Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are the two common neurodegenerative diseases that have been associated with the GGGGCC·GGCCCC repeat RNA expansion in a noncoding region of C9orf72. It has been previously reported that unconventional repeat-associated non-ATG (RAN) translation of GGGGCC·GGCCCC repeats produces five types of dipeptide-repeat proteins (referred to as RAN proteins): poly-glycine-alanine (GA), poly-glycine-proline (GP), poly-glycine-arginine (GR), poly-proline-arginine (PR) and poly-proline-alanine (PA). Although protein aggregates of RAN proteins have been found in patients, it is unclear whether RAN protein aggregation induces neurotoxicity. In the present study, we aimed to understand the biological properties of all five types of RAN proteins. Surprisingly, our results showed that none of these RAN proteins was aggregate-prone in our cellular model and that the turnover of these RAN proteins was not affected by the ubiquitin-proteasome system or autophagy. Moreover, poly-GR and poly-PR, but not poly-GA, poly-GP or poly-PA, localized to the nucleolus and induced the translocation of the key nucleolar component nucleophosmin, leading to nucleolar stress and cell death. This poly-GR- and poly-PR-mediated defect in nucleolar function was associated with the suppression of ribosomal RNA synthesis and the impairment of stress granule formation. Taken together, the results of the present study suggest a simple model of the molecular mechanisms underlying RAN translation-mediated cytotoxicity in C9orf72-linked ALS/FTD in which nucleolar stress, but not protein aggregation, is the primary contributor to C9orf72-linked neurodegeneration.

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A GGGGCC hexanucleotide repeat expansion in intron 1 of chromosome 9 open reading frame 72 ( C9ORF72 ) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Repeat-associated non-ATG translation of dipeptide repeat proteins (DPRs) contributes to the neuropathological features of c9FTD/ALS. Among the five DPRs, arginine-rich poly-PR are reported to be the most toxic. Here, we generate a transgenic mouse line that expresses poly-PR (GFP-PR 28 ) specifically in neurons. GFP-PR 28 homozygous mice show decreased survival time, while the heterozygous mice show motor imbalance, decreased brain weight, loss of Purkinje cells and lower motor neurons, and inflammation in the cerebellum and spinal cord. Transcriptional analysis shows that in the cerebellum, GFP-PR 28 heterozygous mice show differential expression of genes related to synaptic transmission. Our findings show that GFP-PR 28 transgenic mice partly model neuropathological features of c9FTD/ALS, and show a role for poly-PR in neurodegeneration.

GGGGCC repeat expansion inC9orf72 is the most common genetic cause in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two neurodegenerative disorders in association with aging. Bidirectional repeat expansions in the noncoding region of C9orf72 have been shown to produce dipeptide repeat (DPR) proteins through repeat-associated non-ATG (RAN) translation and to reduce the expression level of the C9orf72 gene product, C9orf72 protein. Mechanisms underlying C9orf72-linked neurodegeneration include expanded RNA repeat gain of function, DPR toxicity, and C9orf72 protein loss of function. In the current study,we focus on the cellular function of C9orf72 protein. We report that C9orf72 can regulate lysosomal biogenesis and autophagy at the transcriptional level. We show that loss of C9orf72 leads to striking accumulation of lysosomes, autophagosomes, and autolysosomes in cells, which is associated with suppressed mTORC1 activity and enhanced nuclear translocation of MiT/TFE family members MITF, TFE3, and TFEB, three master regulators of lysosomal biogenesis and autophagy. We demonstrate that the DENN domain of C9orf72 specifically binds to inactive Rag GTPases, but not active Rag GTPases, thereby affecting the function of Rag/raptor/mTOR complex and mTORC1 activity. Furthermore, active Rag GTPases, but not inactive Rag GTPases or raptor rescued the impaired activity and lysosomal localization of mTORC1 in C9orf72-deficient cells. Taken together, the present study highlights a key role of C9orf72 in lysosomal and autophagosomal regulation, and demonstrates that Rag GTPases and mTORC1 are involved in C9orf72-mediated autophagy.

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Aim: In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury. Methods: Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining. Results: In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 μmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume. Conclusion: Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.