Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43.
ABSTRACT TAR DNA binding protein of 43 kDa (TDP-43) is a major component of the ubiquitin-positive inclusions found in the brain of patients with frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). Here, we report that expression of TDP-43 C-terminal fragments as green fluorescent protein (GFP) fusions in SH-SY5Y cells results in the formation of abnormally phosphorylated and ubiquitinated inclusions that are similar to those found in FTLD-U and ALS. Co-expression of DsRed-tagged full-length TDP-43 with GFP-tagged C-terminal fragments of TDP-43 causes formation of cytoplasmic inclusions positive for both GFP and DsRed. Cells with GFP and DsRed positive inclusions lack normal nuclear staining for endogenous TDP-43. These results suggest that GFP-tagged C-terminal fragments of TDP-43 are bound not only to transfected DsRed-full-length TDP-43 but also to endogenous TDP-43. Endogenous TDP-43 may be recruited to cytoplasmic aggregates of TDP-43 C-terminal fragments, which results in the failure of its nuclear localization and function. Interestingly, expression of GFP-tagged TDP-43 C-terminal fragments harboring pathogenic mutations that cause ALS significantly enhances the formation of inclusions. We also identified cleavage sites of TDP-43 C-terminal fragments deposited in the FTLD-U brains using mass spectrometric analyses. We propose that generation and aggregation of phosphorylated C-terminal fragments of TDP-43 play a primary role in the formation of inclusions and resultant loss of normal TDP-43 localization, leading to neuronal degeneration in TDP-43 proteinopathy.
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ABSTRACT: Ubiquitin-positive, tau- and alpha-synuclein-negative inclusions are hallmarks of frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Although the identity of the ubiquitinated protein specific to either disorder was unknown, we showed that TDP-43 is the major disease protein in both disorders. Pathologic TDP-43 was hyper-phosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments and was recovered only from affected central nervous system regions, including hippocampus, neocortex, and spinal cord. TDP-43 represents the common pathologic substrate linking these neurodegenerative disorders.Science 11/2006; 314(5796):130-3. · 31.20 Impact Factor
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ABSTRACT: TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies.American Journal Of Pathology 08/2008; 173(1):182-94. · 4.52 Impact Factor
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a common, fatal motor neuron disorder with no effective treatment. Approximately 10% of cases are familial ALS (FALS), and the most common genetic abnormality is superoxide dismutase-1 (SOD1) mutations. Most ALS research in the past decade has focused on the neurotoxicity of mutant SOD1, and this knowledge has directed therapeutic strategies. We recently identified TDP-43 as the major pathological protein in sporadic ALS. In this study, we investigated TDP-43 in a larger series of ALS cases (n = 111), including familial cases with and without SOD1 mutations. Ubiquitin and TDP-43 immunohistochemistry was performed on postmortem tissue from sporadic ALS (n = 59), ALS with SOD1 mutations (n = 15), SOD-1-negative FALS (n = 11), and ALS with dementia (n = 26). Biochemical analysis was performed on representative cases from each group. All cases of sporadic ALS, ALS with dementia, and SOD1-negative FALS had neuronal and glial inclusions that were immunoreactive for both ubiquitin and TDP-43. Cases with SOD1 mutations had ubiquitin-positive neuronal inclusions; however, no cases were immunoreactive for TDP-43. Biochemical analysis of postmortem tissue from sporadic ALS and SOD1-negative FALS demonstrated pathological forms of TDP-43 that were absent in cases with SOD1 mutations. These findings implicate pathological TDP-43 in the pathogenesis of sporadic ALS. In contrast, the absence of pathological TDP-43 in cases with SOD1 mutations implies that motor neuron degeneration in these cases may result from a different mechanism, and that cases with SOD1 mutations may not be the familial counterpart of sporadic ALS.Annals of Neurology 06/2007; 61(5):427-34. · 11.19 Impact Factor