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

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2011-05, Vol.286 (21), p.18845-18855
Main Authors: Pesiridis, G. Scott, Tripathy, Kalyan, Tanik, Selçuk, Trojanowski, John Q., Lee, Virginia M.-Y.
Format: Article
Language:English
Subjects:
Active Transport, Cell Nucleus - genetics
Amino Acid Motifs
Amyotrophic Lateral Sclerosis - genetics
Amyotrophic Lateral Sclerosis - metabolism
Amyotrophic Lateral Sclerosis - pathology
Amyotropic Lateral Sclerosis (Lou Gehrig's Disease)
Cell Line
Cell Nucleus - metabolism
Cell Nucleus - pathology
Cytoplasm - genetics
Cytoplasm - metabolism
Cytoplasm - pathology
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Dyneins - genetics
Dyneins - metabolism
Frontotemporal Lobar Degeneration
Frontotemporal Lobar Degeneration - genetics
Frontotemporal Lobar Degeneration - metabolism
Frontotemporal Lobar Degeneration - pathology
Humans
Microtubules - genetics
Microtubules - metabolism
Microtubules - pathology
Molecular Bases of Disease
Neurodegeneration
Protein Degradation
Proteolysis
RNA - genetics
RNA - metabolism
RNA-binding Protein
TDP-43
Tobacco Etch Virus (TEV)
Viral Protease
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M111.231118