Objective: To evaluate the safety and activity of low dose Interleukin-2 (ld IL-2) in Amyotrophic... more Objective: To evaluate the safety and activity of low dose Interleukin-2 (ld IL-2) in Amyotrophic Lateral Sclerosis (ALS). Background: Blood regulatory T-cells (Tregs) level is a strong predictor of ALS disease severity. In auto-immune/auto-inflammatory conditions, ld IL-2 was reported to safely expand Tregs, inducing significant clinical improvements. Design/Methods: This was a double-blind, placebo-controlled, randomized (12 patients/group), parallel-group study of ld IL-2 (1MIU and 2 MIU/days s.c.) for 5 days every 4 weeks for 3 cycles. Assessment times were: before each cycle, 3 days after cycle 1 & 3 (T e max), 3 weeks after 3 rd cycle, and 3 months after last dosing. Primary criteria: Tregs at T e max/cycle-1 (expressed as numbers: Tregn and as % of CD4: Treg%). Secondary criteria: Tregs at all other study times, immuno-inflammatory blood-cells, cytokines/chemokines, functional decline (ALSFRS, Vital Capacity), and neurofilaments (NFs) levels. Safety was assessed from randomiz...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 famili... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.
Degenerative Neurological and Neuromuscular Disease, 2016
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in l... more Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in loss of the upper and lower motor neurons from motor cortex, brainstem, and spinal cord. While the majority of cases are sporadic, approximately 10% show familial inheritance. ALS is usually inherited in an autosomal dominant manner, although autosomal recessive and X-linked inheritance do occur. To date, 24 of the genes at 26 loci have been identified; these include loci linked to ALS and to frontotemporal dementia-ALS, where family pedigrees contain individuals with frontotemporal dementia with/without ALS. The most commonly established genetic causes of familial ALS (FALS) to date are the presence of a hexanucleotide repeat expansion in the C9ORF72 gene (39.3% FALS) and mutation of SOD1, TARDBP, and FUS, with frequencies of 12%-23.5%, 5%, and 4.1%, respectively. However, with the increasing use of next-generation sequencing of small family pedigrees, this has led to an increasing number of genes being associated with ALS. This review provides a comprehensive review on the genetics of ALS and an update of the pathogenic mechanisms associated with these genes. Commonly implicated pathways have been established, including RNA processing, the protein degradation pathways of autophagy and ubiquitin-proteasome system, as well as protein trafficking and cytoskeletal function. Elucidating the role genetics plays in both FALS and sporadic ALS is essential for understanding the subsequent cellular dysregulation that leads to motor neuron loss, in order to develop future effective therapeutic strategies.
BackgroundGenome‐wide association studies have confirmed the APOE ε4 allele as the single‐nucleot... more BackgroundGenome‐wide association studies have confirmed the APOE ε4 allele as the single‐nucleotide polymorphism (SNP) best associated with Alzheimer’s disease. These same studies, however, have found that a considerable number of other SNPs are associated with Alzheimer’s disease. The statistical association yielded by these other SNPs, however, is considerable weaker than that of the APOE ε4. It is unclear, therefore, whether these non‐APOE SNPs have similar effects in APOE ε4 carriers and non‐carriers. In this study, we investigated the association between a polygenic hazard score (PHS) for sporadic Alzheimer’s disease and clinical profiles of a large cohort non‐demented participants stratified by APOE ε4 status.MethodA cohort of 776 participants was shortlisted from the entire ADNI database: 262 cognitively unimpaired participants (185 with a ε3ε3 genotype and 77 with a ε4ε3 genotype) and 514 participants with a diagnosis of MCI (241 with a ε3ε3 genotype, 212 with a ε4ε3 genoty...
Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently be... more Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently been linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), and may be the most common genetic cause of both neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the most common neuropathological subtype of FTLD, characterized by inclusions of TAR DNA binding protein of 43kDa (FTLD-TDP). Previous reports have shown that TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN) mutations, with the major (risk) allele of rs1990622 associating with earlier age at onset of disease. Here we report that rs1990622 genotype affects age at death in a singlesite discovery cohort of FTLD patients with C9orf72 expansions (n=14), with the major allele correlated with later age at death (p=0.024). We replicate this modifier effect in a 30-site international neuropathological cohort of FTLD-TDP patients with C9orf72 expansions (n=75), again finding that the major allele associates with later age at death (p=0.016), as well as later age at onset (p=0.019). In contrast, TMEM106B genotype does not affect age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72 expansions. Intriguingly, the genotype that confers increased risk for developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later age at onset and death in C9orf72 expansion carriers, providing an example of sign epistasis in human neurodegenerative disease.
and Shaw PJ () A Y X TDP truncation leads to an altered metabolic profile in amyotrophic lateral ... more and Shaw PJ () A Y X TDP truncation leads to an altered metabolic profile in amyotrophic lateral sclerosis fibroblasts driven by pyruvate and TCA cycle intermediate alterations. Front. Aging Neurosci. :. doi: .
Amyotrophic lateral sclerosis is a neurodegenerative disease in which death of motoneurons leads ... more Amyotrophic lateral sclerosis is a neurodegenerative disease in which death of motoneurons leads to progressive failure of the neuromuscular system resulting in death frequently within 2-3 years of symptom onset. Focal onset and propagation of the disease symptoms to contiguous motoneuron groups is a striking feature of the human disease progression. Recent work, using mutant superoxide dismutase 1 murine models and in vitro culture systems has indicated that astrocytes are likely to contribute to the propagation of motoneuron injury and disease progression. However, the basis of this astrocyte toxicity and/or failure of motoneuron support has remained uncertain. Using a combination of in vivo and in vitro model systems of superoxide dismutase 1-related amyotrophic lateral sclerosis, linked back to human biosamples, we set out to elucidate how astrocyte properties change in the presence of mutant superoxide dismutase 1 to contribute to motoneuron injury. Gene expression profiling of spinal cord astrocytes from presymptomatic transgenic mice expressing mutant superoxide dismutase 1 revealed two striking changes. First, there was evidence of metabolic dysregulation and, in particular, impairment of the astrocyte lactate efflux transporter, with resultant decrease of spinal cord lactate levels. Second, there was evidence of increased nerve growth factor production and dysregulation of the ratio of pro-nerve growth factor to mature nerve growth factor, favouring p75 receptor expression and activation by neighbouring motoneurons. Functional in vitro studies showed that astrocytes expressing mutant superoxide dismutase 1 are toxic to normal motoneurons. We provide evidence that reduced metabolic support from lactate release and activation of pro-nerve growth factor-p75 receptor signalling are key components of this toxicity. Preservation of motoneuron viability could be achieved by increasing lactate provision to motoneurons, depletion of increased pro-nerve growth factor levels or p75 receptor blockade. These findings are likely to be relevant to human amyotrophic lateral sclerosis, where we have demonstrated increased levels of pro-nerve growth factor in cerebrospinal fluid and increased expression of the p75 receptor by spinal motoneurons. Taken together, these data confirm that altered properties of astrocytes are likely to play a crucial role in the propagation of motoneuron injury in superoxide dismutase 1-related amyotrophic lateral sclerosis and indicate that manipulation of the energy supply to motoneurons as well as inhibition of p75 receptor signalling may represent valuable neuroprotective strategies.
The advent of next generation sequencing, and specifically the sequencing of all RNA molecules in... more The advent of next generation sequencing, and specifically the sequencing of all RNA molecules in a quantitative manner, has recently become an alternative, though expensive method for measuring levels of gene expression. However, this has the potential to add further knowledge and value to the application of gene expression profiling to disease. 3. Uses of gene expression profiling Quantification of the transcriptome has been a useful mechanism for both discovering and defining mechanisms of pathogenesis in ALS (Cox et al 2010; Ferraiuolo et al 2007; Kirby et al 2005; Kirby et al 2011). In particular lists of differentially expressed genes can be usefully converted to functional 'themes' by an enrichment analysis (Hosack et al 2003). Various categorisations exist, including the gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG), which classify genes according to molecular function, biological process, cellular component or a known biological pathway. A frequent application of gene expression profiling has been the development of putative biomarkers via a supervised classification approach (Booij et al 2011; Nagasaka et al 2005; Scherzer et al 2007). The large number of targets quantified simultaneously by gene expression profiling is essential for biomarker discovery, as it allows an unbiased survey of the most informative RNA transcripts. A reliable biomarker(s) for defining pathogenesis and prognosis in ALS has yet to be established, though gene expression profiling is one of the methodologies currently being used to establish an ALS biomarker(s).
We describe an autosomal dominant, multi‐generational, amyotrophic lateral sclerosis (ALS) pedigr... more We describe an autosomal dominant, multi‐generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co‐segregates with a heterozygous p.Y374X nonsense mutation within TDP‐43. Mislocalization of TDP‐43 and formation of insoluble TDP‐43‐positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP‐43 pathology within lower motor neurons, but classical TDP‐43‐positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP‐43 protein expression is reduced compared to wild‐type controls. Despite absence of TDP‐43‐positive inclusions we confirmed deficient TDP‐43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP‐43 protein species but not typical C‐terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP‐43 combined with atypical TDP‐43 protein species and absent C‐terminal fragments extends the molecular phenotypes associated with TDP‐43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP‐43‐mediated toxicity.
Neuropathology and Applied Neurobiology, Jul 7, 2015
Intermediate-length cytosine-adenine-guanine repeat expansions in the ATXN2 gene (which encodes f... more Intermediate-length cytosine-adenine-guanine repeat expansions in the ATXN2 gene (which encodes for Ataxin-2 protein) have been linked to increased risk for motor neurone disease/amyotrophic lateral sclerosis (ALS). We screened DNA from cases for which we had post-mortem brain tissue to enable characterization of the neuropathology associated with this mutation. Polymerase chain reaction and sequencing of DNA from frozen brain tissue on a cohort of 178 amyotrophic lateral sclerosis (ALS) autopsy cases from the north of England and 159 controls was performed. This was followed by tinctorial staining and immunohistochemistry (including for Ataxin-2) on selected blocks from ALS cases with intermediate-length expansions (ATXN2-ALS), sporadic ALS cases and neurologically healthy controls. Four ALS cases with intermediate-length CAG repeat expansions within ATXN2 were identified. One such case also had a mutation of the C9ORF72 gene. All had lower motor neurone depletion, and three out of four cases had transactive response DNA binding protein 43 (TDP-43)-positive neuronal cytoplasmic inclusions (predominantly skein-like). No inclusions of aggregated polyglutamine proteins were identified. Ataxin-2 protein expression was largely granular and cytoplasmic with the most prominent staining observed in larger neurones. Ataxin-2 staining was variable both within and between cases, but no staining pattern that was specific for cases with ATXN2 mutations was seen. Intermediate expansions of the CAG repeat in ATXN2 are associated with ALS. They are mostly associated with TDP-43 proteinopathy, but not with 1C2-positive polyglutamine inclusions. In the nervous system, Ataxin-2 protein expression is predominantly seen in large neurones. There is no consistent histopathological hallmark that is unique to ATXN2-ALS.
Research into amyotrophic lateral sclerosis (ALS) has been stimulated by a series of genetic and ... more Research into amyotrophic lateral sclerosis (ALS) has been stimulated by a series of genetic and molecular pathology discoveries. The hallmark neuronal cytoplasmic inclusions of sporadic ALS (sALS) predominantly comprise a nuclear RNA processing protein, TDP-43 encoded by the gene TARDBP, a discovery that emerged from high throughput analysis of human brain tissue from patients with frontotemporal dementia (FTD) who share a common molecular pathology with ALS. The link between RNA processing and ALS was further strengthened by the discovery that another genetic locus linking familial ALS (fALS) and FTD was due to mutation of the fused in sarcoma (FUS) gene. Of potentially even greater importance it emerges that TDP-43 accumulation and inclusion formation characterises not only most sALS cases but also those that arise from mutations in several genes including TARDBP (predominantly ALS cases) itself, C9ORF72 (ALS and FTD cases), progranulin (predominantly FTD phenotypes), VAPB (predominantly ALS cases) and in some ALS cases with rare genetic variants of uncertain pathogenicity (CHMP2B). "TDP-proteinopathy" therefore now represents a final common pathology associated with changes in multiple genes and opens the possibility of research by triangulation towards key common upstream molecular events. It also delivers final proof of the hypothesis that ALS and most FTD cases are disorders within a common pathology expressed as a clinico-anatomical spectrum. The emergence of TDP-proteinopathy also confirms the view that glial pathology is a crucial facet in this class of neurodegeneration, adding to the established view of non-nerve cell autonomous degeneration of the motor system from previous research on SOD1 fALS. Future research into the mechanisms of TDP-43 and FUS-related neurodegeneration, taking into account the major component of glial pathology now revealed in those disorders will significantly accelerate new discoveries in this field, including target identification for new therapy.
MicroRNAs (miRNAs) are small, abundant RNA molecules that constitute part of the cell's non-codin... more MicroRNAs (miRNAs) are small, abundant RNA molecules that constitute part of the cell's non-coding RNA "dark matter." In recent years, the discovery of miRNAs has revolutionised the traditional view of gene expression and our understanding of miRNA biogenesis and function has expanded. Altered expression of miRNAs is increasingly recognized as a feature of many disease states, including neurodegeneration. Here, we review the emerging role for miRNA dysfunction in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Huntington's disease pathogenesis. We emphasize the complex nature of gene regulatory networks and the need for systematic studies, with larger sample cohorts than have so far been reported, to reveal the most important miRNA regulators in disease. Finally, miRNA diversity and their potential to target multiple pathways, offers novel clinical applications for miRNAs as biomarkers and therapeutic agents in neurodegenerative diseases.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons resulting in ... more Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons resulting in progressive paralysis. Gene expression studies of ALS only rarely identify the same gene pathways as gene association studies. We hypothesised that analysing tissues by matching on degree of disease severity would identify different patterns of gene expression than a traditional case-control comparison. We analysed gene expression changes in four post-mortem CNS regions, stratified by severity of motor neuron loss. An overall case (n = 6) control (n = 3) comparison identified known ALS gene, SOX5 as showing differential expression (log2 fold-change = 0.09, p = 5.5x10-5). Analyses stratified by disease severity identified expression changes in C9orf72 (p = 2.77x10-3), MATR3 (p = 3.46x10-3) and VEGFA (p = 8.21x10-4), both implicated in ALS through genetic studies, as well as changes in other genes in pathways involving RNA processing and immune response. These findings suggest analysis of gene expression stratified by disease severity can identify major ALS genes and may be more efficient than traditional case-control comparison.
Amyotrophic lateral sclerosis & frontotemporal degeneration, Jun 2, 2020
This is a repository copy of The involvement of regulatory T cells in amyotrophic lateral scleros... more This is a repository copy of The involvement of regulatory T cells in amyotrophic lateral sclerosis and their therapeutic potential.
Mutations in CHCHD10 have recently been reported as a cause of amyotrophic lateral sclerosis (ALS... more Mutations in CHCHD10 have recently been reported as a cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To address the genetic contribution of CHCHD10 to ALS we have screened a cohort of 425 UK ALS+/-FTD patients and 576 local controls in all coding exons of CHCHD10 by Sanger sequencing. We identified a previously reported Pro34Ser variant that was also present in neurologically healthy controls (p=0.58). Our results suggest that CHCHD10 is not a primary cause of ALS in UK cases.
A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to ... more 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 þ / ÀFTD from five European cohorts (total n ¼ 1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n ¼ 434) and controls (n ¼ 856). Haplotypes were analysed using PLINK and aged using DMLE þ. In a London clinic cohort, the HREM was the most common mutation in familial ALS þ / ÀFTD: 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 þ / ÀFTD 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, Po10 À8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.
Although our knowledge regarding the physiological mechanisms activated in response to exercise h... more Although our knowledge regarding the physiological mechanisms activated in response to exercise has substantially increased in the past decade, the specific genes contributing to the activation and the development of an adaptive response to exercise have remained largely unknown. The availability of muscular biopsies and blood from healthy individuals enabled the study of the response to physical activity in these human tissues (Connolly et al. 2004; Timmons and Sundberg 2006); however, the effects of exercise on the CNS could only be investigated in animal models. These studies, mainly focussed on the hippocampus (Gomez-Pinilla et al. 2002) and the spinal cord (Ying et al. 2005), often use models of forced training (Macias et al. 2007), which have the advantage of being standardized and easy to quantify, but use aversive stimuli to induce exercise and are performed in conditions far from the normal murine pattern of nocturnal activity. These factors may have major confounding effects on both the physiologic responses to exercise and the molecular mechanisms underlying these responses. To identify the specific adaptive transcriptional changes taking place in the neuromuscular system in response to exercise training, we used a model of voluntary wheel
Transcriptome study in neurodegenerative disease has advanced considerably in the past 5 years. I... more Transcriptome study in neurodegenerative disease has advanced considerably in the past 5 years. Increasing scientific rigour and improved analytical tools have led to more-reproducible data. Many transcriptome analysis platforms assay the expression of the entire genome, enabling a complete biological context to be captured. Gene expression profiling (GEP) is, therefore, uniquely placed to discover pathways of disease pathogenesis, potential therapeutic targets, and biomarkers. This Review summarizes microarray human GEP studies in the common neurodegenerative diseases amyotrophic lateral sclerosis (ALS), Parkinson disease (PD) and Alzheimer disease (AD). Several interesting reports have compared pathological gene expression in different patient groups, disease stages and anatomical areas. In all three diseases, GEP has revealed dysregulation of genes related to neuroinflammation. In ALS and PD, gene expression related to RNA splicing and protein turnover is disrupted, and several studies in ALS support involvement of the cytoskeleton. GEP studies have implicated the ubiquitin-proteasome system in PD pathogenesis, and have provided evidence of mitochondrial dysfunction in PD and AD. Lastly, in AD, a possible role for dysregulation of intracellular signalling pathways, including calcium signalling, has been highlighted. This Review also provides a discussion of methodological considerations in microarray sample preparation and data analysis.
Objective: To evaluate the safety and activity of low dose Interleukin-2 (ld IL-2) in Amyotrophic... more Objective: To evaluate the safety and activity of low dose Interleukin-2 (ld IL-2) in Amyotrophic Lateral Sclerosis (ALS). Background: Blood regulatory T-cells (Tregs) level is a strong predictor of ALS disease severity. In auto-immune/auto-inflammatory conditions, ld IL-2 was reported to safely expand Tregs, inducing significant clinical improvements. Design/Methods: This was a double-blind, placebo-controlled, randomized (12 patients/group), parallel-group study of ld IL-2 (1MIU and 2 MIU/days s.c.) for 5 days every 4 weeks for 3 cycles. Assessment times were: before each cycle, 3 days after cycle 1 & 3 (T e max), 3 weeks after 3 rd cycle, and 3 months after last dosing. Primary criteria: Tregs at T e max/cycle-1 (expressed as numbers: Tregn and as % of CD4: Treg%). Secondary criteria: Tregs at all other study times, immuno-inflammatory blood-cells, cytokines/chemokines, functional decline (ALSFRS, Vital Capacity), and neurofilaments (NFs) levels. Safety was assessed from randomiz...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 famili... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.
Degenerative Neurological and Neuromuscular Disease, 2016
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in l... more Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in loss of the upper and lower motor neurons from motor cortex, brainstem, and spinal cord. While the majority of cases are sporadic, approximately 10% show familial inheritance. ALS is usually inherited in an autosomal dominant manner, although autosomal recessive and X-linked inheritance do occur. To date, 24 of the genes at 26 loci have been identified; these include loci linked to ALS and to frontotemporal dementia-ALS, where family pedigrees contain individuals with frontotemporal dementia with/without ALS. The most commonly established genetic causes of familial ALS (FALS) to date are the presence of a hexanucleotide repeat expansion in the C9ORF72 gene (39.3% FALS) and mutation of SOD1, TARDBP, and FUS, with frequencies of 12%-23.5%, 5%, and 4.1%, respectively. However, with the increasing use of next-generation sequencing of small family pedigrees, this has led to an increasing number of genes being associated with ALS. This review provides a comprehensive review on the genetics of ALS and an update of the pathogenic mechanisms associated with these genes. Commonly implicated pathways have been established, including RNA processing, the protein degradation pathways of autophagy and ubiquitin-proteasome system, as well as protein trafficking and cytoskeletal function. Elucidating the role genetics plays in both FALS and sporadic ALS is essential for understanding the subsequent cellular dysregulation that leads to motor neuron loss, in order to develop future effective therapeutic strategies.
BackgroundGenome‐wide association studies have confirmed the APOE ε4 allele as the single‐nucleot... more BackgroundGenome‐wide association studies have confirmed the APOE ε4 allele as the single‐nucleotide polymorphism (SNP) best associated with Alzheimer’s disease. These same studies, however, have found that a considerable number of other SNPs are associated with Alzheimer’s disease. The statistical association yielded by these other SNPs, however, is considerable weaker than that of the APOE ε4. It is unclear, therefore, whether these non‐APOE SNPs have similar effects in APOE ε4 carriers and non‐carriers. In this study, we investigated the association between a polygenic hazard score (PHS) for sporadic Alzheimer’s disease and clinical profiles of a large cohort non‐demented participants stratified by APOE ε4 status.MethodA cohort of 776 participants was shortlisted from the entire ADNI database: 262 cognitively unimpaired participants (185 with a ε3ε3 genotype and 77 with a ε4ε3 genotype) and 514 participants with a diagnosis of MCI (241 with a ε3ε3 genotype, 212 with a ε4ε3 genoty...
Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently be... more Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently been linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), and may be the most common genetic cause of both neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the most common neuropathological subtype of FTLD, characterized by inclusions of TAR DNA binding protein of 43kDa (FTLD-TDP). Previous reports have shown that TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN) mutations, with the major (risk) allele of rs1990622 associating with earlier age at onset of disease. Here we report that rs1990622 genotype affects age at death in a singlesite discovery cohort of FTLD patients with C9orf72 expansions (n=14), with the major allele correlated with later age at death (p=0.024). We replicate this modifier effect in a 30-site international neuropathological cohort of FTLD-TDP patients with C9orf72 expansions (n=75), again finding that the major allele associates with later age at death (p=0.016), as well as later age at onset (p=0.019). In contrast, TMEM106B genotype does not affect age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72 expansions. Intriguingly, the genotype that confers increased risk for developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later age at onset and death in C9orf72 expansion carriers, providing an example of sign epistasis in human neurodegenerative disease.
and Shaw PJ () A Y X TDP truncation leads to an altered metabolic profile in amyotrophic lateral ... more and Shaw PJ () A Y X TDP truncation leads to an altered metabolic profile in amyotrophic lateral sclerosis fibroblasts driven by pyruvate and TCA cycle intermediate alterations. Front. Aging Neurosci. :. doi: .
Amyotrophic lateral sclerosis is a neurodegenerative disease in which death of motoneurons leads ... more Amyotrophic lateral sclerosis is a neurodegenerative disease in which death of motoneurons leads to progressive failure of the neuromuscular system resulting in death frequently within 2-3 years of symptom onset. Focal onset and propagation of the disease symptoms to contiguous motoneuron groups is a striking feature of the human disease progression. Recent work, using mutant superoxide dismutase 1 murine models and in vitro culture systems has indicated that astrocytes are likely to contribute to the propagation of motoneuron injury and disease progression. However, the basis of this astrocyte toxicity and/or failure of motoneuron support has remained uncertain. Using a combination of in vivo and in vitro model systems of superoxide dismutase 1-related amyotrophic lateral sclerosis, linked back to human biosamples, we set out to elucidate how astrocyte properties change in the presence of mutant superoxide dismutase 1 to contribute to motoneuron injury. Gene expression profiling of spinal cord astrocytes from presymptomatic transgenic mice expressing mutant superoxide dismutase 1 revealed two striking changes. First, there was evidence of metabolic dysregulation and, in particular, impairment of the astrocyte lactate efflux transporter, with resultant decrease of spinal cord lactate levels. Second, there was evidence of increased nerve growth factor production and dysregulation of the ratio of pro-nerve growth factor to mature nerve growth factor, favouring p75 receptor expression and activation by neighbouring motoneurons. Functional in vitro studies showed that astrocytes expressing mutant superoxide dismutase 1 are toxic to normal motoneurons. We provide evidence that reduced metabolic support from lactate release and activation of pro-nerve growth factor-p75 receptor signalling are key components of this toxicity. Preservation of motoneuron viability could be achieved by increasing lactate provision to motoneurons, depletion of increased pro-nerve growth factor levels or p75 receptor blockade. These findings are likely to be relevant to human amyotrophic lateral sclerosis, where we have demonstrated increased levels of pro-nerve growth factor in cerebrospinal fluid and increased expression of the p75 receptor by spinal motoneurons. Taken together, these data confirm that altered properties of astrocytes are likely to play a crucial role in the propagation of motoneuron injury in superoxide dismutase 1-related amyotrophic lateral sclerosis and indicate that manipulation of the energy supply to motoneurons as well as inhibition of p75 receptor signalling may represent valuable neuroprotective strategies.
The advent of next generation sequencing, and specifically the sequencing of all RNA molecules in... more The advent of next generation sequencing, and specifically the sequencing of all RNA molecules in a quantitative manner, has recently become an alternative, though expensive method for measuring levels of gene expression. However, this has the potential to add further knowledge and value to the application of gene expression profiling to disease. 3. Uses of gene expression profiling Quantification of the transcriptome has been a useful mechanism for both discovering and defining mechanisms of pathogenesis in ALS (Cox et al 2010; Ferraiuolo et al 2007; Kirby et al 2005; Kirby et al 2011). In particular lists of differentially expressed genes can be usefully converted to functional 'themes' by an enrichment analysis (Hosack et al 2003). Various categorisations exist, including the gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG), which classify genes according to molecular function, biological process, cellular component or a known biological pathway. A frequent application of gene expression profiling has been the development of putative biomarkers via a supervised classification approach (Booij et al 2011; Nagasaka et al 2005; Scherzer et al 2007). The large number of targets quantified simultaneously by gene expression profiling is essential for biomarker discovery, as it allows an unbiased survey of the most informative RNA transcripts. A reliable biomarker(s) for defining pathogenesis and prognosis in ALS has yet to be established, though gene expression profiling is one of the methodologies currently being used to establish an ALS biomarker(s).
We describe an autosomal dominant, multi‐generational, amyotrophic lateral sclerosis (ALS) pedigr... more We describe an autosomal dominant, multi‐generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co‐segregates with a heterozygous p.Y374X nonsense mutation within TDP‐43. Mislocalization of TDP‐43 and formation of insoluble TDP‐43‐positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP‐43 pathology within lower motor neurons, but classical TDP‐43‐positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP‐43 protein expression is reduced compared to wild‐type controls. Despite absence of TDP‐43‐positive inclusions we confirmed deficient TDP‐43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP‐43 protein species but not typical C‐terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP‐43 combined with atypical TDP‐43 protein species and absent C‐terminal fragments extends the molecular phenotypes associated with TDP‐43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP‐43‐mediated toxicity.
Neuropathology and Applied Neurobiology, Jul 7, 2015
Intermediate-length cytosine-adenine-guanine repeat expansions in the ATXN2 gene (which encodes f... more Intermediate-length cytosine-adenine-guanine repeat expansions in the ATXN2 gene (which encodes for Ataxin-2 protein) have been linked to increased risk for motor neurone disease/amyotrophic lateral sclerosis (ALS). We screened DNA from cases for which we had post-mortem brain tissue to enable characterization of the neuropathology associated with this mutation. Polymerase chain reaction and sequencing of DNA from frozen brain tissue on a cohort of 178 amyotrophic lateral sclerosis (ALS) autopsy cases from the north of England and 159 controls was performed. This was followed by tinctorial staining and immunohistochemistry (including for Ataxin-2) on selected blocks from ALS cases with intermediate-length expansions (ATXN2-ALS), sporadic ALS cases and neurologically healthy controls. Four ALS cases with intermediate-length CAG repeat expansions within ATXN2 were identified. One such case also had a mutation of the C9ORF72 gene. All had lower motor neurone depletion, and three out of four cases had transactive response DNA binding protein 43 (TDP-43)-positive neuronal cytoplasmic inclusions (predominantly skein-like). No inclusions of aggregated polyglutamine proteins were identified. Ataxin-2 protein expression was largely granular and cytoplasmic with the most prominent staining observed in larger neurones. Ataxin-2 staining was variable both within and between cases, but no staining pattern that was specific for cases with ATXN2 mutations was seen. Intermediate expansions of the CAG repeat in ATXN2 are associated with ALS. They are mostly associated with TDP-43 proteinopathy, but not with 1C2-positive polyglutamine inclusions. In the nervous system, Ataxin-2 protein expression is predominantly seen in large neurones. There is no consistent histopathological hallmark that is unique to ATXN2-ALS.
Research into amyotrophic lateral sclerosis (ALS) has been stimulated by a series of genetic and ... more Research into amyotrophic lateral sclerosis (ALS) has been stimulated by a series of genetic and molecular pathology discoveries. The hallmark neuronal cytoplasmic inclusions of sporadic ALS (sALS) predominantly comprise a nuclear RNA processing protein, TDP-43 encoded by the gene TARDBP, a discovery that emerged from high throughput analysis of human brain tissue from patients with frontotemporal dementia (FTD) who share a common molecular pathology with ALS. The link between RNA processing and ALS was further strengthened by the discovery that another genetic locus linking familial ALS (fALS) and FTD was due to mutation of the fused in sarcoma (FUS) gene. Of potentially even greater importance it emerges that TDP-43 accumulation and inclusion formation characterises not only most sALS cases but also those that arise from mutations in several genes including TARDBP (predominantly ALS cases) itself, C9ORF72 (ALS and FTD cases), progranulin (predominantly FTD phenotypes), VAPB (predominantly ALS cases) and in some ALS cases with rare genetic variants of uncertain pathogenicity (CHMP2B). "TDP-proteinopathy" therefore now represents a final common pathology associated with changes in multiple genes and opens the possibility of research by triangulation towards key common upstream molecular events. It also delivers final proof of the hypothesis that ALS and most FTD cases are disorders within a common pathology expressed as a clinico-anatomical spectrum. The emergence of TDP-proteinopathy also confirms the view that glial pathology is a crucial facet in this class of neurodegeneration, adding to the established view of non-nerve cell autonomous degeneration of the motor system from previous research on SOD1 fALS. Future research into the mechanisms of TDP-43 and FUS-related neurodegeneration, taking into account the major component of glial pathology now revealed in those disorders will significantly accelerate new discoveries in this field, including target identification for new therapy.
MicroRNAs (miRNAs) are small, abundant RNA molecules that constitute part of the cell's non-codin... more MicroRNAs (miRNAs) are small, abundant RNA molecules that constitute part of the cell's non-coding RNA "dark matter." In recent years, the discovery of miRNAs has revolutionised the traditional view of gene expression and our understanding of miRNA biogenesis and function has expanded. Altered expression of miRNAs is increasingly recognized as a feature of many disease states, including neurodegeneration. Here, we review the emerging role for miRNA dysfunction in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Huntington's disease pathogenesis. We emphasize the complex nature of gene regulatory networks and the need for systematic studies, with larger sample cohorts than have so far been reported, to reveal the most important miRNA regulators in disease. Finally, miRNA diversity and their potential to target multiple pathways, offers novel clinical applications for miRNAs as biomarkers and therapeutic agents in neurodegenerative diseases.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons resulting in ... more Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons resulting in progressive paralysis. Gene expression studies of ALS only rarely identify the same gene pathways as gene association studies. We hypothesised that analysing tissues by matching on degree of disease severity would identify different patterns of gene expression than a traditional case-control comparison. We analysed gene expression changes in four post-mortem CNS regions, stratified by severity of motor neuron loss. An overall case (n = 6) control (n = 3) comparison identified known ALS gene, SOX5 as showing differential expression (log2 fold-change = 0.09, p = 5.5x10-5). Analyses stratified by disease severity identified expression changes in C9orf72 (p = 2.77x10-3), MATR3 (p = 3.46x10-3) and VEGFA (p = 8.21x10-4), both implicated in ALS through genetic studies, as well as changes in other genes in pathways involving RNA processing and immune response. These findings suggest analysis of gene expression stratified by disease severity can identify major ALS genes and may be more efficient than traditional case-control comparison.
Amyotrophic lateral sclerosis & frontotemporal degeneration, Jun 2, 2020
This is a repository copy of The involvement of regulatory T cells in amyotrophic lateral scleros... more This is a repository copy of The involvement of regulatory T cells in amyotrophic lateral sclerosis and their therapeutic potential.
Mutations in CHCHD10 have recently been reported as a cause of amyotrophic lateral sclerosis (ALS... more Mutations in CHCHD10 have recently been reported as a cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To address the genetic contribution of CHCHD10 to ALS we have screened a cohort of 425 UK ALS+/-FTD patients and 576 local controls in all coding exons of CHCHD10 by Sanger sequencing. We identified a previously reported Pro34Ser variant that was also present in neurologically healthy controls (p=0.58). Our results suggest that CHCHD10 is not a primary cause of ALS in UK cases.
A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to ... more 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 þ / ÀFTD from five European cohorts (total n ¼ 1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n ¼ 434) and controls (n ¼ 856). Haplotypes were analysed using PLINK and aged using DMLE þ. In a London clinic cohort, the HREM was the most common mutation in familial ALS þ / ÀFTD: 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 þ / ÀFTD 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, Po10 À8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.
Although our knowledge regarding the physiological mechanisms activated in response to exercise h... more Although our knowledge regarding the physiological mechanisms activated in response to exercise has substantially increased in the past decade, the specific genes contributing to the activation and the development of an adaptive response to exercise have remained largely unknown. The availability of muscular biopsies and blood from healthy individuals enabled the study of the response to physical activity in these human tissues (Connolly et al. 2004; Timmons and Sundberg 2006); however, the effects of exercise on the CNS could only be investigated in animal models. These studies, mainly focussed on the hippocampus (Gomez-Pinilla et al. 2002) and the spinal cord (Ying et al. 2005), often use models of forced training (Macias et al. 2007), which have the advantage of being standardized and easy to quantify, but use aversive stimuli to induce exercise and are performed in conditions far from the normal murine pattern of nocturnal activity. These factors may have major confounding effects on both the physiologic responses to exercise and the molecular mechanisms underlying these responses. To identify the specific adaptive transcriptional changes taking place in the neuromuscular system in response to exercise training, we used a model of voluntary wheel
Transcriptome study in neurodegenerative disease has advanced considerably in the past 5 years. I... more Transcriptome study in neurodegenerative disease has advanced considerably in the past 5 years. Increasing scientific rigour and improved analytical tools have led to more-reproducible data. Many transcriptome analysis platforms assay the expression of the entire genome, enabling a complete biological context to be captured. Gene expression profiling (GEP) is, therefore, uniquely placed to discover pathways of disease pathogenesis, potential therapeutic targets, and biomarkers. This Review summarizes microarray human GEP studies in the common neurodegenerative diseases amyotrophic lateral sclerosis (ALS), Parkinson disease (PD) and Alzheimer disease (AD). Several interesting reports have compared pathological gene expression in different patient groups, disease stages and anatomical areas. In all three diseases, GEP has revealed dysregulation of genes related to neuroinflammation. In ALS and PD, gene expression related to RNA splicing and protein turnover is disrupted, and several studies in ALS support involvement of the cytoskeleton. GEP studies have implicated the ubiquitin-proteasome system in PD pathogenesis, and have provided evidence of mitochondrial dysfunction in PD and AD. Lastly, in AD, a possible role for dysregulation of intracellular signalling pathways, including calcium signalling, has been highlighted. This Review also provides a discussion of methodological considerations in microarray sample preparation and data analysis.
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