Potentiation of Amyotrophic Lateral Sclerosis (ALS)-associated TDP-43 Aggregation by the Proteasome-targeting Factor, Ubiquilin 1

Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2009; 284(12):8083-92. DOI: 10.1074/jbc.M808064200
Source: PubMed


TDP-43 (43-kDa TAR DNA-binding domain protein) is a major constituent of ubiquitin-positive cytoplasmic aggregates present
in neurons of patients with fronto-temporal lobular dementia and amyotrophic lateral sclerosis (ALS). The pathologic significance
of TDP-43 aggregation is not known; however, dominant mutations in TDP-43 cause a subset of ALS cases, suggesting that misfolding
and/or altered trafficking of TDP-43 is relevant to the disease process. Here, we show that the presenilin-binding protein
ubiquilin 1 (UBQLN) plays a role in TDP-43 aggregation. TDP-43 interacted with UBQLN both in yeast and in vitro, and the carboxyl-terminal ubiquitin-associated domain of UBQLN was both necessary and sufficient for binding to polyubiquitylated
forms of TDP-43. Overexpression of UBQLN recruited TDP-43 to detergent-resistant cytoplasmic aggregates that colocalized with
the autophagosomal marker, LC3. UBQLN-dependent aggregation required the UBQLN UBA domain, was mediated by non-overlapping
regions of TDP-43, and was abrogated by a mutation in UBQLN previously linked to Alzheimer disease. Four ALS-associated alleles
of TDP-43 also coaggregated with UBQLN, and the extent of aggregation correlated with in vitro UBQLN binding affinity. Our findings suggest that UBQLN is a polyubiquitin-TDP-43 cochaperone that mediates the autophagosomal
delivery and/or proteasome targeting of TDP-43 aggregates.

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    • "Approximately, 10% of ALS cases are of familial origin among them 20% due to a mutation in the gene encoding the Cu/Zn superoxide dismutase 1 (SOD1) [211] [212] and around 34% are due to a hexanucleotide expansion in C9ORF72 gene [213] [214]. C9ORF72 is also involved in the development of another ND, the fronto-temporal dementia, which can be associated with ALS [215] [216]. Therapy using antisense oligonucleotides that target repeats containing related-RNA is a rational approach [217]. "
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    • "Among these studies, one immunofluorescence staining analysis has shown that depletion of functional multivesicular body (MVBs), required for the macroautophagy degradation pathway, results in the accumulation of the endogenous TDP-43 in the cytoplasm as ubiquitylated species (Filimonenko et al., 2007). In another study, overexpression of ubiquilin 1 (UBQLN), a proteasome-targeting factor, results in an increased amount of insoluble full-length TDP-43 protein and ubiquitylated aggregates consisting of endogenous TDP-43 with the autophagosomal marker LC3 (Kim et al., 2009). Notably, under normal conditions, the cellular concentration of the TDP-43 protein is autoregulated through a negative-feedback loop (Ayala et al., "
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    ABSTRACT: TDP-43 is a pathological signature protein of neurodegenerative diseases with TDP-43 proteinopathies including FTLD-TDP and ALS-TDP. These TDP-43 proteinopathies are characterized with cytoplasmic insoluble TDP-43(+) aggregates in the diseased cells, the formation of which requires the seeding of TDP-25 fragment generated by caspase cleavage of TDP-43. We have investigated the metabolism and mis-metabolism of TDP-43 in cultured cells and found that the endogenous and exogenously over-expressed TDP-43 are degraded not only by ubiquitin proteasome system (UPS) and macroautophagy (MA), but also by the chaperone-mediated autophagy (CMA) mediated through interaction between Hsc70 and ubiquitinated TDP-43. Furthermore, proteolytic cleavage of TDP-43 by caspase(s) is a necessary intermediate step for degradation of a majority of the TDP-43 protein, with the TDP-25/TDP-35 fragments being the main substrates. Finally, we have determined the threshold level of the TDP-25 fragment that is necessary for formation of the cytosolic TDP-43(+) aggregates in cells containing the full-length TDP-43 at an elevated level close to that found in patients with TDP-43 proteinopathies. A comprehensive model of the metabolism and mis-metabolism of TDP-43 in relation to these findings is presented.
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    • "Similar patterns were observed for transfected HEK 293T and SHSY5Y cells (data not shown). Consistent with the previous studies [24,34], the WT or MT hTDP-43 could be seen to form cytosolic aggregates in only a few percent of the transfected cells (data not shown). "
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    ABSTRACT: Background TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined. Results By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT. Conclusions Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general.
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