C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner

Leskelä, Stina; Huber, Nadine; Rostalski, Hannah; Natunen, Teemu; Remes, Anne M.; Takalo, Mari; Hiltunen, Mikko; Haapasalo, Annakaisa

doi:10.3390/cells8101233

Cited by 19 publications

(22 citation statements)

References 42 publications

(95 reference statements)

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“…As reported in other studies, we also presented supporting evidence that C9orf72 protein levels are positively correlated with those of SMCR8 in cultured cells [ 26 , 28 , 29 , 32 , 47 , 54 , 65 , 103 ]. Furthermore, we now show that SMCR8 protein expression is reduced in the brains of C9ALS patients compared with unaffected controls (and as also recently noted by [ 25 ].…”

Section: Discussionsupporting

confidence: 91%

“…Despite its strong association with protein-degradation factors, SMCR8 overexpression does not stimulate degradation of C9orf72 protein with which it is in complex. Contrarily, multiple studies in cells and knockout mice have shown that protein but not RNA levels of SMCR8 and C9orf72 are positively correlated, suggesting that in complex the two proteins stabilize and protect each other from degradation [ 26 , 28 , 29 , 32 , 47 , 54 , 65 , 103 ]. On the other hand, increased SMCR8 protein reportedly has little effect on WDR41 levels in KO mice or cells [ 32 , 35 ].…”

Section: Resultsmentioning

confidence: 99%

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C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

Goodier

Soares

Pereira

et al. 2020

acta neuropathol commun

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A pathogenic GGGCCC hexanucleotide expansion in the first intron/promoter region of the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS). The C9orf72 gene product forms a complex with SMCR8 (Smith-Magenis Syndrome Chromosome Region, Candidate 8) and WDR41 (WD Repeat domain 41) proteins. Recent studies have indicated roles for the complex in autophagy regulation, vesicle trafficking, and immune response in transgenic mice, however a direct connection with ALS etiology remains unclear. With the aim of increasing understanding of the multi-functional C9orf72-SMCR8-WDR41 complex, we determined by mass spectrometry analysis the proteins that directly associate with SMCR8. SMCR8 protein binds many components of the ubiquitin-proteasome system, and we demonstrate its poly-ubiquitination without obvious degradation. Evidence is also presented for localization of endogenous SMCR8 protein to cytoplasmic stress granules. However, in several cell lines we failed to reproduce previous observations that C9orf72 protein enters these granules. SMCR8 protein associates with many products of genes associated with various Mendelian neurological disorders in addition to ALS, implicating SMCR8-containing complexes in a range of neuropathologies. We reinforce previous observations that SMCR8 and C9orf72 protein levels are positively linked, and now show in vivo that SMCR8 protein levels are greatly reduced in brain tissues of C9orf72 gene expansion carrier individuals. While further study is required, these data suggest that SMCR8 protein level might prove a useful biomarker for the C9orf72 expansion in ALS.

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Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

Goodier

Soares

Pereira

et al. 2020

acta neuropathol commun

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“…Degradation of one of these adaptor proteins, p62, is used as another marker for autophagy. Also, it has been proposed that the C9orf72 proteins are involved in the initial phase of autophagy ( 53 , 54 ).…”

Section: Resultsmentioning

confidence: 99%

BV-2 Microglial Cells Overexpressing C9orf72 Hexanucleotide Repeat Expansion Produce DPR Proteins and Show Normal Functionality but No RNA Foci

et al. 2020

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Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 ( C9orf72 ) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia.

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“…Interestingly, upon prolonged oxidative stress almost all the mutant C9ORF72 fibroblasts formed filamentous and round shapes P-TDP-43 inclusions in comparison with control and mTDP-43 fibroblasts, supporting an important involvement of C9ORF72, a regulator of endosomal and vesicular trafficking, in autophagy [50]. Remarkably, it has been demonstrated that C9ORF72 colocalizes with Ubiquilin-2 and LC3-positive vesicles and the ratio of the autophagosome markers LC3II/LC3I is altered in C9ORF72 knocked-down cells [51]. Additionally, C9ORF72 protein was recently described to form a complex with the autophagy receptor p62, which associates with symmetrically methylated arginine proteins enriched in SG, controlling SG removal by autophagy [13].…”

Section: Discussionmentioning

confidence: 76%

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

Gumina

Lenzi

Bossolasco

et al. 2020

Preprint

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Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43). The RNAbinding protein TDP-43 is also a component of stress granules (SG), cytoplasmic foci forming to arrest translation under sub-lethal stress conditions. Although commonly considered as distinct structures, a link between SG and pathological TDP-43 inclusions may occur despite evidence that TDP-43 pathology directly arises from SG is still under debate. Primary fibroblasts and iPSC-derived neurons (iPSC-N) from ALS patients carrying mutations in TARDBP (n=3) and C9ORF72 (n=3) genes and from healthy controls (n=3) were exposed to oxidative stress by sodium arsenite. SG formation and cell response to stress was evaluated and quantified by immunofluorescence and electron microscopy analyses. We found that, not only an acute, but also a chronic oxidative insult, better mimicking a persistent condition of stress as in neurodegeneration, is able to induce SG formation in primary fibroblasts and iPSC-N. Importantly, only upon chronic stress, we observed TDP-43 recruitment into SG and the formation of distinct P-TDP-43 aggregates, very similar to the abnormal inclusions observed in ALS/FTD autoptic brains. Moreover, in fibroblasts, cell response to stress was different in control compared with mutant ALS cells, probably due to their different vulnerability. A quantitative analysis revealed also differences in terms of number of SG-forming cells and SG size, suggesting a different composition of foci in acute and chronic stress. In condition of prolonged stress, SG and P-TDP-43 aggregate formation was concomitant with p62 increase and autophagy dysregulation in both ALS fibroblasts and iPSC-N, as confirmed by immunofluorescence and ultrastructural analyses. We found that exposure to a chronic oxidative insult promotes the formation of both SG and P-TDP-43 aggregates in patient-derived cells, reinforcing the idea that SG fail to properly disassemble, interfering with the protein quality control system. Moreover, we obtained a disease cell model recapitulating ALS/FTD P-TDP-43 aggregates, which represents an invaluable bioassay to study TDP-43 pathology and develop therapeutic strategies aimed at disaggregating or preventing the formation of pathological inclusions.

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C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner

Cited by 19 publications

References 42 publications

C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

BV-2 Microglial Cells Overexpressing C9orf72 Hexanucleotide Repeat Expansion Produce DPR Proteins and Show Normal Functionality but No RNA Foci

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

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