Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.

TDP-43 protein is dysregulated in several neurodegenerative diseases, which often have a multifactorial nature and may have extrinsic stressors as a "second hit." TDP-43 undergoes reversible nuclear condensation in stressed cells including neurons. Here, we demonstrate that stress-inducible nuclear TDP-43 condensates are RNA-depleted, non-liquid assemblies distinct from the known nuclear bodies. Their formation requires TDP-43 oligomerization and ATP and is inhibited by RNA. Using a confocal nanoscanning assay, we find that amyotrophic lateral sclerosis (ALS)-linked mutations alter stress-induced TDP-43 condensation by changing its affinity to liquid-like ribonucleoprotein assemblies. Stress-induced nuclear condensation transiently inactivates TDP-43, leading to loss of interaction with its protein binding partners and loss of function in splicing. Splicing changes are especially prominent and persisting for STMN2 RNA, and STMN2 protein becomes rapidly depleted early during stress. Our results point to early pathological changes to TDP-43 in the nucleus and support therapeutic modulation of stress response in ALS.

Non-coding genome contribution to ALS.

The majority of amyotrophic lateral sclerosis (ALS) is caused by a complex gene-environment interaction. Despite high estimates of heritability, the genetic basis of disease in the majority of ALS patients are unknown. This limits the development of targeted genetic therapies which require an understanding of patient-specific genetic drivers. There is good evidence that the majority of these missing genetic risk factors are likely to be found within the non-coding genome. However, a major challenge in the discovery of non-coding risk variants is determining which variants are functional in which specific CNS cell type. We summarise current discoveries of ALS-associated genetic drivers within the non-coding genome and we make the case that improved cell-specific annotation of genomic function is required to advance this field, particularly via single-cell epigenetic profiling and spatial transcriptomics. We highlight the example of TBK1 where an apparent paradox exists between pathogenic coding variants which cause loss of protein function, and protective non-coding variants which cause reduced gene expression; the paradox is resolved when it is understood that the non-coding variants are acting primarily via change in gene expression within microglia, and the effect of coding variants is most prominent in neurons. We propose that cell-specific functional annotation of ALS-associated genetic variants will accelerate discovery of the genetic architecture underpinning disease in the vast majority of patients.

Deep learning modeling of rare noncoding genetic variants in human motor neurons defines CCDC146 as a therapeutic target for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study, we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs, this method prioritizes functional noncoding variants using deep learning, links cis-regulatory elements (CREs) to target genes using epigenomics data, and integrates these data through gene-level burden tests to identify survival-modifying variants, CREs, and genes. We apply this approach to analyze 6,715 ALS genomes, and pinpoint four novel rare noncoding variants associated with survival, including chr7:76,009,472:C>T linked to CCDC146. CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes, including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO), showing no toxicity, completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72. ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases.

Rare and common genetic determinants of mitochondrial function determine severity but not risk of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.

Atypical TDP-43 protein expression in an ALS pedigree carrying a p.Y374X truncation mutation in TARDBP.

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.

Unbiased metabolome screen leads to personalized medicine strategy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a rapidly progressive neurodegenerative disease that affects 1/350 individuals in the United Kingdom. The cause of amyotrophic lateral sclerosis is unknown in the majority of cases. Two-sample Mendelian randomization enables causal inference between an exposure, such as the serum concentration of a specific metabolite, and disease risk. We obtained genome-wide association study summary statistics for serum concentrations of 566 metabolites which were population matched with a genome-wide association study of amyotrophic lateral sclerosis. For each metabolite, we performed Mendelian randomization using an inverse variance weighted estimate for significance testing. After stringent Bonferroni multiple testing correction, our unbiased screen revealed three metabolites that were significantly linked to the risk of amyotrophic lateral sclerosis: Estrone-3-sulphate and bradykinin were protective, which is consistent with literature describing a male preponderance of amyotrophic lateral sclerosis and a preventive effect of angiotensin-converting enzyme inhibitors which inhibit the breakdown of bradykinin. Serum isoleucine was positively associated with amyotrophic lateral sclerosis risk. All three metabolites were supported by robust Mendelian randomization measures and sensitivity analyses; estrone-3-sulphate and isoleucine were confirmed in a validation amyotrophic lateral sclerosis genome-wide association study. Estrone-3-sulphate is metabolized to the more active estradiol by the enzyme 17ß-hydroxysteroid dehydrogenase 1; further, Mendelian randomization demonstrated a protective effect of estradiol and rare variant analysis showed that missense variants within HSD17B1, the gene encoding 17ß-hydroxysteroid dehydrogenase 1, modify risk for amyotrophic lateral sclerosis. Finally, in a zebrafish model of C9ORF72-amyotrophic lateral sclerosis, we present evidence that estradiol is neuroprotective. Isoleucine is metabolized via methylmalonyl-CoA mutase encoded by the gene MMUT in a reaction that consumes vitamin B12. Multivariable Mendelian randomization revealed that the toxic effect of isoleucine is dependent on the depletion of vitamin B12; consistent with this, rare variants which reduce the function of MMUT are protective against amyotrophic lateral sclerosis. We propose that amyotrophic lateral sclerosis patients and family members with high serum isoleucine levels should be offered supplementation with vitamin B12.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase ( OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.

Genome-wide identification of the genetic basis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases.

A review of Mendelian randomization in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a relatively common and rapidly progressive neurodegenerative disease that, in the majority of cases, is thought to be determined by a complex gene-environment interaction. Exponential growth in the number of performed genome-wide association studies combined with the advent of Mendelian randomization is opening significant new opportunities to identify environmental exposures that increase or decrease the risk of amyotrophic lateral sclerosis. Each of these discoveries has the potential to shape new therapeutic interventions. However, to do so, rigorous methodological standards must be applied in the performance of Mendelian randomization. We have reviewed Mendelian randomization studies performed in amyotrophic lateral sclerosis to date. We identified 20 Mendelian randomization studies, including evaluation of physical exercise, adiposity, cognitive performance, immune function, blood lipids, sleep behaviours, educational attainment, alcohol consumption, smoking and type 2 diabetes mellitus. We have evaluated each study using gold standard methodology supported by the Mendelian randomization literature and the STROBE-Mendelian randomization checklist. Where discrepancies exist between Mendelian randomization studies, we suggest the underlying reasons. A number of studies conclude that there is a causal link between blood lipids and risk of amyotrophic lateral sclerosis; replication across different datasets and even different populations adds confidence. For other putative risk factors, such as smoking and immune function, Mendelian randomization studies have provided cause for doubt. We highlight the use of positive control analyses in choosing exposure single nucleotide polymorphisms (SNPs) to make up the Mendelian randomization instrument, use of SNP clumping to avoid false positive results due to SNPs in linkage and the importance of multiple testing correction. We discuss the implications of survival bias for study of late age of onset diseases such as amyotrophic lateral sclerosis and make recommendations to mitigate this potentially important confounder. For Mendelian randomization to be useful to the amyotrophic lateral sclerosis field, high methodological standards must be applied to ensure reproducibility. Mendelian randomization is already an impactful tool, but poor-quality studies will lead to incorrect interpretations by a field that includes non-statisticians, wasted resources and missed opportunities.

Membrane lipid raft homeostasis is directly linked to neurodegeneration.

Age-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD) are an unmet health need, with significant economic and societal implications, and an ever-increasing prevalence. Membrane lipid rafts (MLRs) are specialised plasma membrane microdomains that provide a platform for intracellular trafficking and signal transduction, particularly within neurons. Dysregulation of MLRs leads to disruption of neurotrophic signalling and excessive apoptosis which mirrors the final common pathway for neuronal death in ALS, PD and AD. Sphingomyelinase (SMase) and phospholipase (PL) enzymes process components of MLRs and therefore play central roles in MLR homeostasis and in neurotrophic signalling. We review the literature linking SMase and PL enzymes to ALS, AD and PD with particular attention to attractive therapeutic targets, where functional manipulation has been successful in preclinical studies. We propose that dysfunction of these enzymes is upstream in the pathogenesis of neurodegenerative diseases and to support this we provide new evidence that ALS risk genes are enriched with genes involved in ceramide metabolism (P=0.019, OR = 2.54, Fisher exact test). Ceramide is a product of SMase action upon sphingomyelin within MLRs, and it also has a role as a second messenger in intracellular signalling pathways important for neuronal survival. Genetic risk is necessarily upstream in a late age of onset disease such as ALS. We propose that manipulation of MLR structure and function should be a focus of future translational research seeking to ameliorate neurodegenerative disorders.

Advances in the genetic classification of amyotrophic lateral sclerosis.

PURPOSE OF REVIEW: Amyotrophic lateral sclerosis (ALS) is an archetypal complex disease wherein disease risk and severity are, for the majority of patients, the product of interaction between multiple genetic and environmental factors. We are in a period of unprecedented discovery with new large-scale genome-wide association study (GWAS) and accelerating discovery of risk genes. However, much of the observed heritability of ALS is undiscovered and we are not yet approaching elucidation of the total genetic architecture, which will be necessary for comprehensive disease subclassification. RECENT FINDINGS: We summarize recent developments and discuss the future. New machine learning models will help to address nonlinear genetic interactions. Statistical power for genetic discovery may be boosted by reducing the search-space using cell-specific epigenetic profiles and expanding our scope to include genetically correlated phenotypes. Structural variation, somatic heterogeneity and consideration of environmental modifiers represent significant challenges which will require integration of multiple technologies and a multidisciplinary approach, including clinicians, geneticists and pathologists. SUMMARY: The move away from fully penetrant Mendelian risk genes necessitates new experimental designs and new standards for validation. The challenges are significant, but the potential reward for successful disease subclassification is large-scale and effective personalized medicine.

Physical exercise is a risk factor for amyotrophic lateral sclerosis: Convergent evidence from Mendelian randomisation, transcriptomics and risk genotypes.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a universally fatal neurodegenerative disease. ALS is determined by gene-environment interactions and improved understanding of these interactions may lead to effective personalised medicine. The role of physical exercise in the development of ALS is currently controversial. METHODS: First, we dissected the exercise-ALS relationship in a series of two-sample Mendelian randomisation (MR) experiments. Next we tested for enrichment of ALS genetic risk within exercise-associated transcriptome changes. Finally, we applied a validated physical activity questionnaire in a small cohort of genetically selected ALS patients. FINDINGS: We present MR evidence supporting a causal relationship between genetic liability to frequent and strenuous leisure-time exercise and ALS using a liberal instrument (multiplicative random effects IVW, p=0.01). Transcriptomic analysis revealed that genes with altered expression in response to acute exercise are enriched with known ALS risk genes (permutation test, p=0.013) including C9ORF72, and with ALS-associated rare variants of uncertain significance. Questionnaire evidence revealed that age of onset is inversely proportional to historical physical activity for C9ORF72-ALS (Cox proportional hazards model, Wald test p=0.007, likelihood ratio test p=0.01, concordance=74%) but not for non-C9ORF72-ALS. Variability in average physical activity was lower in C9ORF72-ALS compared to both non-C9ORF72-ALS (F-test, p=0.002) and neurologically normal controls (F-test, p=0.049) which is consistent with a homogeneous effect of physical activity in all C9ORF72-ALS patients. INTERPRETATION: Our MR approach suggests a positive causal relationship between ALS and physical exercise. Exercise is likely to cause motor neuron injury only in patients with a risk-genotype. Consistent with this we have shown that ALS risk genes are activated in response to exercise. In particular, we propose that G4C2-repeat expansion of C9ORF72 predisposes to exercise-induced ALS. FUNDING: We acknowledge support from the Wellcome Trust (JCK, 216596/Z/19/Z), NIHR (PJS, NF-SI-0617-10077; IS-BRC-1215-20017) and NIH (MPS, CEGS 5P50HG00773504, 1P50HL083800, 1R01HL101388, 1R01-HL122939, S10OD025212, P30DK116074, and UM1HG009442).

Simultaneous ALS and SCA2 associated with an intermediate-length ATXN2 CAG-repeat expansion.

Spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS) share a common molecular basis: both are associated with CAG-repeat expansion of ATXN2 and TDP-43-positive neuronal cytoplasmic inclusions. To date, the two disorders are viewed as clinically distinct with ALS resulting from 30-33 CAG-repeats and SCA2 from >34 CAG-repeats. We describe a 67-year old with a 32 CAG-repeat expansion of ATXN2 who presented with simultaneous symptoms of ALS and SCA2. Our case demonstrates that the clinical dichotomy between SCA2 and ATXN2-ALS is false. We suggest instead that CAG-repeat expansion length determines the timing of SCA2 clinical symptoms relative to onset of ALS; consistent with this age of onset of SCA2 but not ATXN2-ALS, is dependent upon expansion length. Review of the literature and our local cohort provides evidence for occurrence of ALS in late stage SCA2, which may be under-recognised by clinicians who think of the two diseases as distinct.

Rare Variant Burden Analysis within Enhancers Identifies CAV1 as an ALS Risk Gene.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. CAV1 and CAV2 organize membrane lipid rafts (MLRs) important for cell signaling and neuronal survival, and overexpression of CAV1 ameliorates ALS phenotypes in vivo. Genome-wide association studies localize a large proportion of ALS risk variants within the non-coding genome, but further characterization has been limited by lack of appropriate tools. By designing and applying a pipeline to identify pathogenic genetic variation within enhancer elements responsible for regulating gene expression, we identify disease-associated variation within CAV1/CAV2 enhancers, which replicate in an independent cohort. Discovered enhancer mutations reduce CAV1/CAV2 expression and disrupt MLRs in patient-derived cells, and CRISPR-Cas9 perturbation proximate to a patient mutation is sufficient to reduce CAV1/CAV2 expression in neurons. Additional enrichment of ALS-associated mutations within CAV1 exons positions CAV1 as an ALS risk gene. We propose CAV1/CAV2 overexpression as a personalized medicine target for ALS.

Preclinical models of disease and multimorbidity with focus upon cardiovascular disease and dementia.

The use of animal models is fundamental to furthering our understanding of human disease mechanisms, as well as identifying potential therapeutic targets. Diseases of ageing often involve multiple body systems; however, multi-systemic features are not fully recapitulated in the many of the animal models available. Therefore, combining pre-clinical models to better reflect the multimorbidities observed at the clinical level is critical. This review will highlight some of the key pre-clinical experimental models associated with cardiovascular (atherosclerosis, coronary heart disease), cerebrovascular (stroke, vascular dementia), metabolic (obesity, type-2 diabetes mellitus) and neurological (amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's, epilepsy) diseases, and whether these models encompass known multimorbidities. In addition to this, we discuss established pre-clinical models that combine two or more conditions, within the context of dementia.

Concurrent sodium channelopathies and amyotrophic lateral sclerosis supports shared pathogenesis.

Amyotrophic lateral sclerosis (ALS) is an invariably fatal adult-onset neurodegenerative disorder; approximately 10% of ALS is monogenic but all ALS exhibits significant heritability. The skeletal muscle sodium channelopathies are a group of inherited, non-dystrophic ion channel disorders caused by heterozygous point mutations in the SCN4A gene, leading to clinical manifestations of congenital myotonia, paramyotonia, and periodic paralysis syndromes. We provide clinical and genetic evidence of concurrence of these two rare disorders which implies a possible shared underlying pathophysiology in two patients. We then identify an enrichment of ALS-associated mutations in another sodium channel, SCN7A, from whole genome sequencing data of 4495 ALS patients and 1925 controls passing multiple testing correction (67 variants, p = 0.0002, Firth logistic regression). These findings suggest dysfunctional sodium channels may play a role upstream in the pathogenesis of ALS in a subset of patients, potentially opening the door to novel personalized medicine approaches.

Disrupted glycosylation of lipids and proteins is a cause of neurodegeneration.

Glycosyltransferases represent a large family of enzymes that catalyse the biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates. A number of studies have implicated glycosyltransferases in the pathogenesis of neurodegenerative diseases but differentiating cause from effect has been difficult. We have recently discovered that mutations proximal to the substrate binding site of glycosyltransferase 8 domain containing 1 (GLT8D1) are associated with familial amyotrophic lateral sclerosis (ALS). We demonstrated that ALS-associated mutations reduce activity of the enzyme suggesting a loss-of-function mechanism that is an attractive therapeutic target. Our work is the first evidence that isolated dysfunction of a glycosyltransferase is sufficient to cause a neurodegenerative disease, but connection between neurodegeneration and genetic variation within glycosyltransferases is not new. Previous studies have identified associations between mutations in UGT8 and sporadic ALS, and between ST6GAL1 mutations and conversion of mild cognitive impairment into clinical Alzheimer's disease. In this review we consider potential mechanisms connecting glycosyltransferase dysfunction to neurodegeneration. The most prominent candidates are ganglioside synthesis and impaired addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) groups to proteins important for axonal and synaptic function. Special consideration is given to examples where genetic mutations within glycosyltransferases are associated with neurodegeneration in recognition of the fact that these changes are likely to be upstream causes present from birth.

Mutations in the Glycosyltransferase Domain of GLT8D1 Are Associated with Familial Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder without effective neuroprotective therapy. Known genetic variants impair pathways, including RNA processing, axonal transport, and protein homeostasis. We report ALS-causing mutations within the gene encoding the glycosyltransferase GLT8D1. Exome sequencing in an autosomal-dominant ALS pedigree identified p.R92C mutations in GLT8D1, which co-segregate with disease. Sequencing of local and international cohorts demonstrated significant ALS association in the same exon, including additional rare deleterious mutations in conserved amino acids. Mutations are associated with the substrate binding site, and both R92C and G78W changes impair GLT8D1 enzyme activity. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish consistent with ALS. Relative toxicity of mutations in model systems mirrors clinical severity. In conclusion, we have linked ALS pathophysiology to inherited mutations that diminish the activity of a glycosyltransferase enzyme.