Dataset Information

Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies.

ABSTRACT: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be ?55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U.

SUBMITTER: Cascella R

PROVIDER: S-EPMC5016682 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies.

Cascella Roberta R Capitini Claudia C Fani Giulia G Dobson Christopher M CM Cecchi Cristina C Chiti Fabrizio F

The Journal of biological chemistry 20160721 37

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopat ...[more]

PMID: 27445339

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Project description:We review the literature on Tau and TDP-43 proteinopathies in aged human brains and the relevant underlying pathogenetic cascades. Complex interacting pathways are implicated in Alzheimer's disease and related dementias (ADRD), wherein multiple proteins tend to misfold in a manner that is "reactive," but, subsequently, each proteinopathy may contribute strongly to the clinical symptoms. Tau proteinopathy exists in brains of individuals across a broad spectrum of primary underlying conditions-e.g., developmental, traumatic, and inflammatory/infectious diseases. TDP-43 proteinopathy is also expressed in a wide range of clinical disorders. Although TDP-43 proteinopathy was first described in the central nervous system of patients with amyotrophic lateral sclerosis (ALS) and in subtypes of frontotemporal dementia (FTD/FTLD), TDP-43 proteinopathy is also present in chronic traumatic encephalopathy, cognitively impaired persons in advanced age with hippocampal sclerosis, Huntington's disease, and other diseases. We list known Tau and TDP-43 proteinopathies. There is also evidence of cellular co-localization between Tau and TDP-43 misfolded proteins, suggesting common pathways or protein interactions facilitating misfolding in one protein by the other. Multiple pleiotropic gene variants can alter risk for Tau or TDP-43 pathologies, and certain gene variants (e.g., APOE ε4, Huntingtin triplet repeats) are associated with increases of both Tau and TDP-43 proteinopathies. Studies of genetic risk factors have provided insights into multiple nodes of the pathologic cascades involved in Tau and TDP-43 proteinopathies. Variants from a specific gene can be either a low-penetrant risk factor for a group of diseases, or alternatively, a different variant of the same gene may be a disease-driving allele that is associated with a relatively aggressive and early-onset version of a clinically and pathologically specific disease type. Overall, a complex but enlightening paradigm has emerged, wherein both Tau and TDP-43 proteinopathies are linked to numerous overlapping upstream influences, and both are associated with multiple downstream pathologically- and clinically-defined deleterious effects.

Project description:Accumulation of hyperphosphorylated, ubiquitinated and N-terminally truncated TAR DNA-binding protein (TDP-43) is the pathological hallmark lesion in most familial and sporadic forms of FTLD-U and ALS, which can be subsumed as TDP-43 proteinopathies. In order to get more insight into the role of abnormal phosphorylation in the disease process, the identification of specific phosphorylation sites and the generation of phosphorylation-specific antibodies are mandatory. Here, we developed and characterized novel rat monoclonal antibodies (1D3 and 7A9) raised against phosphorylated S409/410 of TDP-43. These antibodies were used to study the presence of S409/410 phosphorylation by immunohistochemistry and biochemical analysis in a large series of 64 FTLD-U cases with or without motor neuron disease including familial cases with mutations in progranulin (n = 5), valosin-containing protein (n = 4) and linkage to chromosome 9p (n = 4), 18 ALS cases as well as other neurodegenerative diseases with concomitant TDP-43 pathology (n = 5). Our data demonstrate that phosphorylation of S409/410 of TDP-43 is a highly consistent feature in pathologic inclusions in the whole spectrum of sporadic and familial forms of TDP-43 proteinopathies. Physiological nuclear TDP-43 was not detectable with these mAbs by immunohistochemistry and by immunoblot analyses. While the accumulation of phosphorylated C-terminal fragments was a robust finding in the cortical brain regions of FTLD-U and ALS, usually being much more abundant than the phosphorylated full-length TDP-43 band, spinal cord samples revealed a predominance of full-length TDP-43 over C-terminal fragments. This argues for a distinct TDP-43 species composition in inclusions in cortical versus spinal cord cells. Overall, these mAbs are powerful tools for the highly specific detection of disease-associated abnormal TDP-43 species and will be extremely useful for the neuropathological routine diagnostics of TDP-43 proteinopathies and for the investigation of emerging cellular and animal models for TDP-43 proteinopathies.

Dataset Information

Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies.

Publications

Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies.

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