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Synucleinopathies, which include multiple system atrophy (MSA), Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies (DLB), are human neurodegenerative diseases 1 . Existing treatments are at best symptomatic. These diseases are characterized by the presence of, and believed to be caused by the formation of, filamentous inclusions of α-synuclein in brain cells 2 , 3 . However, the structures of α-synuclein filaments from the human brain are unknown. Here, using cryo-electron microscopy, we show that α-synuclein inclusions from the brains of individuals with MSA are made of two types of filament, each of which consists of two different protofilaments. In each type of filament, non-proteinaceous molecules are present at the interface of the two protofilaments. Using two-dimensional class averaging, we show that α-synuclein filaments from the brains of individuals with MSA differ from those of individuals with DLB, which suggests that distinct conformers or strains characterize specific synucleinopathies. As is the case with tau assemblies 4 , 5 , 6 , 7 , 8 – 9 , the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, which has implications for understanding the mechanisms of aggregate propagation and neurodegeneration in the human brain. These findings have diagnostic and potential therapeutic relevance, especially because of the unmet clinical need to be able to image filamentous α-synuclein inclusions in the human brain. Cryo-electron microscopy reveals the structures of α-synuclein filaments from the brains of individuals with multiple system atrophy.

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone. The identification of pathogenic mutations within prion-like domains (PrLDs) of the RNA-binding proteins hnRNPA2B1 and hnRNPA1 add to our understanding of how mutations in these proteins lead to degenerative disease, and highlight the potential importance of PrLDs in degenerative diseases of the nervous system, muscle and bone. Disease link to prion-like RNA-binding protein How do mutations in RNA-binding proteins cause human disease, and neurodegeneration in particular? Hong Joo Kim et al . have identified mutations in two RNA-binding proteins, hnRNPA2B1 and hnRNPA1, in two families with inclusion body myopathy with frontotemporal dementia. Both of the mutations lie within a highly conserved part of a protein domain that has similarities to prion proteins, and a tendency to aggregate. This aggregation is enhanced by the mutations. The mutated prion-like domain of hnRNPA2 can functionally replace that of a yeast prion protein and reproduce its prion-like behaviour. These findings have relevance to the pathogenesis of degenerative diseases and proteinopathies such as amyotrophic lateral sclerosis.

Cytoplasmic inclusions containing α-synuclein (α-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although α-Syn fibrils can be generated from recombinant α-Syn protein in vitro, the production of fibrillar α-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular α-Syn aggregation can be triggered by the introduction of exogenously produced recombinant α-Syn fibrils into cultured cells engineered to overexpress α-Syn. Unlike unassembled α-Syn, these α-Syn fibrils \"seeded\" recruitment of endogenous soluble α-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular α-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of α-Syn fibrils can alter this balance by acting as seeds for aggregation.

Pathogenic variants in valosin-containing protein (VCP) cause multisystem proteinopathy (MSP), a disease characterized by multiple clinical phenotypes including inclusion body myopathy, Paget's disease of the bone, and frontotemporal dementia (FTD). How such diverse phenotypes are driven by pathogenic VCP variants is not known. We found that these diseases exhibit a common pathologic feature: ubiquitinated intranuclear inclusions affecting myocytes, osteoclasts, and neurons. Moreover, knock-in cell lines harboring MSP variants show a reduction in nuclear VCP. Given that MSP is associated with neuronal intranuclear inclusions comprised of TDP-43 protein, we developed a cellular model whereby proteostatic stress results in the formation of insoluble intranuclear TDP-43 aggregates. Consistent with a loss of nuclear VCP function, cells harboring MSP variants or cells treated with VCP inhibitor exhibited decreased clearance of insoluble intranuclear TDP-43 aggregates. Moreover, we identified 4 compounds that activate VCP primarily by increasing D2 ATPase activity, where pharmacologic VCP activation appears to enhance clearance of insoluble intranuclear TDP-43 aggregate. Our findings suggest that VCP function is important for nuclear protein homeostasis, that impaired nuclear proteostasis may contribute to MSP, and that VCP activation may be a potential therapeutic by virtue of enhancing the clearance of intranuclear protein aggregates.

ObjectivesTo evaluate the validity, reliability, responsiveness and meaningful change threshold of the Inclusion Body Myositis (IBM) Functional Rating Scale (FRS).MethodsData from a large 20-month multicentre, randomised, double-blind, placebo-controlled trial in IBM were used. Convergent validity was tested using Spearman correlation with other health outcomes. Discriminant (known groups) validity was assessed using standardised effect sizes (SES). Internal consistency was tested using Cronbach’s alpha. Intrarater reliability in stable patients and equivalence of face-to-face and telephone administration were tested using intraclass correlation coefficients (ICCs) and Bland-Altman plots. Responsiveness was assessed using standardised response mean (SRM). A receiver operator characteristic (ROC) curve anchor-based approach was used to determine clinically meaningful IBMFRS change.ResultsAmong the 150 patients, mean (SD) IBMFRS total score was 27.4 (4.6). Convergent validity was supported by medium to large correlations (rs modulus: 0.42–0.79) and discriminant validity by moderate to large group differences (SES=0.51–1.59). Internal consistency was adequate (overall Cronbach’s alpha: 0.79). Test–retest reliability (ICCs=0.84–0.87) and reliability of telephone versus face-to-face administration (ICCs=0.93–0.95) were excellent, with Bland-Altman plots showing good agreement. Responsiveness in the worsened group defined by various external constructs was large at both 12 (SRM=−0.76 to −1.49) and 20 months (SRM=−1.12 to −1.57). In ROC curve analysis, a drop in at least two IBMFRS total score points was shown to represent a meaningful decline.ConclusionsWhen administered by trained raters, the IBMFRS is a reliable, valid and responsive tool that can be used to evaluate the impact of IBM and its treatment on physical function, with a 2-point reduction representing meaningful decline.Trial registration numberNCT02753530.

Inclusion body myositis (IBM) is often viewed as an enigmatic disease with uncertain pathogenic mechanisms and confusion around diagnosis, classification and prospects for treatment. Its clinical features (finger flexor and quadriceps weakness) and pathological features (invasion of myofibres by cytotoxic T cells) are unique among muscle diseases. Although IBM T cell autoimmunity has long been recognized, enormous attention has been focused for decades on several biomarkers of myofibre protein aggregates, which are present in <1% of myofibres in patients with IBM. This focus has given rise, together with the relative treatment refractoriness of IBM, to a competing view that IBM is not an autoimmune disease. Findings from the past decade that implicate autoimmunity in IBM include the identification of a circulating autoantibody (anti-cN1A); the absence of any statistically significant genetic risk factor other than the common autoimmune disease 8.1 MHC haplotype in whole-genome sequencing studies; the presence of a marked cytotoxic T cell signature in gene expression studies; and the identification in muscle and blood of large populations of clonal highly differentiated cytotoxic CD8+ T cells that are resistant to many immunotherapies. Mounting evidence that IBM is an autoimmune T cell-mediated disease provides hope that future therapies directed towards depleting these cells could be effective.Inclusion body myositis (IBM) is a slowly progressive disease characterized by unique clinical and pathological features. This Review discusses the historical and clinical aspects of IBM and the mounting evidence arguing for the important pathogenic function of the immune system.

Neuronal cytoplasmic inclusions (NCIs) containing phosphorylated TDP-43 (p-TDP-43) are the pathological hallmarks of motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and FTLD-TDP. The vast majority of NCIs in the brain and spinal cord also label for ubiquitin and p62, however, we have previously reported a subset of TDP-43 proteinopathy patients who have unusual and abundant p62 positive, TDP-43 negative inclusions in the cerebellum and hippocampus. Here we sought to determine whether these cases carry the hexanucleotide repeat expansion in C9orf72 . Repeat primer PCR was performed in 36 MND/ALS, FTLD-MND/ALS and FTLD-TDP cases and four controls. Fourteen individuals with the repeat expansion were detected. In all the 14 expansion mutation cases there were abundant globular and star-shaped p62 positive NCIs in the pyramidal cell layer of the hippocampus, the vast majority of which were p-TDP-43 negative. p62 positive NCIs were also abundant in the cerebellar granular and molecular layers in all cases and in Purkinje cells in 12/14 cases but they were only positive for p-TDP-43 in the granular layer of one case. Abundant p62 positive, p-TDP-43 negative neuronal intranuclear inclusions (NIIs) were seen in 12/14 cases in the pyramidal cell layer of the hippocampus and in 6/14 cases in the cerebellar granular layer. This unusual combination of inclusions appears pathognomonic for C9orf72 repeat expansion positive MND/ALS and FTLD-TDP which we believe form a pathologically distinct subset of TDP-43 proteinopathies. Our results suggest that proteins other than TDP-43 are binding p62 and aggregating in response to the mutation which may play a mechanistic role in neurodegeneration.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult 1 – 8 , but skin biopsy enables its ante-mortem diagnosis 9 – 12 . The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1–q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC , a gene that evolved by segmental duplication, causes a human disease. Long-read sequencing identifies a GGC repeat expansion in NOTCH2NLC that is associated with neuronal intranuclear inclusion disease, a progressive neurodegenerative disorder. The expansion results in abnormal anti-sense transcripts that could contribute to disease pathogenesis.

ObjectiveTo elucidate the common and distinct clinical features of immune-mediated necrotising myopathy (IMNM), also known as necrotising autoimmune myopathy associated with autoantibodies against signal recognition particle (SRP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR).MethodsWe examined a cohort of 460 patients with idiopathic inflammatory myopathies (IIMs) through a muscle biopsy-oriented registration study in Japan. Study entry was strictly determined by the comprehensive histological assessment to exclude other neuromuscular disorders. Anti-SRP and anti-HMGCR antibodies were detected by RNA immunoprecipitation and ELISA, respectively.ResultsOf 460 patients with IIM, we diagnosed 73 (16%) as having inclusion body myositis (IBM). Of 387 patients with IIMs other than IBM, the frequencies of anti-SRP and anti-HMGCR antibodies were 18% and 12%, respectively. One patient had both autoantibodies. Severe limb muscle weakness, neck weakness, dysphagia, respiratory insufficiency and muscle atrophy were more frequently observed in patients with anti-SRP antibodies than in those with anti-HMGCR antibodies. Serum creatine levels were markedly higher in the patients with autoantibodies than in those without. Histology was characterised by necrosis and regeneration of muscle fibres and was consistent with IMNM except in 1 HMGCR-positive IBM patient. Most patients were initially treated with corticosteroids; however, additional immunosuppressive drugs were required, especially in the patients with anti-SRP antibodies. Rates of unsatisfactory neurological outcome were similar in the 2 autoantibody groups.ConclusionsAnti-SRP antibodies are associated with severe neurological symptoms, more so than are anti-HMGCR antibodies. Although these autoantibodies are independent serological markers associated with IMNM, patients bearing either share common characteristics.

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.