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The N Termini of TAR DNA-Binding Protein 43 (TDP43) C-Terminal Fragments Influence Degradation, Aggregation Propensity, and Morphology.

ABSTRACT: Fragments of the TAR DNA-binding protein 43 (TDP43) are major components of intracellular aggregates associated with amyotrophic lateral sclerosis and frontotemporal dementia. A variety of C-terminal fragments (CTFs) exist, with distinct N termini; however, little is known regarding their differences in metabolism and aggregation dynamics. Previously, we found that specific CTFs accumulate in the absence of the Arg/N-end rule pathway of the ubiquitin proteasome system (UPS) and that their degradation requires arginyl-tRNA protein transferase 1 (ATE1). Here, we examined two specific CTFs of TDP43 (TDP43219 and TDP43247), which are ?85% identical and differ at their N termini by 28 amino acids. We found that TDP43247 is degraded primarily by the Arg/N-end rule pathway, whereas degradation of TDP43219 continues in the absence of ATE1. These fragments also differ in their aggregation propensities and form morphologically distinct aggregates. This work reveals that the N termini of otherwise similar CTFs have profound effects on fragment behavior and may influence clinical outcomes in neurodegeneration associated with aggregation.

SUBMITTER: Kasu YAT

PROVIDER: S-EPMC6146831 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The N Termini of TAR DNA-Binding Protein 43 (TDP43) C-Terminal Fragments Influence Degradation, Aggregation Propensity, and Morphology.

Kasu Yasar Arfat T YAT Alemu Samrawit S Lamari Angela A Loew Nicole N Brower Christopher S CS

Molecular and cellular biology 20180914 19

Fragments of the TAR DNA-binding protein 43 (TDP43) are major components of intracellular aggregates associated with amyotrophic lateral sclerosis and frontotemporal dementia. A variety of C-terminal fragments (CTFs) exist, with distinct N termini; however, little is known regarding their differences in metabolism and aggregation dynamics. Previously, we found that specific CTFs accumulate in the absence of the Arg/N-end rule pathway of the ubiquitin proteasome system (UPS) and that their degrad ...[more]

PMID: 29987190

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Project description:Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the aggregate-associated diseases, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. In fact, although understanding the processes that cause the disease is still an open challenge, its relationship with protein aggregation is widely known. In particular, human TDP-43, an RNA/DNA binding protein, is a major component of the pathological cytoplasmic inclusions observed in ALS patients. Indeed, the deposition of the phosphorylated full-length TDP-43 in spinal cord cells has been widely studied. Moreover, it has also been shown that the brain cortex presents an accumulation of phosphorylated C-terminal fragments (CTFs). Even if it is debated whether the aggregation of CTFs represents a primary cause of ALS, it is a hallmark of TDP-43 related neurodegeneration in the brain. Here, we investigate the CTFs aggregation process, providing a computational model of interaction based on the evaluation of shape complementarity at the molecular interfaces. To this end, extensive Molecular Dynamics (MD) simulations were conducted for different types of protein fragments, with the aim of exploring the equilibrium conformations. Adopting a newly developed approach based on Zernike polynomials, able to find complementary regions in the molecular surface, we sampled a large set of solvent-exposed portions of CTFs structures as obtained from MD simulations. Our analysis proposes and assesses a set of possible association mechanisms between the CTFs, which could drive the aggregation process of the CTFs. To further evaluate the structural details of such associations, we perform molecular docking and additional MD simulations to propose possible complexes and assess their stability, focusing on complexes whose interacting regions are both characterized by a high shape complementarity and involve β3 and β5 strands at their interfaces.

Project description:Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. Using genome-wide linkage analysis and whole exome sequencing in an extended five-generation family with an autosomal dominant rimmed vacuole myopathy, we identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43p.Trp385IlefsTer10) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43Trp385IlefsTer10 does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43p.Trp385IlefsTer10 behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43p.Trp385IlefsTer10 showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43p.Trp385IlefsTer10 is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. Our study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology.

Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson's, 8 Alzheimer's disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson's and Alzheimer's disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

Dataset Information

The N Termini of TAR DNA-Binding Protein 43 (TDP43) C-Terminal Fragments Influence Degradation, Aggregation Propensity, and Morphology.

Publications

The N Termini of TAR DNA-Binding Protein 43 (TDP43) C-Terminal Fragments Influence Degradation, Aggregation Propensity, and Morphology.

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