TAR DNA-binding protein (TDP-43, also known as TARDBP) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients post mortem. TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.
COBISS.SI-ID: 27414567
TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.
COBISS.SI-ID: 27582247
This is an invited scientific commentary by the elite journal Brain on their publication ‘Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation carriers’ by Freischmidt et al. Defective RNA processing has occupied centre stage in the pathogenesis of amyotrophic lateral sclerosis (ALS) since the identification of TARDBP (also known as TDP-43) inclusions in 95% of cases and pathogenic mutations in RNA processing genes such as TARDBP, FUS and MATR3 (Sreedharan et al., 2008; Vance et al., 2009; Johnson et al., 2014). FUS and TARDBP are known to regulate mRNA transcription, splicing, stability and transport (Tollervey et al., 2011; Rogelj et al., 2012) but they are also part of the large Drosha complex that regulates microRNA (miRNA) biogenesis (Gregory et al., 2004). Dysregulation of miRNA expression has been shown in many cancers and more recently in Alzheimer’s disease and is predicted to play a mechanistic role and/or be an indirect biomarker of disease. Freischmidt et al. (2014) report that levels of a specific subset of miRNAs are reduced in the serum of patients with familial and sporadic ALS, and that these reductions are even detectable in presymptomatic carriers of pathogenic ALS mutations. If these results can be replicated in larger cohorts then this will become a landmark study.
COBISS.SI-ID: 28055335