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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration.

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Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. The mutations in the parkin gene can lead to a loss of function of parkin and cause autosomal recessive juvenile onset parkinsonism. Recently, parkin was reported to be involved in the regulation of mitophagy. Here, we identify the Bcl-2, an anti-apoptotic and autophagy inhibitory protein, as a substrate for parkin. Parkin directly binds to Bcl-2 via its C terminus and mediates the mono-ubiquitination of Bcl-2, which increases the steady-state levels of Bcl-2. Overexpression of parkin, but not its ligase-deficient forms, decreases autophagy marker LC3 conversion, whereas knockdown of parkin increases LC3 II levels. In HeLa cells, a parkin-deficient cell line, knockdown of parkin does not change LC3 conversion. Moreover, overexpression of parkin enhances the interactions between Bcl-2 and Beclin 1. Our results provide evidence that parkin mono-ubiquitinates Bcl-2 and regulates autophagy via Bcl-2. Parkinson disease (PD)2 is the second most common neurodegenerative disorder after Alzheimer disease (1) and is characterized by a distinct set of motor symptoms including tremor, muscle rigidity, postural instability, and bradykinesia (2). Although the cause of PD is poorly understood, there is evidence that both environmental factors and genetic factors contribute to its development. Recently, several genes have been reported to be associated with the pathogenesis of familial forms of PD. Mutations in the parkin gene (PARK2; OMIM600116) cause autosomal recessive juvenile onset parkinsonism (3). It has been shown that mutations in parkin account for nearly 50% of patients with the early onset familial PD cases (3-6) and more than 15% of sporadic PD cases with early onset (7).Parkin is a 465-amino acid protein that contains an ubiquitin-like domain at its N terminus and two RING finger domains separated by an in-between-ring domain at its C terminus. Similar to other RING finger-containing proteins, parkin is an E3 ubiquitin ligase. Parkin ubiquitinates several target proteins and enhances their degradation via the ubiquitin-proteasome system (8, 9). Ubiquitination of a substrate is usually processed by a multi-step involving the sequential activity of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin-protein ligase (10). It was reported that parkin can selectively interact with the E2 enzymes, UbcH7 and UbcH8 (9,11,12). A number of protein substrates for parkin have been identified, including synphilin-1 (13, 14), CDCrel-1 and 2a (12, 15), Pael-R (16), synaptotagmin XI (17), ␣-and ␤-tubulin (18), RanBP2 (19), cyclin E (20), the aminoacyl-tRNA synthetase cofactor p38/AIMP2 (21, 22), Eps15 (23), and far upstream sequence element-binding protein 1 (24). Within these substrates, p38/AIMP2 and far upstream sequence element-binding protein 1 were reported to be accumulated in brains of parkin null mice, MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treated mice, and sporadic PD cases (22,24)...