ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS

Abstract

Dominant mutations in two functionally related DNA/RNA-binding proteins, trans-activating response region (TAR) DNA-binding protein with a molecular mass of 43 KDa (TDP-43) and fused in sarcoma/ translocation in liposarcoma (FUS/TLS), cause an inherited form of ALS that is accompanied by nuclear and cytoplasmic aggregates containing TDP-43 or FUS/TLS. Using isogenic cell lines expressing wild-type or ALS-linked TDP-43 mutants and fibroblasts from a human patient, pulse-chase radiolabeling of newly synthesized proteins is used to determine, surprisingly, that ALS-linked TDP-43 mutant polypeptides are more stable than wild-type TDP-43. Tandem-affinity purification and quantitative mass spectrometry are used to identify TDP-43 complexes not only with heterogeneous nuclear ribonucleoproteins family proteins, as expected, but also with components of Drosha microprocessor complexes, consistent with roles for TDP-43 in both mRNA processing and microRNA biogenesis. A fraction of TDP-43 is shown to be complexed with FUS/TLS, an interaction substantially enhanced by TDP-43 mutants. Taken together, abnormal stability of mutant TDP-43 and its enhanced binding to normal FUS/TLS imply a convergence of pathogenic pathways from mutant TDP-43 and FUS/TLS in ALS.mass spectrometry | protein stability | amyotrophic lateral sclerosis | microRNA | ribonucleoproteins

Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability

“…Although TDP-43 M337V and control neurons expressed equivalent levels of TARDBP mRNA, the mutant cells had significantly higher levels of soluble and detergent-resistant TDP-43. Previously, increased stability of mutant TDP-43 proteins had only been observed in isogenic transformed cell lines (41). Our findings suggest that differences in TDP-43 protein levels result from a posttranslational mechanism rather than from transcriptional differences.…”

The cleavage pattern of TDP-43 determines its rate of clearance and cytotoxicity

“…Indeed, the expression of TDP-43 is elevated in neurons that have differentiated from patient-derived iPS cells that carry the M337V mutation 24 . Furthermore, TDP-43 proteins that are mutated have longer half-lives than the wild-type protein and these longer half-lives correlate with a more rapid onset of the disease 10,25 . In this study, we have demonstrated that the speed of clearance of full-length TDP-43 is a critical determinant of the severity of cytotoxicity and that the onset of cell death induced by the overexpression of TDP-43 occurs earlier than that of the formation of CTF aggregates, which might enhance cytotoxicity.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…Consistent with this hypothesis, both TDP43 and FUS were co-purified with the Drosha miRNA processing complex from human cells [81]. Subsequent studies confirmed these interactions [82], and demonstrated that TDP43, but not FUS, associates with Dicer, an essential cytoplasmic miRNA processing complex [83]. TDP43 binds directly to some miRNAs, and silencing of TDP43 significantly reduced the abundance of several miRNAs (miR-132-5p/3p, miR-143-5p/3p, and miR-574-3p) in human cell lines [84], suggesting that TDP43 is involved in the production or maturation of distinct miRNA species.…”

“…In addition, cytoplasmic accumulations of FUS associate with and sequester protein arginine methyltransferase 1, resulting in deficient methylation and acetylation of nucleosome components [65], thereby affecting global gene expression. TDP43 binds to a diverse set of RNA processing proteins, including several hnRNPs, splicing factors, ribosomal proteins, and nucleosome subunits [77,82,116]. Many of the factors associated with TDP43 and FUS are components of cytoplasmic RNA stress granules [poly(A)-binding protein 1, cytotoxic granule-associated RNA binding protein] or miRNA processing complexes (Drosha, Dicer).…”