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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.

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.

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.

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.

Frontotemporal dementia with inclusion body myopathy and Paget disease of bone is a rare, autosomal-dominant disorder caused by mutations in the gene valosin-containing protein (VCP). The CNS pathology is characterized by a novel pattern of ubiquitin pathology distinct from sporadic and familial frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) without VCP mutations. TAR DNA binding protein 43 (TDP-43) was recently identified as a major disease protein in the ubiquitin-positive inclusions of sporadic and familial FTLD-U. To determine whether the ubiquitin pathology associated with mutations in VCP is characterized by the accumulation of TDP-43, we analyzed TDP-43 in the CNS pathology of five patients with VCP gene mutations. Accumulations of TDP-43 colocalized with ubiquitin pathology in inclusion body myopathy and Paget disease of bone, including both intranuclear inclusions and dystrophic neurites. Similar to FTLD-U, phosphorylated TDP-43 was detected only in insoluble brain extracts from affected brain regions. Identification of TDP-43, but not VCP, within ubiquitin-positive inclusions supports the hypothesis that VCP gene mutations lead to a dominant negative loss or alteration of VCP function culminating in impaired degradation of TDP-43. TDP-43 is a common pathologic substrate linking a variety of distinct patterns of FTLD-U pathology caused by different genetic alterations.

One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.

Frontotemporal lobar degeneration (FTLD) is pathologically subdivided based on the presence of particular pathological proteins that are identified in inclusion bodies observed post-mortem. The FTLD-FUS subgroup is defined by the presence of the fused in sarcoma protein (FUS) in pathological inclusions. FUS is a heterogeneous nuclear ribonucleoprotein (hnRNP) protein and a member of the FET (FUS, EWS, TAF15) protein family. It shuttles between the nucleus and cytoplasm, and has been implicated in many cellular functions including translation, splicing, and RNA transport. EWS, TAF15 and the nuclear import receptor transportin have been shown to co-accumulate with FUS in neuronal inclusions specifically in FTLD-FUS, with transportin-positive inclusions most frequently observed. Here, we report the identification of hnRNP R and hnRNP Q in neuronal cytoplasmic and intranuclear inclusions in the frontal cortex and hippocampus of FTLD-FUS patients, as frequently as transportin. hnRNP R and hnRNP Q were not found in the characteristic pathological inclusions observed in FTLD-TDP (subtypes A-C). Additionally, we studied the expression of hnRNP R in the frontal and temporal cortices from patients with FTLD and found significantly increased expression of the heterogeneous nuclear ribonucleoprotein R in several FTLD disease groups. Our identification of the frequent presence of hnRNP R and hnRNP Q in FTLD-FUS inclusions suggests a potential role for these hnRNPs in FTLD-FUS pathogenesis and supports the role of dysfunctional RNA metabolism in FTLD.

The term frontotemporal lobar degeneration (FTLD) describes a group of disorders that are subdivided by the presence of one of a number of pathological proteins identified in the inclusion bodies observed post-mortem. The FUS variant is defined by the presence of the fused in sarcoma protein (FUS) in the pathological inclusions. However, similar to other FTLDs, the disease pathogenesis of FTLD-FUS remains largely poorly understood. Here we present data that the protein transportin1 (TRN1) is abundant in the FUS-positive inclusions. TRN1, the protein product of the TNP01 gene, is responsible for shuttling proteins containing an M9 nuclear localisation signal between the nuclear and cytoplasmic compartments. RNA interacting proteins, including FUS, have been implicated as targets of TRN1. Using TRN1 immunohistochemistry and Western blotting in this study, we investigated 13 cases of FTLD-FUS including 6 cases with neuronal intermediate filament inclusion disease (NIFID) and 7 atypical frontotemporal lobar degeneration with ubiquitinated inclusion (aFTLD-U) cases. The data from our immunohistochemical studies show that FUS-immunoreactive inclusions are also strongly labelled with the anti-TRN1 antibody and double-label immunofluorescence studies indicate good co-localisation between the FUS and TRN1 pathologies. Our biochemical investigations demonstrate that urea-soluble TRN1 is present in aFTLD-U and NIFID, but not in normal control brains. These findings implicate abnormalities of FUS transport in the pathogenesis of FTLD-FUS.

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.

Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1 , we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 ( NOTCH2NLC ) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361 / NUTM2B-AS1 and LRP12 , respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases. Whole-genome sequencing identifies noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and oculopharyngeal myopathy with leukoencephalopathy, three disorders with overlapping clinical features and neuroimaging findings.