Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

Abstract

The presence of lower molecular weight species comprising the C-terminal region of TAR DNA-binding protein 43 (TDP-43) is a characteristic of TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we have identified a novel splice variant of TDP-43 that is upregulated in ALS and generates a 35-kDa N-terminally truncated species through use of an alternate translation initiation codon (ATGMet85), denoted here as Met85-TDP-35. Met85-TDP-35 expressed ectopically in human neuroblastoma cells exhibited reduced solubility, cytoplasmic distribution, and aggregation. Furthermore, Met85-TDP-35 sequestered full-length TDP-43 from the nucleus to form cytoplasmic aggregates. Expression of Met85-TDP-35 in primary motor neurons resulted in the formation of Met85-TDP-35-positive cytoplasmic aggregates and motor neuron death. A neo-epitope antibody specific for Met85-TDP-35 labeled the 35-kDa lower molecular weight species on immunoblots of urea-soluble extracts from ALS-FTLD disease-affected tissues and co-labeled TDP-43-positive inclusions in ALS spinal cord sections, confirming the physiological relevance of this species. These results show that the 35-kDa low molecular weight species in ALS-FTLD can be generated from an abnormal splicing event and use of a downstream initiation codon and may represent a mechanism by which TDP-43 elicits its pathogenicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1412-5) contains supplementary material, which is available to authorized users.

TDP-43/FUS in motor neuron disease: Complexity and challenges

“…Overexpression of TDP-43 causes asymmetric interactions between TDP-43 and its nascent RNA to stall RNA polymerase II (RNA Pol II), leading to transcript degradation and maintaining the autoregulation of TDP-43 protein level (Avendano-Vazquez et al, 2012). Interestingly, TDP-35, a second splice variant of TDP-43, has been found to be expressed in the brains of ALS patients, through the use of the downstream start codon ATG Met85 (Xiao et al, 2015). Expression of TDP-35 in primary neurons causes cytoplasmic aggregation and neuronal death (Xiao et al, 2015).…”

“…Interestingly, TDP-35, a second splice variant of TDP-43, has been found to be expressed in the brains of ALS patients, through the use of the downstream start codon ATG Met85 (Xiao et al, 2015). Expression of TDP-35 in primary neurons causes cytoplasmic aggregation and neuronal death (Xiao et al, 2015). …”

Fluid-Based Biomarkers for Amyotrophic Lateral Sclerosis

“…One challenge may be that as TDP-43 is a self-aggregating protein, it may self-assemble and generate structures that limit access of the detection antibody when present in biofluids. In addition, TDP-43 has been shown to be subject to alternative splicing causing truncations of the N-or C-terminus, leading to splice variants linked to ALS [96]. TDP-43 has been detected as 2 distinct species via Western blot: a 45-kDa (phosphorylated form) and 28-kDa isoform [95].…”

Deciphering spreading mechanisms in amyotrophic lateral sclerosis

“…Similarly, cleavage of TDP-43 leads to generation of C-terminal fragments (25 kDa in size), which are enriched in the insoluble fraction of pathological tissue with TDP-43 truncation due to caspase-3 [38]. More recently, formation of TDP-43 truncated form was shown to be generated by abnormal splicing using an alternate translation initiation codon (ATGMet85) with this isoform being associated with pathological inclusion bodies [39]. TDP-43 acetylation switch was also demonstrated to participate in its toxicity and aggregation [40].…”