TDP-43 Is Intrinsically Aggregation-prone, and Amyotrophic Lateral Sclerosis-linked Mutations Accelerate Aggregation and Increase Toxicity

Abstract

Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS FTLD-U patients. The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations.TDP-43 is a ubiquitously expressed and highly conserved metazoan nuclear protein (1), which contains two RNA recognition motifs (RRMs) 3 and a glycine-rich region in its C-terminal domain (see Fig. 1A). TDP-43 function is uncertain, but it likely plays important roles in pre-mRNA splicing and transcriptional repression (2, 3). In ALS and FTLD-U, TDP-43 is depleted from the nucleus and accumulates in ubiquitinated cytoplasmic inclusions (4). These and other situations of TDP-43 pathology, including some forms of Alzheimer and Parkinson diseases, are now known as TDP-43 proteinopathies (5). Importantly, mutations in the TDP-43 gene (TARDBP) are linked to sporadic and non-SOD1 familial ALS, implying that TDP-43 abnormalities are likely one cause of disease (6 -11). However, despite this synthesis of pathology and genetics, the mechanisms by which TDP-43 might contribute to disease remain unknown and controversial (12, 13).A key unresolved question is whether TDP-43 is inherently aggregation-prone or whether TDP-43 is sequestered by other aggregated components and is merely a marker of disease (13-16). Indeed, multiple proteins aside from TDP-43 are found in Sarkosyl-insoluble fractions from FTLD-U patients (14). Moreover, deconvolution imaging reveals that TDP-43 appears to be excluded from some regions of the ubiquitinated inclusions in ALS (15).Here, we assess TDP-43 aggregation in the absence of other components. We then define which domains of TDP-43 are important for this process and determine the direct effects of several ALS-linked TDP-43 mutations on TDP-43 misfolding and toxicity. Our findings bring to light several intrinsic properties of TDP-43 and ALS-linked TDP-43 mutants that likely play important roles in the aberrant TDP-43 proteostasis (17) that contributes to the pathogenesis of ALS,...

Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA*

“…Although TDP-43 mutants appeared grossly normal in gel filtration and FUS/TLS interaction assays, C-terminal fragments of TDP-43, which have received attention as possible ALS disease determinants, showed altered complex forming potential in all of the cell lines tested (15)(16)(17)34). Specifically, the major 35-kDa TDP-43 fragment, p35 , was excluded from the RNA-dependent TDP-43L complexes but retained its ability to interact with FUS/TLS (Fig.…”

“…Virtually all ALS-associated mutations in TDP-43 occur in an unstructured Gly-rich domain that binds to heterogeneous ribonucleoprotein A/B complexes (12)(13)(14). ALS-associated mutants of TDP-43 are hyperphosphorylated, ubiquitylated, aggregationprone, and cleaved into 25-and 35-kDa C-terminal fragments that exhibit cytotoxicity in cellulo (15)(16)(17)(18). TDP-43 is degraded by proteasome and autophagy-dependent pathways, which may be mediated in part through its association with the ubiquitin-binding protein Ubiquilin (19 -22).…”

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

“…Overexpression of CTFs of varying lengths in cultured cells produces cytoplasmic aggregates that are ubiquitinated and phosphorylated, recapitulating biochemical properties of authentic . Experiments performed in vitro demonstrate that CTFs readily aggregate and form fibrillar structures (19,20). However, CTF expression in transgenic flies is well tolerated without neurotoxicity or motor phenotypes (21,22).…”

Aggregation party in ALS

“…3 In a new report published in Nature, his group identified 13 yeast genes whose overexpression increased toxicity caused by TDP-43 compared with that seen in vector-treated controls. Among these was pbp1, which encodes an mRNA-associated protein that strongly resembles human ataxin 2 (ATXN2; SCA2).…”