Toxic gain-of-function mechanisms in C9orf72 ALS-FTD neurons drive autophagy and lysosome dysfunction.

Hexanucleotide repeat expansions in the C9orf72 gene are the primary genetic cause for both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases. Significant advances in the elucidation of the disease mechanisms responsible for C9orf72 ALS-FTD have revealed both a toxic gain-of-function and a loss-of-function mechanism as possible underlying disease cause. As the differential contribution of both gain and loss of function in C9orf72 ALS-FTD pathogenesis remains debated, we investigated disease mechanisms in motor neurons derived from both authentic human patient C9orf72 ALS-FTD iPSCs as well as a C9orf72 knockout iPSC line. We found that patient neurons presented with less motile and enlarged lysosomes, a decrease in autophagic flux and an increase in SQSTM1/p62 puncta and insoluble TARDBP/TDP-43 species. Importantly, we found that C9orf72 knockout barely has any influence on these phenotypes and mainly results in impaired endosomal maturation. Together, our data suggest that toxic gain-of-function, rather than loss-of-function, mechanisms in C9orf72 ALS-FTD impair the autophagy-lysosome system in neurons.

TUBA4A downregulation as observed in ALS post-mortem motor cortex causes ALS-related abnormalities in zebrafish.

Disease-associated variants of TUBA4A (alpha-tubulin 4A) have recently been identified in familial ALS. Interestingly, a downregulation of TUBA4A protein expression was observed in familial as well as sporadic ALS brain tissue. To investigate whether a decreased TUBA4A expression could be a driving factor in ALS pathogenesis, we assessed whether TUBA4A knockdown in zebrafish could recapitulate an ALS-like phenotype. For this, we injected an antisense oligonucleotide morpholino in zebrafish embryos targeting the zebrafish TUBA4A orthologue. An antibody against synaptic vesicle 2 was used to visualize motor axons in the spinal cord, allowing the analysis of embryonic ventral root projections. Motor behavior was assessed using the touch-evoked escape response. In post-mortem ALS motor cortex, we observed reduced TUBA4A levels. The knockdown of the zebrafish TUBA4A orthologue induced a motor axonopathy and a significantly disturbed motor behavior. Both phenotypes were dose-dependent and could be rescued by the addition of human wild-type TUBA4A mRNA. Thus, TUBA4A downregulation as observed in ALS post-mortem motor cortex could be modeled in zebrafish and induced a motor axonopathy and motor behavior defects reflecting a motor neuron disease phenotype, as previously described in embryonic zebrafish models of ALS. The rescue with human wild-type TUBA4A mRNA suggests functional conservation and strengthens the causal relation between TUBA4A protein levels and phenotype severity. Furthermore, the loss of TUBA4A induces significant changes in post-translational modifications of tubulin, such as acetylation, detyrosination and polyglutamylation. Our data unveil an important role for TUBA4A in ALS pathogenesis, and extend the relevance of TUBA4A to the majority of ALS patients, in addition to cases bearing TUBA4A mutations.

European Academy of Neurology (EAN) guideline on the management of amyotrophic lateral sclerosis in collaboration with European Reference Network for Neuromuscular Diseases (ERN EURO-NMD).

BACKGROUND: This update of the guideline on the management of amyotrophic lateral sclerosis (ALS) was commissioned by the European Academy of Neurology (EAN) and prepared in collaboration with the European Reference Network for Neuromuscular Diseases (ERN EURO-NMD) and the support of the European Network for the Cure ALS (ENCALS) and the European Organization for Professionals and Patients with ALS (EUpALS). METHODS: Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology was used to assess the effectiveness of interventions for ALS. Two systematic reviewers from Cochrane Response supported the guideline panel. The working group identified a total of 26 research questions, performed systematic reviews, assessed the quality of the available evidence, and made specific recommendations. Expert consensus statements were provided where insufficient evidence was available. RESULTS: A guideline mapping effort revealed only one other ALS guideline that used GRADE methodology (a National Institute for Health and Care Excellence [NICE] guideline). The available evidence was scarce for many research questions. Of the 26 research questions evaluated, the NICE recommendations could be adapted for 8 questions. Other recommendations required updates of existing systematic reviews or de novo reviews. Recommendations were made on currently available disease-modifying treatments, multidisciplinary care, nutritional and respiratory support, communication aids, psychological support, treatments for common ALS symptoms (e.g., muscle cramps, spasticity, pseudobulbar affect, thick mucus, sialorrhea, pain), and end-of-life management. CONCLUSIONS: This update of the guideline using GRADE methodology provides a framework for the management of ALS. The treatment landscape is changing rapidly, and further updates will be prepared when additional evidence becomes available.

The ALSFRS-R Summit: a global call to action on the use of the ALSFRS-R in ALS clinical trials.

The Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) was developed more than 25 years ago as an instrument to monitor functional change over time in patients with ALS. It has since been revised and extended to meet the needs of high data quality in ALS trials (ALSFRS-R), however a full re-validation of the scale was not completed. Despite this, the scale has remained a primary outcome measure in clinical trials. We convened a group of clinical trialists to discuss and explore opportunities to improve the scale and propose alternative measures. In this meeting report, we present a call to action on the use of the ALSFRS-Revised scale in clinical trials, focusing on the need for (1) harmonization of the ALSFRS-R administration globally, (2) alignment on a set of recommendations for clinical trial design and statistical analysis plans (SAPs), and (3) use of additional outcome measures.

PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic, which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach, we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila. Consistent with a conserved role in disease pathogenesis, pharmacological inhibition of both proteins rescued disease-relevant phenotypes, including mitochondrial trafficking defects and neuromuscular junction failure, in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies, mice, and human iPSC-sMNs, we observed reduced GSK3 inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore, we found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs, and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover, increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity, confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3, and kinesin-1, that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease.

Specialized multidisciplinary care improves ALS survival in Belgium: a population-based retrospective study.

ALS is a neurodegenerative disease characterized by loss of motor neurons, resulting in progressive weakness and wasting of muscles. The average survival time is 2-5 years, mostly due to respiratory failure. Since current therapies can prolong survival time by only a few months, multidisciplinary care remains the cornerstone of the management of ALS. At the ALS Expert Centre of University Hospitals Leuven, a large proportion of Belgian ALS patients are seen for diagnosis and a significant number is also in follow-up with the multidisciplinary team. In this retrospective study, we compared the outcome of incident patients who were in follow-up at our site with patients who were not in follow-up. We included 659 patients of which 557 (84.5%) received specialized care at the ALS Expert Centre. After adjusting for clinically relevant prognostic parameters, multidisciplinary follow-up significantly prolonged survival (p = 0.004; HR = 0.683; CI 95% [0.528 - 0.884]). This increase in survival is mainly driven by patients with spinal onset (p = 0.035; HR = 0.746; CI 95% [0.568 - 0.980]), since no significant increased survival time was observed in patients with bulbar onset (p = 0.28; HR = 0.778; CI 95% [0.495 - 1.223]). These data confirm that multidisciplinary follow-up contributes to a better outcome of patients, emphasizing the importance of multidisciplinary specialized care in ALS.

The sense of antisense therapies in ALS.

Treatment of patients with amyotrophic lateral sclerosis (ALS) has entered a new era now that encouraging results about antisense oligonucleotides (ASOs) are becoming available and a first ASO therapy for ALS has been approved by the FDA. Moreover, there is hope not only that ALS can be stopped but also that symptoms can be reversed. Until now, degrading ASOs seemed to be successful mostly for rarer forms of familial ALS. However, the first attempts to correct mis-splicing events in sporadic ALS are underway, as well as a clinical trial examining interference with a genetic modifier. In this review, we discuss the current status of using ASOs in ALS and the possibilities and pitfalls of this therapeutic strategy.

Assessing the use of HL7 FHIR for implementing the FAIR guiding principles: a case study of the MIMIC-IV Emergency Department module.

Objectives: To provide a real-world example on how and to what extent Health Level Seven Fast Healthcare Interoperability Resources (FHIR) implements the Findable, Accessible, Interoperable, and Reusable (FAIR) guiding principles for scientific data. Additionally, presents a list of FAIR implementation choices for supporting future FAIR implementations that use FHIR. Materials and methods: A case study was conducted on the Medical Information Mart for Intensive Care-IV Emergency Department (MIMIC-ED) dataset, a deidentified clinical dataset converted into FHIR. The FAIRness of this dataset was assessed using a set of common FAIR assessment indicators. Results: The FHIR distribution of MIMIC-ED, comprising an implementation guide and demo data, was more FAIR compared to the non-FHIR distribution. The FAIRness score increased from 60 to 82 out of 95 points, a relative improvement of 37%. The most notable improvements were observed in interoperability, with a score increase from 5 to 19 out of 19 points, and reusability, with a score increase from 8 to 14 out of 24 points. A total of 14 FAIR implementation choices were identified. Discussion: Our work examined how and to what extent the FHIR standard contributes to FAIR data. Challenges arose from interpreting the FAIR assessment indicators. This study stands out for providing a real-world example of a dataset that was made more FAIR using FHIR. Conclusion: To the best of our knowledge, this is the first study that formally assessed the conformance of a FHIR dataset to the FAIR principles. FHIR improved the accessibility, interoperability, and reusability of MIMIC-ED. Future research should focus on implementing FHIR in research data infrastructures.

Prognostic value of motor and extramotor involvement in ALS.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in upper and lower motor neuron loss. ALS often has a focal onset of weakness, which subsequently spreads to other body regions. Survival is limited to two to five years after disease onset, often due to respiratory failure. Cognitive impairment is present in approximately 30% to 50% of patients and in 10%-15% of patients, the clinical criteria of frontotemporal dementia (FTD) are met. METHODS: In this retrospective single-center ALS cohort study, we examined the occurrence of cognitive and behavioral impairment in relation to motor impairment at disease presentation and studied its impact on survival. RESULTS: The degree of lower motor neuron involvement was associated with a worse survival, but there was no effect for upper motor neuron involvement. Patients who were cognitively normal had a significantly better survival compared to patients with cognitive or behavioral impairment and to patients with comorbid FTD. There was no significant difference regarding survival between patients with FTD and patients with cognitive or behavioral impairment. CONCLUSIONS: The extent of motor and extramotor involvement in patients with ALS at disease presentation holds complementary prognostic information.

A randomized double-blind clinical trial on safety and efficacy of tauroursodeoxycholic acid (TUDCA) as add-on treatment in patients affected by amyotrophic lateral sclerosis (ALS): the statistical analysis plan of TUDCA-ALS trial.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a highly debilitating neurodegenerative condition. Despite recent advancements in understanding the molecular mechanisms underlying ALS, there have been no significant improvements in therapeutic options for ALS patients in recent years. Currently, there is no cure for ALS, and the only approved treatment in Europe is riluzole, which has been shown to slow the disease progression and prolong survival by approximately 3 months. Recently, tauroursodeoxycholic acid (TUDCA) has emerged as a promising and effective treatment for neurodegenerative diseases due to its neuroprotective activities. METHODS: The ongoing TUDCA-ALS study is a double-blinded, parallel arms, placebo-controlled, randomized multicenter phase III trial with the aim to assess the efficacy and safety of TUDCA as add-on therapy to riluzole in patients with ALS. The primary outcome measure is the treatment response defined as a minimum of 20% improvement in the ALS Functional Rating Scale-Revised (ALSFRS-R) slope during the randomized treatment period (18 months) compared to the lead-in period (3 months). Randomization will be stratified by country. Primary analysis will be conducted based on the intention-to-treat principle through an unadjusted logistic regression model. Patient recruitment commenced on February 22, 2019, and was closed on December 23, 2021. The database will be locked in September 2023. DISCUSSION: This paper provides a comprehensive description of the statistical analysis plan in order to ensure the reproducibility of the analysis and avoid selective reporting of outcomes and data-driven analysis. Sensitivity analyses have been included in the protocol to assess the impact of intercurrent events related to the coronavirus disease 2019. By focusing on clinically meaningful and robust outcomes, this trial aims to determine whether TUDCA can be effective in slowing the disease progression in patients with ALS. TRIAL REGISTRATION: ClinicalTrials.gov NCT03800524 . Registered on January 11, 2019.

SorCS2 binds progranulin to regulate motor neuron development.

Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.

TDP-43 pathology is associated with increased tau burdens and seeding.

BACKGROUND: Most Alzheimer's Disease (AD) cases also exhibit limbic predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC), besides amyloid-ß plaques and neurofibrillary tangles (NFTs) containing hyperphosphorylated tau (p-tau). LATE-NC is characterized by cytoplasmic aggregates positive for pathological TDP-43 and is associated with more severe clinical outcomes in AD, compared to AD cases lacking TDP-43 pathology TDP-43: AD(LATE-NC-). Accumulating evidence suggests that TDP-43 and p-tau interact and exhibit pathological synergy during AD pathogenesis. However, it is not yet fully understood how the presence of TDP-43 affects p-tau aggregation in symptomatic AD. METHODS: In this study, we investigated the impact of TDP-43 proteinopathy on p-tau pathology with different approaches: histologically, in a human post-mortem cohort (n = 98), as well as functionally using a tau biosensor cell line and TDP-43A315T transgenic mice. RESULTS: We found that AD cases with comorbid LATE-NC, AD(LATE-NC+), have increased burdens of pretangles and/or NFTs as well as increased brain levels of p-tau199, compared to AD(LATE-NC-) cases and controls. The burden of TDP-43 pathology was also correlated with the Braak NFT stages. A tau biosensor cell line treated with sarkosyl-insoluble, brain-derived homogenates from AD(LATE-NC+) cases displayed exacerbated p-tau seeding, compared to control and AD(LATE-NC-)-treated cells. Consistently, TDP-43A315T mice injected with AD(LATE-NC+)-derived extracts also exhibited a more severe hippocampal seeding, compared to the remaining experimental groups, albeit no TDP-43 aggregation was observed. CONCLUSIONS: Our findings extend the current knowledge by supporting a functional synergy between TDP-43 and p-tau. We further demonstrate that TDP-43 pathology worsens p-tau aggregation in an indirect manner and increases its seeding potential, probably by increasing p-tau levels. This may ultimately contribute to tau-driven neurotoxicity and cell death. Because most AD cases present with comorbid LATE-NC, this study has an impact on the understanding of TDP-43 and tau pathogenesis in AD and LATE, which account for the majority of dementia cases worldwide. Moreover, it highlights the need for the development of a biomarker that detects TDP-43 during life, in order to properly stratify AD and LATE patients.

A toxic gain-of-function mechanism in C9orf72 ALS impairs the autophagy-lysosome pathway in neurons.

BACKGROUND: Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS. METHODS: In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO). RESULTS: Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients. CONCLUSIONS: Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS.

Peptides From the Variable Domain of Immunoglobulin G as Biomarkers in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND AND OBJECTIVES: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinically heterogeneous immune-mediated disease. Diagnostic biomarkers for CIDP are currently lacking. Peptides derived from the variable domain of circulating immunoglobulin G (IgG) have earlier been shown to be shared among patients with the same immunologic disease. Because humoral immune factors are hypothesized to be involved in the pathogenesis of CIDP, we evaluated IgG variable domain-derived peptides as diagnostic biomarkers in CIDP (primary objective) and whether IgG-derived peptides could cluster objective clinical entities in CIDP (secondary objective). METHODS: IgG-derived peptides were determined in prospectively collected sera of patients with CIDP and neurologic controls by means of mass spectrometry. Peptides of interest were selected through statistical analysis in a discovery cohort followed by sequence determination and confirmation. Diagnostic performance was evaluated for individual selected peptides and for a multipeptide model incorporating selected peptides, followed by performance reassessment in a validation cohort. Clustering of patients with CIDP based on IgG-derived peptides was evaluated through unsupervised sparse principal component analysis followed by k-means clustering. RESULTS: Sixteen peptides originating from the IgG variable domain were selected as candidate biomarkers in a discovery cohort of 44 patients with CIDP and 29 neurologic controls. For all 16 peptides, univariate logistic regressions and ROC curve analysis demonstrated increasing peptide abundances to associate with increased odds for CIDP (area under the curves [AUCs] ranging from 64.6% to 79.6%). When including age and sex in the logistic regression models, this remained the case for 13/16 peptides. A model composed of 5/16 selected peptides showed strong discriminating performance between patients with CIDP and controls (AUC 91.5%; 95% CI 84.6%-98.4%; p < 0.001). In the validation cohort containing 45 patients and 43 controls, 2/16 peptides demonstrated increasing abundances to associate with increased odds for CIDP, while the five-peptide model demonstrated an AUC of 61.2% (95% CI 49.3%-73.2%; p = 0.064). Peptide-based patient clusters did not associate with clinical features. DISCUSSION: IgG variable domain-derived peptides showed a valid source for diagnostic biomarkers in CIDP, albeit with challenges toward replication. Our proof-of-concept findings warrant further study of IgG-derived peptides as biomarkers in more homogeneous cohorts of patients with CIDP and controls. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the pattern of serum IgG-derived peptide clusters may help differentiate between patients with CIDP and those with other peripheral neuropathies.

PRECISION ALS-an integrated pan European patient data platform for ALS.

Amyotrophic Lateral Sclerosis (ALS) is an incurable neurodegenerative condition. Despite significant advances in pre-clinical models that enhance understanding of disease pathobiology, translation of candidate drugs to effective human therapies has been disappointing. There is increasing recognition of the need for a precision medicine approach toward drug development, as many failures in translation can be attributed in part to disease heterogeneity in humans. PRECISION-ALS is an academic industry collaboration between clinicians, Computer Scientists, Information engineers, technologists, data scientists and industry partners that will address the key clinical, computational, data science and technology associated research questions to generate a sustainable precision medicine based approach toward new drug development. Using extant and prospectively collected population based clinical data across nine European sites, PRECISION-ALS provides a General Data Protection Regulation (GDPR) compliant framework that seamlessly collects, processes and analyses research-quality multimodal and multi-sourced clinical, patient and caregiver journey, digitally acquired data through remote monitoring, imaging, neuro-electric-signaling, genomic and biomarker datasets using machine learning and artificial intelligence. PRECISION-ALS represents a first-in-kind modular transferable pan-European ICT framework for ALS that can be easily adapted to other regions that face similar precision medicine related challenges in multimodal data collection and analysis.

Genetic variability in sporadic amyotrophic lateral sclerosis.

With the advent of gene therapies for amyotrophic lateral sclerosis (ALS), there is a surge in gene testing for this disease. Although there is ample experience with gene testing for C9orf72, SOD1, FUS and TARDBP in familial ALS, large studies exploring genetic variation in all ALS-associated genes in sporadic ALS (sALS) are still scarce. Gene testing in a diagnostic setting is challenging, given the complex genetic architecture of sALS, for which there are genetic variants with large and small effect sizes. Guidelines for the interpretation of genetic variants in gene panels and for counselling of patients are lacking. We aimed to provide a thorough characterization of genetic variability in ALS genes by applying the American College of Medical Genetics and Genomics (ACMG) criteria on whole genome sequencing data from a large cohort of 6013 sporadic ALS patients and 2411 matched controls from Project MinE. We studied genetic variation in 90 ALS-associated genes and applied customized ACMG-criteria to identify pathogenic and likely pathogenic variants. Variants of unknown significance were collected as well. In addition, we determined the length of repeat expansions in C9orf72, ATXN1, ATXN2 and NIPA1 using the ExpansionHunter tool. We found C9orf72 repeat expansions in 5.21% of sALS patients. In 50 ALS-associated genes, we did not identify any pathogenic or likely pathogenic variants. In 5.89%, a pathogenic or likely pathogenic variant was found, most commonly in SOD1, TARDBP, FUS, NEK1, OPTN or TBK1. Significantly more cases carried at least one pathogenic or likely pathogenic variant compared to controls (odds ratio 1.75; P-value 1.64 × 10-5). Isolated risk factors in ATXN1, ATXN2, NIPA1 and/or UNC13A were detected in 17.33% of cases. In 71.83%, we did not find any genetic clues. A combination of variants was found in 2.88%. This study provides an inventory of pathogenic and likely pathogenic genetic variation in a large cohort of sALS patients. Overall, we identified pathogenic and likely pathogenic variants in 11.13% of ALS patients in 38 known ALS genes. In line with the oligogenic hypothesis, we found significantly more combinations of variants in cases compared to controls. Many variants of unknown significance may contribute to ALS risk, but diagnostic algorithms to reliably identify and weigh them are lacking. This work can serve as a resource for counselling and for the assembly of gene panels for ALS. Further characterization of the genetic architecture of sALS is necessary given the growing interest in gene testing in ALS.

Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival.

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

Differences in Cerebral Glucose Metabolism in ALS Patients with and without C9orf72 and SOD1 Mutations.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons. In 10% of patients, the disorder runs in the family. Our aim was to study the impact of ALS-causing gene mutations on cerebral glucose metabolism. Between October 2010 and October 2022, 538 patients underwent genetic testing for mutations with strong evidence of causality for ALS and 18F-2-fluoro-2-deoxy-D-glucose-PET (FDG PET), at University Hospitals Leuven. We identified 48 C9orf72-ALS and 22 SOD1-ALS patients. After propensity score matching, two cohorts of 48 and 21 matched sporadic ALS patients, as well as 20 healthy controls were included. FDG PET images were assessed using a voxel-based and volume-of-interest approach. We observed widespread frontotemporal involvement in all ALS groups, in comparison to healthy controls. The degree of relative glucose metabolism in SOD1-ALS in motor and extra-motor regions did not differ significantly from matched sporadic ALS patients. In C9orf72-ALS, we found more pronounced hypometabolism in the peri-rolandic region and thalamus, and hypermetabolism in the medulla extending to the pons, in comparison to matched sporadic ALS patients. Our study revealed C9orf72-dependent differences in glucose metabolism in the peri-rolandic region, thalamus, and brainstem (i.e., medulla, extending to the pons) in relation to matched sporadic ALS patients.

Multiomics and machine-learning identify novel transcriptional and mutational signatures in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal and incurable neurodegenerative disease that mainly affects the neurons of the motor system. Despite the increasing understanding of its genetic components, their biological meanings are still poorly understood. Indeed, it is still not clear to which extent the pathological features associated with amyotrophic lateral sclerosis are commonly shared by the different genes causally linked to this disorder. To address this point, we combined multiomics analysis covering the transcriptional, epigenetic and mutational aspects of heterogenous human induced pluripotent stem cell-derived C9orf72-, TARDBP-, SOD1- and FUS-mutant motor neurons as well as datasets from patients' biopsies. We identified a common signature, converging towards increased stress and synaptic abnormalities, which reflects a unifying transcriptional program in amyotrophic lateral sclerosis despite the specific profiles due to the underlying pathogenic gene. In addition, whole genome bisulphite sequencing linked the altered gene expression observed in mutant cells to their methylation profile, highlighting deep epigenetic alterations as part of the abnormal transcriptional signatures linked to amyotrophic lateral sclerosis. We then applied multi-layer deep machine-learning to integrate publicly available blood and spinal cord transcriptomes and found a statistically significant correlation between their top predictor gene sets, which were significantly enriched in toll-like receptor signalling. Notably, the overrepresentation of this biological term also correlated with the transcriptional signature identified in mutant human induced pluripotent stem cell-derived motor neurons, highlighting novel insights into amyotrophic lateral sclerosis marker genes in a tissue-independent manner. Finally, using whole genome sequencing in combination with deep learning, we generated the first mutational signature for amyotrophic lateral sclerosis and defined a specific genomic profile for this disease, which is significantly correlated to ageing signatures, hinting at age as a major player in amyotrophic lateral sclerosis. This work describes innovative methodological approaches for the identification of disease signatures through the combination of multiomics analysis and provides novel knowledge on the pathological convergencies defining amyotrophic lateral sclerosis.