Abstract Background Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. Methods We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. Results One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. Conclusion Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP’s surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.
SUMMARY Uptake and spread of proteopathic seeds, such as αS, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase αS seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of αS seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to αS since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.
ABSTRACT Aggregates of the RNA binding protein TDP-43 are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are neurodegenerative disorders with overlapping clinical, genetic and pathological features. Mutations in the TDP-43 gene are causative of ALS, supporting its central role in pathogenesis. The process of TDP-43 aggregation remains poorly understood and whether this includes formation of intermediate complexes is unknown. We characterized aggregates derived from purified TDP-43 as a function of time and analyzed them under semi-denaturing conditions. Our assays identified oligomeric complexes at the initial time points prior to the formation of large aggregates, suggesting that ordered oligomerization is an intermediate step of TDP-43 aggregation. In addition, we analyzed liquid-liquid phase separation of TDP-43 and detected similar oligomeric assembly upon the maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, ALS-linked mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that the aggregates generated from purified protein seed intracellular aggregation, which is detected by established markers of TDP-43 pathology. Remarkably, cytoplasmic aggregate propagation is detected earlier with A315T and M337V and is 50% more widespread than with wild-type aggregates. Our findings provide evidence for a controlled process of TDP-43 self-assembly into intermediate structures that provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.
Fidelity of mRNA translation is vital for the structure and function of proteins and for the maintenance of physiological processes in living organisms. Mutations in human transfer RNAs (tRNAs), tRNA modifying enzymes, translational factors, ribosomal proteins, and aminoacyl-tRNA synthetases (AaRSs) and related enzymes cause tumorigenesis, Diamond-Blackfan anemia, type 2 diabetes and a myriad of neurological disorders and diseases. Particularly intriguing is the importance of the integrity of gene translation for the development of the human brain. Recently, four mutations in the human gene encoding the terminal catalytic enzyme of selenocysteine synthesis, O-phosphoseryl-tRNASec:selenocysteinyl-tRNASec synthase (SepSecS), have been causatively linked to the development of severe neurological disorders in children. The affected individuals experienced progressive atrophy in the cerebrum and cerebellum, severe spasticity, profound mental retardation and rarely lived past 12-13 years of age. In spite of the detailed genetic studies and clinical profiles, the mechanism by which these disorders develop and the role SepSecS plays in the process are not clear. Herein, we determined the architecture, stoichiometry and arrangement in solution of the SepSecS-tRNASec binary complex, which is critical for synthesis of selenocysteine and selenoproteins, and hence for the integrity of the human selenoproteome. We also present, for the first time, a detailed biophysical characterization of the pathological SepSecS mutants and propose a rational mechanism that governs the development of brain pathologies. Our results further our understanding of the role of SepSecS in selenoprotein synthesis, extend the list of neurological disorders elicited by protein and enzyme aggregation, and suggest that mutations in components of translational machinery could induce both early and late onset neurological disorders and diseases. The findings presented in this doctoral work will facilitate future in vitro and cell based experiments aimed at deciphering the exact sequence of events that lead to the developmental failures observed in patients harboring detrimental SEPSECS mutations.
We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate.We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency.Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain.These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation.
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