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

ArticleinJournal of Biological Chemistry 286(21):18845-55 · March 2011with13 Reads
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.
    • "However, this capacity to aggregate is not enhanced by disease-linked mutations affecting the NLS (Ju et al. 2011; Sun et al. 2011). Over-expression of TDP-43 prion-like domain, induced protein accumulation and cell toxicity (Furukawa et al. 2004; Johnson et al. 2009; Zhang et al. 2009; Liu-Yesucevitz et al. 2010; Pesiridis et al. 2011), whereas deletion of the same domain from TDP-43 (Johnson et al. 2009; Furukawa et al. 2011) or FUS (Sun et al. 2011) interfered with aggregate formation, indicating their requirement for efficient seeding. While short recombinant or synthetic peptides derived from the prion-like domain can acquire toxic amyloid forms, purified full-length TDP-43 does not appear to have a classic amyloid structure, but rather forms small pore-like oligomers and short fibrils (Johnson et al. 2009; Couthouis et al. 2011; Shimonaka et al. 2016), which can cluster together in large complexes with resemblance to pathological inclusions (Fang et al. 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: Frontotemporal dementia is a devastating neurodegenerative disease causing stark alterations in personality and language. Characterized by severe atrophy of the frontal and temporal brain lobes, frontotemporal dementia (FTD) shows extreme heterogeneity in clinical presentation, genetic causes, and pathological findings. Like most neurodegenerative diseases, the initial symptoms of FTD are subtle, but increase in severity over time, as the disease progresses. Clinical progression is paralleled by exacerbation of pathological findings and the involvement of broader brain regions, which currently lack mechanistic explanation. Yet, a flurry of studies indicate that protein aggregates accumulating in neurodegenerative diseases can act as propagating entities, amplifying their pathogenic conformation, in a way similar to infectious prions. In this prion-centric view, FTD can be divided into three subtypes, TDP-43 or FUS proteinopathy and tauopathy. Here, we review the current evidence that FTD-linked pathology propagates in a prion-like manner and discuss the implications of these findings for disease progression and heterogeneity. Frontotemporal dementia (FTD) is a progressive neurodegenerative disease causing severe personality dysfunctions, characterized by profound heterogeneity. Accumulation of tau, TDP-43 or FUS cytoplasmic aggregates characterize molecularly distinct and non-overlapping FTD subtypes. Here, we discuss the current evidence suggesting that prion-like propagation and cell-to-cell spread of each of these cytoplasmic aggregates may underlie disease progression and heterogeneity. This article is part of the Frontotemporal Dementia special issue.
    Full-text · Article · Aug 2016
    • "A distinctive feature of this study was that the direct deactivation of a protein by TEVp cleavage takes place on a much faster time scale than gene deletion or RNA interference. A tetracycline-inducible TEVp was used to study the role of C-terminal fragments (CTFs) of the ribonucleoprotein TDP-43 (Pesiridis et al., 2011), which are found in cytoplasmic inclusions, together with full-lenght TDP-43, in neurons of some detrimental diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). The introduction of TS in TDP-43 allowed monitoring of CTFs formation and aggregation after induction of soluble or nuclear TEVp variants. "
    [Show abstract] [Hide abstract] ABSTRACT: About thirty years ago, studies on the RNA genome of Tobacco Etch Virus revealed the presence of an efficient and specific protease, called Tobacco Etch Virus protease (TEVp), that was part of the Nuclear Inclusion a (NIa) enzyme. TEVp is an efficient and specific protease of 27kDa that has become a valuable biotechnological tool. Nowadays TEVp is a unique endopeptidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. A number of TEVp mutants with different rate of cleavage, stability and specificity have been reported. Similarly, a panel of different target cleavage sites, derived from the canonical ENLYFQ-G/S site, has been established. In this review we describe these aspects of TEVp and some of its multiple applications. A particular focus is on the use and molecular biology of TEVp in living cells and organisms.
    Article · Jun 2016
    • "Aggregates of TDP-35 are found in ALS spinal cord sections, and are associated with neuronal death in motor neurons when accumulating in the cytoplasm (Medina et al., 2014). Relocation of TDP-43 from the nucleus to the cytoplasm may occur by sequestration of soluble TDP-43 by small, insoluble cytosolic TDP aggregates, thereby depleting TDP-43 from the RNA shuttling system (Pesiridis et al., 2011). Such loss of function of nuclear TDP-35 could result in dysregulation of exon splicing as well as RNA metabolism (Lagier-Tourenne et al., 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: The progression of many neurodegenerative diseases is assumed to be caused by misfolding of specific characteristic diseases related proteins, resulting in aggregation and fibril formation of these proteins. Protein misfolding associated age related diseases, although different in disease manifestations, share striking similarities. In all cases, one disease protein aggregates and loses its function or additionally shows a toxic gain of function. However, the clear link between these individual amyloid-like protein aggregates and cellular toxicity is often still uncertain. The similar features of protein misfolding and aggregation in this group of proteins, all involved in age related neurodegenerative diseases, results in high interest in characterization of their structural properties. We review here recent findings on structural properties of some age related disease proteins, in the context of their biological importance in disease.
    Full-text · Article · Mar 2016
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