A "Two-hit" Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport

Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(21):18845-55. DOI: 10.1074/jbc.M111.231118
Source: PubMed


Carboxyl-terminal fragments (CTFs) of TDP-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar
degeneration and amyotrophic lateral sclerosis, but the biological significance of these CTFs and how they are generated remain
enigmatic. To address these issues, we engineered mammalian cells with an inducible tobacco etch virus (TEV) protease that
cleaves TDP-43 containing a TEV cleavage site. Regions of TDP-43 flanking the second RNA recognition motif (RRM2) are efficiently
cleaved by TEV, whereas sites within this domain are more resistant to cleavage. CTFs containing RRM2 generated from de novo cleavage of nuclear TDP-43 are transported to the cytoplasm and efficiently cleared, indicating that cleavage alone is not
sufficient to initiate CTF aggregation. However, CTFs rapidly aggregated into stable cytoplasmic inclusions following de novo cleavage when dynein-mediated microtubule transport was disrupted, RNA was depleted, or natively misfolded CTFs were introduced
into these cells. Our data support a “two-hit” mechanism of CTF aggregation dependent on TDP-43 cleavage.

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    • "Furthermore, nuclear accumulation of truncated huntingtin was shown to be toxic to cells [30] [31]. Protein mislocalization is also seen in case of TDP43, a predominant nuclear protein that is abnormally sorted into the cytoplasm in neurodegenerative TDP43 foldopathies [32]. Cytoplasmic inclusions of TDP43 C-terminal fragments have been observed in FTDP brain [33], where the C-terminus of TDP43 is able to induce cytoplasmic localization of the protein and promote its aggregation. "
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    ABSTRACT: Tau protein is a member of microtubule-associated protein family. Under pathological conditions, tau undergoes multiple modifications that lead to the formation of insoluble deposits in neurons, resulting in neuronal dysfunction in several neurodegenerative disorders collectively called tauopathies, with Alzheimer's disease being the most frequent example. This typical cytosolic protein has been shown to translocate into the nucleus and participate in DNA protection upon stress conditions. In our study, we demonstrate that truncated Tau151-391/4R changes its usual behavior and gains constitutive access into the nucleus of both primary rat neurons and human neuroblastoma cells. Our results show that partial/dysregulated nuclear localization of tau results from the removal of the N-terminal (1-150) residues of the protein. Data obtained by cell fractionation data were supported by confocal microscopy analysis of GFP-fused tau proteins. Furthermore, neither addition of the fusion protein, nor increased tau phosphorylation had any effect on the intracellular distribution of truncated tau. Our data further suggest that differential tau phospho-status between cytosolic and nuclear fractions is rather a consequence than a cause of truncated tau nuclear localization. Finally, truncated tau in the nucleus is engaged in interactions with subnuclear structure(s), since it exhibits reduced mobility. We conclude that N-terminal truncation of tau proteins leads to their nonphysiological subcellular distribution as a result of modified tau conformation.
    Full-text · Article · Jan 2015 · Journal of Alzheimer's disease: JAD
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    • "Of note, MAb 138 does detect the normal nuclear TDP-43 in QBI293 cells by ICC (Table 2), albeit to a much less degree or intensity than the other MAbs reported in our study (data not shown) and most published pan-TDP-43 antibodies. Our previous findings that TDP-43 could be extracted biochemically with low-stringency buffers from cultured cells [27], but was only extractable with high-stringency detergent-containing buffer from CNS tissue [1], supports a potential structural difference between nuclear TDP-43 in CNS and cultured cells. Thus, the slight immunoreactivity of MAb 138 with non-pathological TDP-43 in non-neuronal cell culture is difficult to interpret precisely at this time, but we will continue to characterize the basis for its preferential recognition of pathological TDP-43 in a large comprehensive neuropathological survey and further neuronal culture experiments. "
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    ABSTRACT: The RNA/DNA-binding protein, TDP-43, is the key component of ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis (ALS) and the majority of frontotemporal lobar degeneration (FTLD-TDP) referred to collectively as TDP-43 proteinopathies. To further elucidate mechanisms of pathological TDP-43 processing and identify TDP-43 epitopes that could be useful as potential biomarkers of TDP-43 proteinopathies, we developed a panel of novel monoclonal antibodies (MAbs) directed at regions extending across the length of TDP-43. Here, we confirm previous observations that there is no or minimal accumulation of TDP-43 N-terminal domains in neocortical inclusions in human TDP-43 proteinopathy tissues and we identify a subset of these MAbs that are specific for human versus mouse TDP-43. Notably, one of these MAbs recognized an epitope that preferentially detected pathological TDP-43 inclusions with negligible reactivity for normal nuclear TDP-43 resembling anti-phospho-TDP-43 specific antibodies that only bind pathological TDP-43. Hence, we infer that this new MAb recognizes a phosphorylation independent but disease-specific pathologic conformation in abnormal TDP-43. These data suggest that the novel MAbs reported here will be useful for patient-oriented research as well as for studies of animal and cell-based models of TDP-43 proteinopathies including ALS and FTLD-TDP.
    Full-text · Article · Mar 2014
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    • "TDP-43 forms amorphous aggregates when isolated but can assemble into filamentous structures when prompted by an activating or nucleating event indicating that endogenous TDP-43 solubility is likely regulated by its binding partners and that when not sequestered it is vulnerable to either aggregation or polymerization [11], [26], [27], [28]. Similar to Tau in AD and α-synuclein in PD, TDP-43 becomes phosphorylated, truncated and ubiquitinated during disease progression until it reaches its final intractable state, stressing the cells, mislocalized and malfunctioning, and the neurons die [29], [30], [31]. "
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    ABSTRACT: Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10–12 nm filaments though wider 18–20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades.
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