Kelly L. Williams

TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is familial in 10% of cases. We have identified a missense mutation in the gene encoding fused in sarcoma (FUS) in a British kindred, linked to ALS6. In a survey of 197 familial ALS index cases, we identified two further missense mutations in eight families. Postmortem analysis of three cases with FUS mutations showed FUS-immunoreactive cytoplasmic inclusions and predominantly lower motor neuron degeneration. Cellular expression studies revealed aberrant localization of mutant FUS protein. FUS is involved in the regulation of transcription and RNA splicing and transport, and it has functional homology to another ALS gene, TARDBP, which suggests that a common mechanism may underlie motor neuron degeneration.

A yeast functional screen predicts new candidate ALS disease genes

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis

Objective FUS gene mutations were recently identified in familial amyotrophic lateral sclerosis (ALS). The present studies sought to define the clinical, post-mortem and neurophysiological phenotypes in ALS families with FUS mutations and to determine the frequency of FUS mutations in familial and sporadic ALS. Methods FUS was screened for mutations in familial and sporadic ALS cases. Clinical, post-mortem and neurophysiological features of large families with FUS mutations are described. Results and conclusions FUS mutations were evident in 3.2% (4/124) of familial ALS, representing the second most common gene abnormality to be described in familial ALS after SOD1. No mutations were present in 247 sporadic ALS cases. The clinical presentation in 49 affected patients was consistent with a predominantly lower motor neuron disorder, supported by post-mortem findings. Upper motor neuron involvement varied, with Wallerian degeneration of corticospinal tracts present in one post-mortem case but absent in a second case from the same family. Features of cortical hyperexcitability demonstrated upper motor neuron involvement consistent with other forms of familial and sporadic ALS. One case presented with frontotemporal dementia (FTD) indicating that this may be a rare presenting feature in families with FUS mutation. Ubiquitin-positive cytoplasmic skein-like inclusions were present in lower motor neurons, but in contrast to sporadic ALS, no TDP-43 pathology was evident. Mutation-specific clinical features were identified. Patients with a R521C mutation were significantly more likely to develop disease at a younger age, and dropped-head syndrome was a frequent feature. Reduced disease penetrance was evident among most affected families.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

Sigma nonopioid intracellular receptor 1 mutations cause frontotemporal lobar degeneration-motor neuron disease.

Frontotemporal lobar degeneration (FTLD) is the most common cause of early‐onset dementia. Pathological ubiquitinated inclusion bodies observed in FTLD and motor neuron disease (MND) comprise trans‐activating response element (TAR) DNA binding protein (TDP‐43) and/or fused in sarcoma (FUS) protein. Our objective was to identify the causative gene in an FTLD‐MND pedigree with no mutations in known dementia genes.

Exome sequencing to identify de novo mutations in sporadic ALS trios

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, we assessed the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n = 141 exomes). We found that amino acid–altering de novo mutations were enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex (nBAF) component SS18L1 (also known as CREST). CREST mutations inhibited activity-dependent neurite outgrowth in primary neurons, and CREST associated with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.

UBQLN2/ubiquilin 2 mutation and pathology in familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) shows clinical and pathological overlap with frontotemporal dementia that includes the presence of hallmark ubiquitinated inclusions in affected neurons. Mutations in UBQLN2, which encodes ubiquilin 2, were recently identified in X-linked juvenile and adult-onset ALS and ALS/dementia. As part of an established exome sequencing program to identify disease genes in familial ALS, we identified a novel missense UBQLN2 mutation (c.1460C>T, p.T487I) in 2 apparently unrelated multigenerational ALS families with no evidence of frontotemporal dementia. This mutation segregated with the disease and was absent in 820 healthy controls and all public single nucleotide polymorphism databases. The UBQLN2 p.T487I mutation substitutes a highly conserved residue and is located immediately upstream of a PXX region where all previous mutations have been identified. Immunostaining of spinal cord from a patient with UBQLN2 p.T487I mutation showed colocalization of ubiquilin 2 with ubiquitin in all neuronal inclusions examined and frequent colocalization with TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma protein (FUS). To examine ubiquilin 2 pathology in broader ALS, we showed that ubiquilin 2 pathology also extends to ALS with a FUS mutation. These data further support the importance of ubiquilin 2 in the pathogenesis of ALS.

Mutant FUS induces endoplasmic reticulum stress in amyotrophic lateral sclerosis and interacts with protein disulfide-isomerase.

Mutations in the gene encoding fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), but the mechanisms by which these mutants trigger neurodegeneration remain unknown. Endoplasmic reticulum (ER) stress is increasingly recognized as an important and early pathway to motor neuron death in ALS. FUS is normally located in the nucleus but in ALS, FUS redistributes to the cytoplasm and forms inclusions. In this study, we investigated whether FUS induces ER stress in a motor neuron like cell line (NSC-34). We demonstrate that ER stress is triggered in cells expressing mutant FUS, and this is closely associated with redistribution of mutant FUS to the cytoplasm. Mutant FUS also colocalized with protein disulfide-isomerase (PDI), an important ER chaperone, in NSC-34 cells and PDI was colocalized with FUS inclusions in human ALS lumbar spinal cords, in both sporadic ALS and mutant FUS-linked familial ALS tissues. These findings implicate ER stress in the pathophysiology of FUS, and provide evidence for common pathogenic pathways in ALS linked to the ER.

Arts syndrome is caused by loss-of-function mutations in PRPS1

Arts syndrome is an X-linked disorder characterized by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy. Linkage analysis in a Dutch family and an Australian family suggested that the candidate gene maps to Xq22.1-q24. Oligonucleotide microarray expression profiling of fibroblasts from two probands of the Dutch family revealed reduced expression levels of the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1). Subsequent sequencing of PRPS1 led to the identification of two different missense mutations, c.455T-->C (p.L152P) in the Dutch family and c.398A-->C (p.Q133P) in the Australian family. Both mutations result in a loss of phosphoribosyl pyrophosphate synthetase 1 activity, as was shown in silico by molecular modeling and was shown in vitro by phosphoribosyl pyrophosphate synthetase activity assays in erythrocytes and fibroblasts from patients. This is in contrast to the gain-of-function mutations in PRPS1 that were identified previously in PRPS-related gout. The loss-of-function mutations of PRPS1 likely result in impaired purine biosynthesis, which is supported by the undetectable hypoxanthine in urine and the reduced uric acid levels in serum from patients. To replenish low levels of purines, treatment with S-adenosylmethionine theoretically could have therapeutic efficacy, and a clinical trial involving the two affected Australian brothers is currently underway.