Bradley Smith

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.

Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS

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.

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways

New players in Lou Gehrigs disease Amyotrophic lateral sclerosis (ALS), often referred to as “Lou Gehrigs disease,” is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Cirulli et al. sequenced the expressed genes of nearly 3000 ALS patients and compared them with those of more than 6000 controls (see the Perspective by Singleton and Traynor). They identified several proteins that were linked to disease in patients. One such protein, TBK1, is implicated in innate immunity and autophagy and may represent a therapeutic target. Science, this issue p. 1436; see also p. 1422 Analysis of the expressed genes of nearly 2900 patients with amyotrophic lateral sclerosis and about 6400 controls reveals a disease predisposition–associated gene. [Also see Perspective by Singleton and Traynor] Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

p62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS

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.

Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

Summary The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

Chromosome 9p21 in sporadic amyotrophic lateral sclerosis in the UK and seven other countries: a genome-wide association study

Summary Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons that results in progressive weakness and death from respiratory failure, commonly within about 3 years. Previous studies have shown association of a locus on chromosome 9p with ALS and linkage with ALS–frontotemporal dementia. We aimed to test whether this genomic region is also associated with ALS in an independent set of UK samples, and to identify risk factors associated with ALS in a further genome-wide association study that combined data from the independent analysis with those from other countries. Methods We collected samples from patients with sporadic ALS from 20 UK hospitals and obtained UK control samples from the control groups of the Depression Case Control study, the Bipolar Affective Case Control Study, and the British 1958 birth cohort DNA collection. Genotyping of DNA in this independent analysis was done with Illumina HumanHap550 BeadChips. We then undertook a joint genome-wide analysis that combined data from the independent set with published data from the UK, USA, Netherlands, Ireland, Italy, France, Sweden, and Belgium. The threshold for significance was p=0·05 in the independent analysis, because we were interested in replicating a small number of previously reported associations, whereas the Bonferroni-corrected threshold for significance in the joint analysis was p=2·20×10−7 Findings After quality control, samples were available from 599 patients and 4144 control individuals in the independent set. In this analysis, two single nucleotide polymorphisms in a locus on chromosome 9p21.2 were associated with ALS: rs3849942 (p=2·22×10−6; odds ratio [OR] 1·39, 95% CI 1·21–1·59) and rs2814707 (p=3·32×10−6; 1·38, 1·20–1·58). In the joint analysis, which included samples from 4312 patients with ALS and 8425 control individuals, rs3849942 (p=4·64×10−10; OR 1·22, 95% CI 1·15–1·30) and rs2814707 (p=4·72×10−10; 1·22, 1·15–1·30) were associated with ALS. Interpretation We have found strong evidence of a genetic association of two single nucleotide polymorphisms on chromosome 9 with sporadic ALS, in line with findings from previous independent GWAS of ALS and linkage studies of ALS–frontotemporal dementia. Our findings together with these earlier findings suggest that genetic variation at this locus on chromosome 9 causes sporadic ALS and familial ALS–frontotemporal dementia. Resequencing studies and then functional analysis should be done to identify the defective gene. Funding ALS Therapy Alliance, the Angel Fund, the Medical Research Council, the Motor Neurone Disease Association of Great Britain and Northern Ireland, the Wellcome Trust, and the National Institute for Health Research Dementias and Neurodegenerative Diseases Research Network (DeNDRoN).

Familial amyotrophic lateral sclerosis is associated with a mutation in D-amino acid oxidase

We report a unique mutation in the D-amino acid oxidase gene (R199W DAO) associated with classical adult onset familial amyotrophic lateral sclerosis (FALS) in a three generational FALS kindred, after candidate gene screening in a 14.52 cM region on chromosome 12q22-23 linked to disease. Neuronal cell lines expressing R199W DAO showed decreased viability and increased ubiquitinated aggregates compared with cells expressing the wild-type protein. Similarly, lentiviral-mediated expression of R199W DAO in primary motor neuron cultures caused increased TUNEL labeling. This effect was also observed when motor neurons were cocultured on transduced astrocytes expressing R199W, indicating that the motor neuron cell death induced by this mutation is mediated by both cell autonomous and noncell autonomous processes. DAO controls the level of D-serine, which accumulates in the spinal cord in cases of sporadic ALS and in a mouse model of ALS, indicating that this abnormality may represent a fundamental component of ALS pathogenesis.

The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/−FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/−FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/−FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10−8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.

Synergistic response to oncogenic mutations defines gene class critical to cancer phenotype

Understanding the molecular underpinnings of cancer is of critical importance to the development of targeted intervention strategies. Identification of such targets, however, is notoriously difficult and unpredictable. Malignant cell transformation requires the cooperation of a few oncogenic mutations that cause substantial reorganization of many cell features and induce complex changes in gene expression patterns. Genes critical to this multifaceted cellular phenotype have therefore only been identified after signalling pathway analysis or on an ad hoc basis. Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signalling circuitry suggest that malignant transformation is a highly cooperative process, involving synergy at multiple levels of regulation, including gene expression. Here we show that a large proportion of genes controlled synergistically by loss-of-function p53 and Ras activation are critical to the malignant state of murine and human colon cells. Notably, 14 out of 24 ‘cooperation response genes’ were found to contribute to tumour formation in gene perturbation experiments. In contrast, only 1 in 14 perturbations of the genes responding in a non-synergistic manner had a similar effect. Synergistic control of gene expression by oncogenic mutations thus emerges as an underlying key to malignancy, and provides an attractive rationale for identifying intervention targets in gene networks downstream of oncogenic gain- and loss-of-function mutations.

Activation of AMP-activated protein kinase by human EGF receptor 2/EGF receptor tyrosine kinase inhibitor protects cardiac cells

The human EGF receptor (HER) 2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, and its inhibition may explain the increased incidence of cardiomyopathy associated with the anti-HER2 monoclonal antibody trastuzumab (Genentech, South San Francisco, CA), particularly in patients with prior exposure to cardiotoxic chemotherapies e.g., anthracyclines. Here, we show that GW2974 (HER2/EGF receptor tyrosine kinase inhibitor), but not trastuzumab, activates AMP-activated protein kinase (AMPK), initiating a metabolic stress response in human cardiomyocytes that protects against TNFα-induced cell death. GW2974 stimulates calcium dependent fatty acid oxidation in vitro and in the myocardium of GW2974-treated rodents. Calcium chelation or siRNA-targeted AMPK knockdown blocks GW2974 induced fatty acid oxidation. In addition, inhibition of AMPK by a specific inhibitor resulted in increased killing of cardiomyocytes. Elucidating the effects of HER2-targeted therapies on AMPK may predict for risk of cardiomyopathy and provide a novel HER2-targeted strategy designed to protect myocardium from the pro-apoptotic effects of pro-inflammatory cytokines released in response to cardiac injury by chemotherapy or acute ischemia.