assays and atomic force microscopy. In contrast, recombinant CHCHD10WT inhibits the growth of TDP-43 aggregates. These results in human brains, transgenic mice, and in vitro systems substantiate the role of wild type and mutant CHCHD10 in modulating mitochondrial CHCHD10 and TDP-43 pathogenesis together with associated phenotypes in long-term synaptic plasticity and motor unit physiology in mice and humans.

SSH1 impedes SQSTM1/p62 flux and MAPT/Tau clearance independent of CFL (cofilin) activation.

Accumulation of toxic protein assemblies and damaged mitochondria are key features of neurodegenerative diseases, which arise in large part from clearance defects in the Macroautophagy/autophagy-lysosome system. The autophagy cargo receptor SQSTM1/p62 plays a major role in the clearance of ubiquitinated cargo through Ser403 phosphorylation by multiple kinases. However, no phosphatase is known to physiologically dephosphorylate SQSTM1 on this activating residue. RNAi-mediated knockdown and overexpression experiments using genetically encoded fluorescent reporters and defined mutant constructs in cell lines, primary neurons, and brains show that SSH1, the canonical CFL (cofilin) phosphatase, mediates the dephosphorylation of phospho-Ser403-SQSTM1, thereby impairing SQSTM1 flux and phospho-MAPT/tau clearance. The inhibitory action of SSH1 on SQSTM1 is fully dependent on SQSTM1 Ser403 phosphorylation status and is separable from SSH1-mediated CFL activation. These findings reveal a unique action of SSH1 on SQSTM1 independent of CFL and implicate an inhibitory role of SSH1 in SQSTM1-mediated clearance of autophagic cargo, including phospho-MAPT/tau. Abbreviations: AAV: adeno-associated virus; Aß42O: amyloid ß1-42 oligomers; AD: Alzheimer disease; CA3: cornu Ammonis 3; CSNK2/CK2: casein kinase 2; FCCP: 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile; FTLD: frontotemporal lobar degeneration; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SQSTM1/p62: sequestosome-1; PLA: proximity ligation assay; RFP: red fluorescent protein; RIPA: radioimmunoprecipitation assay; shRNA: short hairpin RNA; siRNA: small interfering RNA; Ser403: Serine403; SSH1: slingshot protein phosphatase 1; TBK1: TANK-binding kinase 1; ULK: unc-51 like kinase 1.

CHCHD10-regulated OPA1-mitofilin complex mediates TDP-43-induced mitochondrial phenotypes associated with frontotemporal dementia.

Mutations in CHCHD10, a gene coding for a mitochondrial protein, are implicated in ALS-FTD spectrum disorders, which are pathologically characterized by transactive response DNA binding protein 43 kDa (TDP-43) accumulation. While both TDP-43 and CHCHD10 mutations drive mitochondrial pathogenesis, mechanisms underlying such phenotypes are unclear. Moreover, despite the disruption of the mitochondrial mitofilin protein complex at cristae junctions in patient fibroblasts bearing the CHCHD10S59L mutation, the role of CHCHD10 variants in mitofilin-associated protein complexes in brain has not been examined. Here, we utilized novel CHCHD10 transgenic mouse variants (WT, R15L, & S59L), TDP-43 transgenic mice, FTLD-TDP patient brains, and transfected cells to assess the interplay between CHCHD10 and TDP-43 on mitochondrial phenotypes. We show that CHCHD10 mutations disrupt mitochondrial OPA1-mitofilin complexes in brain, associated with impaired mitochondrial fusion and respiration. Likewise, CHCHD10 levels and OPA1-mitofilin complexes are significantly reduced in brains of FTLD-TDP patients and TDP-43 transgenic mice. In cultured cells, CHCHD10 knockdown results in OPA1-mitofilin complex disassembly, while TDP-43 overexpression also reduces CHCHD10, promotes OPA1-mitofilin complex disassembly via CHCHD10, and impairs mitochondrial fusion and respiration, phenotypes that are rescued by wild type (WT) CHCHD10. These results indicate that disruption of CHCHD10-regulated OPA1-mitofilin complex contributes to mitochondrial abnormalities in FTLD-TDP and suggest that CHCHD10 restoration could ameliorate mitochondrial dysfunction in FTLD-TDP.

Dual role of cofilin in APP trafficking and amyloid-ß clearance.

The accumulation of amyloid-ß (Aß) plays a pivotal early event in the pathogenesis of Alzheimer's disease (AD). In the brain, neurons produce Aß by the proteolytic processing of amyloid precursor protein (APP) through the endocytic pathway, whereas microglia mediate Aß clearance also via endocytic mechanisms. Previous studies have shown the critical importance of cofilin, a filamentous actin-severing protein, in actin dynamics and pathogen-triggered endocytic processes. Moreover, the binding of Aß42 oligomers to ß1-integrin triggers the cofilin activation, and in turn, cofilin promotes the internalization of surface ß1-integrin. However, a role for cofilin in APP processing and Aß metabolism has not been investigated. In this study, we found that knockdown of cofilin in Chinese hamster ovary 7WD10 cells and primary neurons significantly reduces Aß production by increasing surface APP (sAPP) levels. Expression of active (S3A) but not inactive (S3E) cofilin reduces sAPP levels by enhancing APP endocytosis. Accordingly, Aß deposition in APP and presenilin 1 (PS1) transgenic mice is significantly reduced by genetic reduction of cofilin (APP/PS1;cofilin+/-). However, the reduction of Aß load in APP/PS1;cofilin+/- mice is paradoxically associated with significantly increased ionized calcium-binding adaptor molecule 1-positive microglial activation surrounding Aß deposits. Primary microglia isolated from cofilin+/- mice demonstrate significantly enhanced state of activation and greater ability to uptake and clear Aß42, which is reversed with the active (S3A) but not inactive (S3E) form of cofilin. These results taken together indicate a significant role for cofilin in Aß accumulation via dual and opposing endocytic mechanisms of promoting Aß production in neurons and inhibiting Aß clearance in microglia.-Liu, T., Woo, J.-A. A., Yan, Y., LePochat, P., Bukhari, M. Z., Kang, D. E. Dual role of cofilin in APP trafficking and amyloid-ß clearance.

Activated cofilin exacerbates tau pathology by impairing tau-mediated microtubule dynamics.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. While the accumulation of Aß is pivotal to the etiology of AD, both the microtubule-associated protein tau (MAPT) and the F-actin severing protein cofilin are necessary for the deleterious effects of Aß. However, the molecular link between tau and cofilin remains unclear. In this study, we found that cofilin competes with tau for direct microtubule binding in vitro, in cells, and in vivo, which inhibits tau-induced microtubule assembly. Genetic reduction of cofilin mitigates tauopathy and synaptic defects in Tau-P301S mice and movement deficits in tau transgenic C. elegans. The pathogenic effects of cofilin are selectively mediated by activated cofilin, as active but not inactive cofilin selectively interacts with tubulin, destabilizes microtubules, and promotes tauopathy. These results therefore indicate that activated cofilin plays an essential intermediary role in neurotoxic signaling that promotes tauopathy.

Cofilin-mediated Neuronal Apoptosis via p53 Translocation and PLD1 Regulation.

Amyloid ß (Aß) accumulation is an early event in the pathogenesis of Alzheimer's disease (AD), leading to mitochondrial and synaptic dysfunction, tau accumulation, and eventual neuronal death. While the p53 apoptotic pathway has clearly been associated with Aß deposits and neuronal apoptosis, the critical upstream factors contributing to p53 activation in AD are not well understood. We have previously shown that cofilin activation plays a pivotal role in Aß-induced mitochondrial and synaptic dysfunction. In this study, we show that activated cofilin (S3A) preferentially forms a complex with p53 and promotes its mitochondrial and nuclear localization, resulting in transcription of p53-responsive genes and promotion of apoptosis. Conversely, reduction of endogenous cofilin by knockdown or genetic deficiency inhibits mitochondrial and nuclear translocation of p53 in cultured cells and in APP/PS1 mice. This cofilin-p53 pro-apoptotic pathway is subject to negative regulation by PLD1 thorough cofilin inactivation and inhibition of cofilin/p53 complex formation. Finally, activated cofilin is unable to induce apoptosis in cells genetically lacking p53. These findings taken together indicate that cofilin coopts and requires the nuclear and mitochondrial pro-apoptotic p53 program to induce and execute apoptosis, while PLD1 functions in a regulatory multi-brake capacity in this pathway.

Loss of function CHCHD10 mutations in cytoplasmic TDP-43 accumulation and synaptic integrity.

Although multiple CHCHD10 mutations are associated with the spectrum of familial and sporadic frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) diseases, neither the normal function of endogenous CHCHD10 nor its role in the pathological milieu (that is, TDP-43 pathology) of FTD/ALS have been investigated. In this study, we made a series of observations utilizing Caenorhabditis elegans models, mammalian cell lines, primary neurons and mouse brains, demonstrating that CHCHD10 normally exerts a protective role in mitochondrial and synaptic integrity as well as in the retention of nuclear TDP-43, whereas FTD/ALS-associated mutations (R15L and S59L) exhibit loss of function phenotypes in C. elegans genetic complementation assays and dominant negative activities in mammalian systems, resulting in mitochondrial/synaptic damage and cytoplasmic TDP-43 accumulation. As such, our results provide a pathological link between CHCHD10-associated mitochondrial/synaptic dysfunction and cytoplasmic TDP-43 inclusions.