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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia (FTD), with patients sometimes experiencing elements of both conditions (ALS-FTSD). For mutations associated with ALS-FTSD, such as theC9orf72hexanucleotide repeat expansion (HRE), the factors influencing where an individual may lie on this spectrum require further characterisation. Here, using NanoString molecular barcoding with a panel of 770 neuroinflammatory genes, we interrogate inflammatory dysregulation at the level of gene expression. We identified 20 dysregulated neuroinflammatory genes in the motor cortex of deeply clinically phenotyped C9-ALSpost-mortemcases, with enrichment of microglial and inflammatory response gene sets. Our analyses also revealed two distinct ALS-related neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. Two genes with significant correlations to available clinical metrics were selected for validation:FKBP5andBDNF. FKBP5 and its signalling partner, NF-κB, appeared to have a cell-type-specific staining distribution, with activated (i.e., nuclear) NF-κB immunoreactivity in C9-ALS. Expression ofBDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™in situhybridisation. Finally, we compared NPS between C9-ALS cases and those from deeply clinically phenotyped sporadic ALS (sALS) and SOD1-ALS cohorts, with NPS1 and NPS2 appearing across all cohorts. A subset of these signatures was also detected in publicly available RNA-sequencing data from independent C9-ALS and sALS cohorts, underscoring the relevance of these pathways across cohorts. Our findings highlight the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within genetic and sporadic cohorts.

Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS. We examined tissue from 13 people who went on to develop ALS; including sporadic ALS (n = 12) and C9orf72 hexanucleotide repeat expansion (n = 1). The tissue cohort consisted of 68 formalin-fixed paraffin embedded samples from 21 surgical cases (some patients having more than one case over their lifetimes), from 8 organ systems, which we examined for evidence of phosphorylated TDP-43 (pTDP-43) pathology. We identified pTDP-43 aggregates in multiple cell types of the GI tract, including macrophages and dendritic cells within the lamina propria; as well as ganglion/neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis and in some instances preceded neurological symptom onset by more than 10 years. These data imply that patients with microscopically unexplained non-CNS symptoms could have occult protein aggregation that could be detected many years prior to neurological involvement.

Clinical heterogeneity observed across patients with amyotrophic lateral sclerosis (ALS) is a known complicating factor in identifying potential therapeutics, even within cohorts with the same mutation, such as C9orf72 hexanucleotide repeat expansions (HREs). Thus, further understanding of pathways underlying this heterogeneity is essential for appropriate ALS trial stratification and the meaningful assessment of clinical outcomes. It has been shown that both inflammation and protein misfolding can influence ALS pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. However, there has yet to be a systematic and quantitative assessment of immunohistochemical markers to interrogate the potential relevance of these pathways in an unbiased manner. To investigate this, we extensively characterised features of commonly used glial activation and protein misfolding stains in thousands of images of post-mortem tissue from a heterogeneous cohort of deeply clinically profiled patients with a C9orf72 HRE. Using a random forest model, we show that microglial staining features are the most accurate classifiers of disease status in our panel and that clinicopathological relationships exist between microglial activation status, TDP-43 pathology, and language dysfunction. Furthermore, we detected spatially resolved changes in fused in sarcoma (FUS) staining, suggesting that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Interestingly, no one feature alone significantly impacted the predictiveness of the model, indicating that the collective examination of all features, or a combination of several features, is what allows the model to be predictive. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9-ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological relationships within and between cohorts. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

IntroductionMotor neuron disease (MND) is a rapidly fatal neurodegenerative disease. Despite decades of research and clinical trials there remains no cure and only one globally approved drug, riluzole, which prolongs survival by 2–3 months. Recent improved mechanistic understanding of MND heralds a new translational era with many potential targets being identified that are ripe for clinical trials. Motor Neuron Disease Systematic Multi-Arm Adaptive Randomised Trial (MND-SMART) aims to evaluate the efficacy of drugs efficiently and definitively in a multi-arm, multi-stage, adaptive trial. The first two drugs selected for evaluation in MND-SMART are trazodone and memantine.Methods and analysisInitially, up to 531 participants (177/arm) will be randomised 1:1:1 to oral liquid trazodone, memantine and placebo. The coprimary outcome measures are the Amyotrophic Lateral Sclerosis Functional Rating Scale Revised (ALSFRS-R) and survival. Comparisons will be conducted in four stages. The decision to continue randomising to arms after each stage will be made by the Trial Steering Committee who receive recommendations from the Independent Data Monitoring Committee. The primary analysis of ALSFRS-R will be conducted when 150 participants/arm, excluding long survivors, have completed 18 months of treatment; if positive the survival effect will be inferentially analysed when 113 deaths have been observed in the placebo group. The trial design ensures that other promising drugs can be added for evaluation in planned trial adaptations. Using this novel trial design reduces time, cost and number of participants required to definitively (phase III) evaluate drugs and reduces exposure of participants to potentially ineffective treatments.Ethics and disseminationMND-SMART was approved by the West of Scotland Research Ethics Committee on 2 October 2019. (REC reference: 19/WS/0123) Results of the study will be submitted for publication in a peer-reviewed journal and a summary provided to participants.Trial registration numbersEuropean Clinical Trials Registry (2019-000099-41); NCT04302870.

Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in amyotrophic lateral sclerosis (ALS), where the key pathological cell-type, the motor neuron (MN), has an axon extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. To address this, we undertook cellular and molecular interrogation of multiple patient-derived induced pluripotent stem cell lines and patient autopsy samples harbouring the most common ALS causing mutation, C9orf72. Using paired mutant and isogenic expansion-corrected controls, we show that C9orf72 MNs have shorter axons, impaired fast axonal transport of mitochondrial cargo, and altered mitochondrial bioenergetic function. RNAseq revealed reduced gene expression of mitochondrially encoded electron transport chain transcripts, with neuropathological analysis of C9orf72-ALS post-mortem tissue importantly confirming selective dysregulation of the mitochondrially encoded transcripts in ventral horn spinal MNs, but not in corresponding dorsal horn sensory neurons, with findings reflected at the protein level. Mitochondrial DNA copy number was unaltered, both in vitro and in human post-mortem tissue. Genetic manipulation of mitochondrial biogenesis in C9orf72 MNs corrected the bioenergetic deficit and also rescued the axonal length and transport phenotypes. Collectively, our data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function.

Aims: The mechanisms underlying the selective degeneration of motor neurones in amyotrophic lateral sclerosis (ALS) are poorly understood. The aim of this study was to implement spatially resolved RNA sequencing in human post mortem cortical tissue from an ALS patient harbouring the C9orf72 hexanucleotide repeat expansion to identify dysregulated transcripts that may account for differential vulnerabilities of distinct (i) cell types and (ii) brain regions in the pathogenesis of ALS.Methods: Using spatial transcriptomics (ST) we analysed the transcriptome of post mortem brain tissue, with spatial resolution down to 100 μm. Validation of these findings was then performed using BaseScope, an adapted, in situ hybridization technique with single‐transcript single‐cell‐resolution, providing extensive regional and cell‐type specific confirmation of these dysregulated transcripts. The validation cohort was then extended to include multiple post mortem brain regions and spinal cord tissue from an extended cohort of C9orf72, sporadic ALS (sALS) and SOD1 ALS cases.Results: We identified sixteen dysregulated transcripts of proteins that have roles within six disease‐related pathways. Furthermore, these complementary molecular pathology techniques converged to identify two spatially dysregulated transcripts, GRM3 and USP47, that are commonly dysregulated across sALS, SOD1 and C9orf72 cases alike.Conclusions: This study presents the first description of ST in human post mortem cortical tissue from an ALS patient harbouring the C9orf72 hexanucleotide repeat expansion. These data taken together highlight the importance of preserving spatial resolution, facilitating the identification of genes whose dysregulation may in part underlie regional susceptibilities to ALS, crucially highlighting potential therapeutic and diagnostic targets.

ObjectiveNeurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS.DesignWe requested all surgical specimens of non-CNS tissue taken during life from 48 people with ALS, for whom evidence of the characteristic proteinopathy associated with ALS had been identified in the CNS after death (i.e., the pathological cytoplasmic accumulation of phosphorylated TDP-43 (pTDP-43) aggregates). Of the 48 patients, 13 had sufficient tissue for evaluation: 12 patients with sporadic ALS and 1 patient with a C9orf72 hexanucleotide repeat expansion. The final cohort consisted of 68 formalin-fixed paraffin embedded tissue samples from 22 surgical cases (some patients having more than one case over their lifetimes), representing 8 organ systems, which we examined for evidence of pTDP-43 pathology. The median age of tissue removal was 62.4 years old and median tissue removal to death was 6.3 years.ResultsWe identified pTDP-43 aggregates in multiple cell types of the GI tract (i.e., colon and gallbladder), including macrophages and dendritic cells within the lamina propria, as well as neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis (median=3years) and, in some instances, preceded neurological symptom onset by more than 10 years.ConclusionThese data imply that patients with non-CNS symptoms may have occult protein aggregation that could be detected many years prior to neurological involvement.SummaryNeurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of TDP-43 pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS. We identified pTDP-43 aggregates in multiple cell types of the GI tract (i.e., colon and gallbladder), and within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis (median=24months) and, in some instances, preceded neurological symptom onset by more than 10years. These data imply that patients with non-CNS symptoms may have occult protein aggregation tha could be detected many years prior to neurological involvement.Graphical AbstractGraphical Abstract.Ante-mortem tissue cohort comprised of tissue taken from people with ALS demonstrates non-CNS accumulation of pTDP-43 aggregates prior to symptom onset.Schematic of workflow to identify pTDP-43 aggregates indicative of non-central nervous system (CNS) manifestations of ALS. Lower panel left: cartoon depicting organs and cell types that had evidence of pTDP-43 aggregation in ALS patient non-CNS ante-mortem tissue. Lower panel right: cartoon depicting organs with no evidence of pTDP-43 aggregation in ALS patient non-CNS ante-mortem tissue.

ObjectivesMotor neuron disease (MND) is an incurable progressive neurodegenerative disease with limited treatment options. There is a pressing need for innovation in identifying therapies to take to clinical trial. Here, we detail a systematic and structured evidence-based approach to inform consensus decision making to select the first two drugs for evaluation in Motor Neuron Disease-Systematic Multi-arm Adaptive Randomised Trial (MND-SMART:NCT04302870), an adaptive platform trial. We aim to identify and prioritise candidate drugs which have the best available evidence for efficacy, acceptable safety profiles and are feasible for evaluation within the trial protocol.MethodsWe conducted a two-stage systematic review to identify potential neuroprotective interventions. First, we reviewed clinical studies in MND, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and multiple sclerosis, identifying drugs described in at least one MND publication or publications in two or more other diseases. We scored and ranked drugs using a metric evaluating safety, efficacy, study size and study quality. In stage two, we reviewed efficacy of drugs in MND animal models, multicellular eukaryotic models and human induced pluripotent stem cell (iPSC) studies. An expert panel reviewed candidate drugs over two shortlisting rounds and a final selection round, considering the systematic review findings, late breaking evidence, mechanistic plausibility, safety, tolerability and feasibility of evaluation in MND-SMART.ResultsFrom the clinical review, we identified 595 interventions. 66 drugs met our drug/disease logic. Of these, 22 drugs with supportive clinical and preclinical evidence were shortlisted at round 1. Seven drugs proceeded to round 2. The panel reached a consensus to evaluate memantine and trazodone as the first two arms of MND-SMART.DiscussionFor future drug selection, we will incorporate automation tools, text-mining and machine learning techniques to the systematic reviews and consider data generated from other domains, including high-throughput phenotypic screening of human iPSCs.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are regarded as two ends of a pathogenetic spectrum, termed ALS-frontotemporal spectrum disorder (ALS-FTSD). However, it is currently difficult to predict where on the spectrum an individual will lie, especially for patients with C9orf72 hexanucleotide repeat expansions (HRE), a mutation associated with both ALS and FTD. It has been shown that both inflammation and protein misfolding influence aspects of ALS and ALS-FTSD disease pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. Previous studies have highlighted markers which may influence C9orf72-associated disease presentation in a targeted fashion, though there has yet to be a systematic and quantitative assessment of common immunohistochemical markers to investigate the significance of these pathways in an unbiased manner. Here we report the first extensive digital pathological assessment with random forest modelling of pathological markers often used in neuropathology practice. This study profiles glial activation and protein misfolding in a cohort of deeply clinically profiled post-mortem tissue from patients with a C9orf72 HRE, who either met the criteria for a diagnosis of ALS or ALS-FTSD. We show that microglial immunohistochemical staining features, both morphological and spatial, are the best independent classifiers of disease status and that clinicopathological associations exist between microglial activation status and cognitive dysfunction in ALS-FTSD patients with C9orf72 HRE. Furthermore, we show that spatially resolved changes in FUS staining are also an accurate predictor of disease status, implying that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9orf72 ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological associations between cohorts.

Up to 50% of amyotrophic lateral sclerosis patients present with cognitive deficits in addition to motor dysfunction, but the molecular mechanisms underlying diverse clinical and pathological presentations remain poorly understood. There is therefore an unmet need to identify molecular drivers of cognitive dysfunction to enable better therapeutic targeting and prognostication. To address this, we employed a non-biased approach to identify molecular targets using a deeply phenotyped, clinically stratified cohort of cognitively affected and unaffected brain regions from three brain regions of 13 amyotrophic lateral sclerosis patients with the same cognitive screening test performed during life. Using NanoString molecular barcoding as a sensitive mRNA sequencing technique on post-mortem tissue, we profiled a data-driven panel of 770 genes using the Neuropathology Panel, followed by region and cell type-specific validation using BaseScope in situ hybridisation and immunohistochemistry. We identified 50 significantly dysregulated genes that are distinct between cognitively affected and unaffected brain regions. Using BaseScope in situ hybridisation, we also demonstrate that macromolecular complex regulation, notably NLRP3 inflammasome modulation, is a potential, therapeutically targetable, pathological correlate of cognitive resilience in ALS. © 2021 The Authors. The Journal of Pathology published by John WileySons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Oligodendrocytes are implicated in amyotrophic lateral sclerosis pathogenesis and display transactive response DNA-binding protein-43 (TDP-43) pathological inclusions. To investigate the cell autonomous consequences of TDP-43 mutations on human oligodendrocytes, we generated oligodendrocytes from patient-derived induced pluripotent stem cell lines harbouring mutations in the TARDBP gene, namely G298S and M337V. Through a combination of immunocytochemistry, electrophysiological assessment via whole-cell patch clamping, and three-dimensional cultures, no differences in oligodendrocyte differentiation, maturation or myelination were identified. Furthermore, expression analysis for monocarboxylate transporter 1 (a lactate transporter) coupled with a glycolytic stress test showed no deficit in lactate export. However, using confocal microscopy, we report TDP-43 mutation-dependent pathological mis-accumulation of TDP-43. Furthermore, using in vitro patch-clamp recordings, we identified functional Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor dysregulation in oligodendrocytes. Together, these findings establish a platform for further interrogation of the role of oligodendrocytes and cellular autonomy in TDP-43 proteinopathy.

Amyotrophic lateral sclerosis (ALS) is characterised by progressive motor neuron degeneration. Although there are over 40 genes associated with causal monogenetic mutations, the majority of ALS patients are not genetically determined. Causal ALS mutations are being increasingly mechanistically studied, though how these mechanisms converge and diverge between the multiple known familial causes of ALS (fALS) and sporadic forms of ALS (sALS) and furthermore between different neuron types, is poorly understood. One common pathway that is implicated in selective motor neuron death is enhanced α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPAR)‐mediated excitoxicity. Specifically, human in vitro and pathological evidence has linked the C9orf72 repeat expansion mutation to a relative increase in the Ca2+‐permeable AMPAR population due to AMPAR subunit dysregulation. Here, we provide the first comparative quantitative assessment of the expression profile of AMPAR subunit transcripts, using BaseScope, in post‐mortem lower motor neurons (spinal cord, anterior horn), upper motor neurons (motor cortex) and neurons of the pre‐frontal cortex in sALS and fALS due to mutations in SOD1 and C9orf72. Our data indicated that AMPAR dysregulation is prominent in lower motor neurons in all ALS cases. However, sALS and mutant C9orf72 cases exhibited GluA1 upregulation whereas mutant SOD1 cases displayed GluA2 down regulation. We also showed that sALS cases exhibited widespread AMPAR dysregulation in the motor and pre‐frontal cortex, though the exact identity of the AMPAR subunit being dysregulated was dependent on brain region. In contrast, AMPAR dysregulation in mutant SOD1 and C9orf72 cases was restricted to lower motor neurons only. Our data highlight the complex dysregulation of AMPAR subunit expression that reflects both converging and diverging mechanisms at play between different brain regions and between ALS cohorts. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Objectives: People with motor neurone disease (pwMND) experience communication impairments due to speech and motor dysfunction. Communication support in the form of Augmentative and Alternative Communication (AAC) in conjunction with Assistive Technology (AT) access methods are available, however, variation in provision care pathways exists across Scotland. We conducted a baseline study of communication support for pwMND in Scotland to inform and improve future service provision. Methods: A cross-sectional population-based study was undertaken. Anonymised demographic and clinical phenotypic data for all pwMND in Scotland were extracted from the Care Audit Research Evaluation of MND (CARE-MND) platform, the National MND Register for Scotland. Additional information for AT loans was provided by the third sector charitable organization MND Scotland (MNDS). Results: In total, 371 pwMND were included, 43% of all pwMND were recorded as having impaired speech (recent ALSFRS-R score assessment ≤3) and 69% had been referred to Speech and Language Therapist (SLT) services, although there was variation in referral time from diagnosis date. AAC equipment had been acquired by 17.3% of all pwMND; most commonly iPads and the LightwriterTM speech generating device. Conclusions: Our data highlight a high prevalence of speech impairment in pwMND irrespective of the subtype diagnosis. We therefore recommend standardized care pathways and earlier access to coordinated SLT and Occupational Therapist services to enable prospective and personalized decision making. Our findings further highlight the need for qualitative research to understand the preferences and impact of such interventions from the perspective of the user and their communication partners.

ObjectiveApproximately 35% of patients with amyotrophic lateral sclerosis (ALS) exhibit mild cognitive deficits in executive functions, language and fluency, without dementia. The precise pathology of these extramotor symptoms has remained unknown. This study aimed to determine the pathological correlate of cognitive impairment in patients with non-demented ALS.MethodsIn-depth neuropathological analysis of 27 patients with non-demented ALS who had undergone cognitive testing (Edinburgh Cognitive and Behaviour ALS Screen (ECAS)) during life. Analysis involved assessing 43 kDa Tar-DNA binding protein (TDP-43) accumulation in brain regions specifically involved in executive functions, language functions and verbal fluency to ascertain whether functional deficits would relate to a specific regional distribution of pathology.ResultsAll patients with cognitive impairment had TDP-43 pathology in extramotor brain regions (positive predictive value of 100%). The ECAS also predicted TDP-43 pathology with 100% specificity in brain regions associated with executive, language and fluency domains. We also detected a subgroup with no cognitive dysfunction, despite having substantial TDP-43 pathology, so called mismatch cases.ConclusionsCognitive impairment as detected by the ECAS is a valid predictor of TDP-43 pathology in non-demented ALS. The profile of mild cognitive deficits specifically predicts regional cerebral involvement. These findings highlight the utility of the ECAS in accurately assessing the pathological burden of disease.

AimsClusterin is a topologically dynamic chaperone protein with the ability to participate in both intra‐ and extacellular proteostasis. Clusterin has been shown to be upregulated in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) and has been shown to protect against TDP‐43 protein misfolding in animal and cell models. Previous studies have demonstrated an association between the pathological burden of TDP‐43 misfolding and cognitive deficits in ALS, demonstrating high specificity, but correspondingly low sensitivity owing to a subset of individuals with no evidence of cognitive deficits despite a high burden of TDP‐43 pathology, called mismatch cases.MethodsHypothesising that differences in the ability to cope with protein misfolding in these cases may be due to differences in expression of protective mechanisms such as clusterin expression, we assessed the spatial expression of clusterin and another chaperone protein, HspB8, in post‐mortem brain tissue of mismatch cases. We employed a modified in situ hybridization technique called BaseScope, with single cell, single transcript resolution.ResultsMismatch cases demonstrated differential spatial expression of clusterin, with a predominantly neuronal pattern, compared to cases with cognitive manifestations of their TDP‐43 pathology who demonstrated a predominantly glial distribution of expression.ConclusionsOur data suggests that, in individuals with TDP‐43 pathology, predominantly neuronal expression of clusterin in extra‐motor brain regions may indicate a cell protective mechanism delaying clinical manifestations such as cognitive dysfunction.

Amyotrophic lateral sclerosis is a progressive and devastating neurodegenerative disease. Despite decades of clinical trials, effective disease-modifying drugs remain scarce. To understand the challenges of trial design and delivery, we performed a systematic review of Phase II, Phase II/III and Phase III amyotrophic lateral sclerosis clinical drug trials on trial registries and PubMed between 2008 and 2019. We identified 125 trials, investigating 76 drugs and recruiting more than 15 000 people with amyotrophic lateral sclerosis. About 90% of trials used traditional fixed designs. The limitations in understanding of disease biology, outcome measures, resources and barriers to trial participation in a rapidly progressive, disabling and heterogenous disease hindered timely and definitive evaluation of drugs in two-arm trials. Innovative trial designs, especially adaptive platform trials may offer significant efficiency gains to this end. We propose a flexible and scalable multi-arm, multi-stage trial platform where opportunities to participate in a clinical trial can become the default for people with amyotrophic lateral sclerosis., Wong et al. reviewed historical approaches to clinical trials in amyotrophic lateral sclerosis. Lack of sensitive outcome measures, limitations in resources and barriers to trial participation were challenges for timely and definitive evaluation of drugs in two-arm trials. They concluded that future trials should be more flexible, scalable and efficient., Graphical Abstract Graphical Abstract

Oligodendrocytes are implicated in amyotrophic lateral sclerosis pathogenesis and display transactive response DNA-binding protein-43 (TDP-43) pathological inclusions. To investigate the cell autonomous consequences of TDP-43 mutations on human oligodendrocytes, we generated oligodendrocytes from patient-derived induced pluripotent stem cell lines harbouring mutations in the

Amyotrophic Lateral Sclerosis (ALS) is incurable, progressively fatal and a highly heterogeneous neuro- degenerative disease. Significant variation exists across the phenotypic presentation and genetic back- ground of the condition. The identification of TDP-43 protein aggregates as a pathological hallmark of ALS has established the condition as a proteinopathy. As TDP-43 is ubiquitously expressed and regulates transcription and splicing, understanding the transcriptional profiles associated with distinct ALS clinical phenotypes, particularly those associated with neuro-resilience, is a critical yet understudied area. We carried out transcriptional profiling on motor cortex samples from a cohort of sporadic ALS cases to determine the transcriptional signatures associated with long and short disease duration. Bioinformatic gene ontology analyses identified unique inflammatory transcriptional profiles associated with these extreme disease phenotypes. Our data demonstrate that inflammatory pathway dysregulation can dis- tinguish between phenotypic extremes in ALS. Characterising the molecular profile of clinically heteroge- neous cases provides vital insight in to the underlying pathophysiology of the condition. Understanding the molecular basis of factors which confer neuro-resilience will facilitate the development of targeted disease therapies. The ability to distinguish between molecular subtypes of ALS will enable the stratifica- tion of future trials to test these therapies and also to advance disease prognostication. 17e.elliott@ed.ac.uk

Purpose of Review Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) spectrum disorder is a rare fatal disease with strong genetic influences. The implementation of short-read sequencing methodologies in increasingly large patient cohorts has rapidly expanded our knowledge of the complex genetic architecture of the disease. We aim to convey the broad history of ALS gene discovery as context for a focused review of 11 ALS gene associations reported over the last 5 years. We also summarize the current level of genetic evidence for all previously reported genes. Recent Findings The history of ALS gene discovery has occurred in at least four identifiable phases, each powered by different technologies and scale of investigation. The most recent epoch, benefitting from population-scale genome data, large international consortia, and low-cost sequencing, has yielded 11 new gene associations. We summarize the current level of genetic evidence supporting these ALS genes, highlighting any genotype-phenotype or genotype-pathology correlations, and discussing preliminary understanding of molecular pathogenesis. This era has also raised uncertainty around prior ALS-associated genes and clarified the role of others. Summary Our understanding of the genetic underpinning of ALS has expanded rapidly over the last 25 years and has led directly to the clinical application of molecularly driven therapies. Ongoing sequencing efforts in ALS will identify new causative and risk factor genes while clarifying the status of genes reported in prior eras of research.

Interventions targeting mitochondrial dysfunction have the potential to extend survival in preclinical models of amyotrophic lateral sclerosis. The aim of this systematic review was to assess the efficacy of targeting mitochondria as a potential therapeutic target in amyotrophic lateral sclerosis. Preclinical studies written in the English language were identified with no restrictions on publication date from PubMed, Medline and EMBASE databases. All studies adopting interventions targeting mitochondria to treat amyotrophic lateral sclerosis in genetic or drug-induced organism models were considered for inclusion. A total of 76 studies were included in the analysis. Survival data were extracted, and the meta-analysis was completed in RevMan 5 software. We show that targeting mitochondrial dysfunction in amyotrophic lateral sclerosis results in a statistically significant improvement in survival (Z = 5.31; P

A significant body of neuropathological research examining the molecular heterogeneity across ALS phenotypes exists and an up to date formal review of these studies is now required. Through conducting a systematic review and meta-analysis of the existing ALS human neuropathology literature we aim to outline the current pathological molecular profile of ALS. A search will be conducted without language or publication date restrictions across three databases; 1. PubMed, 2. MEDLINE, 3. EMBASE. Studies comparing human ALS or ALS-FTD brain and/or spinal cord samples to controls will be selected. The primary outcome measure will be molecular species quantification followed by an assessment of study heterogeneity as a secondary outcome measure. The Systematic Review Facility online screening tool (app.syrf.org.uk) will be used by two independent authors to screen the titles and abstracts based on predefined criteria. Data will be extracted by one reviewer and an assessment of study quality will be conducted against the CAMARADES’ study quality checklist (adapted). Additional study quality assessment and data extraction will be carried out by an independent reviewer for 10% of the selected studies. Where appropriate, standardised mean differences (SMD) will be used to describe outcome measures to enable inter-study comparison. Advances in transcriptional profiling technology have generated a variety of high throughput platforms, ranging from microarray gene chips with a specific range of oligonucleotide probes through to sequencing at a whole genome level. Human pathological studies to date have revolutionised our understanding of the ALS disease aetiology. Here we aim to review the outcomes of these studies across the field as a whole and to consider the impact of methodological heterogeneity on the results presented.

The C9orf72 hexanucleotide repeat expansion is the commonest known genetic mutation in amyotrophic lateral sclerosis. A neuropathological hallmark is the intracellular accumulation of RNA foci. The role that RNA foci play in the pathogenesis of amyotrophic lateral sclerosis is widely debated. Historically, C9orf72 RNA foci have been identified using in situ hybridization. Here, we have implemented BaseScope™, a high-resolution modified in situ hybridization technique. We demonstrate that previous studies have underestimated the abundance of RNA foci in neurons and glia. This improved detection allowed us to investigate the abundance, regional distribution and cell type specificity of sense C9orf72 RNA foci in post-mortem brain and spinal cord tissue of six deeply clinically phenotyped C9orf72 patients and six age- and sex-matched controls. We find a correlation between RNA foci and the accumulation of transactive response DNA-binding protein of 43 kDa in spinal motor neurons (rs = 0.93; P = 0.008), but not in glia or cortical motor neurons. We also demonstrate that there is no correlation between the presence of RNA foci and the accumulation of transactive response DNA binding protein of 43 kDa in extra-motor brain regions. Furthermore, there is no association between the presence of RNA foci and cognitive indices. These results highlight the utility of BaseScope™ in the clinicopathological assessment of the role of sense RNA foci in C9orf72.

Various studies have suggested that a neurotoxic cerebrospinal fluid profile could be implicated in amyotrophic lateral sclerosis. Here, we systematically review the evidence for cerebrospinal fluid cytotoxicity in amyotrophic lateral sclerosis and explore its clinical correlates. We searched the following databases with no restrictions on publication date: PubMed, Embase and Web of Science. All studies that investigated cytotoxicity in vitro following exposure to cerebrospinal fluid from amyotrophic lateral sclerosis patients were considered for inclusion. Meta-analysis could not be performed, and findings were instead narratively summarized. Twenty-eight studies were included in our analysis. Both participant characteristics and study conditions including cerebrospinal fluid concentration, exposure time and culture model varied considerably across studies. Of 22 studies assessing cell viability relative to controls, 19 studies reported a significant decrease following exposure to cerebrospinal fluid from patients with amyotrophic lateral sclerosis, while three early studies failed to observe any difference. Seven of eight studies evaluating apoptosis observed significant increases in the levels of apoptotic markers following exposure to cerebrospinal fluid from patients with amyotrophic lateral sclerosis, with the remaining study reporting a qualitative difference. Although five studies investigated the possible relationship between cerebrospinal fluid cytotoxicity and patient characteristics, such as age, gender and disease duration, none demonstrated an association with any of the factors. In conclusion, our analysis suggests that cerebrospinal fluid cytotoxicity is a feature of sporadic and possibly also of familial forms of amyotrophic lateral sclerosis. Further research is, however, required to better characterize its underlying mechanisms and to establish its possible contribution to amyotrophic lateral sclerosis pathophysiology., Systematic review of in vitro evidence suggests that cerebrospinal fluid cytotoxicity is a feature of sporadic and possibly also of familial forms of amyotrophic lateral sclerosis. Further research is, however, required to elucidate the mechanisms underlying its toxicity, and to establish its possible contribution in overall pathophysiology., Graphical Abstract Graphical Abstract

Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.

BACKGROUND: Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are incurable, progressive and fatal neurodegenerative diseases with patients variably affected clinically by motor, behavior, and cognitive deficits. The accumulation of an RNA-binding protein, TDP-43, is the most significant pathological finding in approximately 95% of ALS cases and 50% of FTD cases, and discovery of this common pathological signature, together with an increasing understanding of the shared genetic basis of these disorders, has led to FTD and ALS being considered as part of a single disease continuum. Given the widespread aggregation and accumulation of TDP-43 in FTD-ALS spectrum disorder, TDP-43 may have potential as a biomarker in these diseases.METHODS: We therefore conducted a systematic review and meta-analysis to evaluate the diagnostic utility of TDP-43 detected in the cerebrospinal fluid (CSF) of patients with FTD-ALS spectrum disorder.RESULTS: From seven studies, our results demonstrate that patients with ALS have a statistically significantly higher level of TDP-43 in CSF (effect size 0.64, 95% CI: 0.1-1.19, p = 0.02).CONCLUSIONS: These data suggest promise for the use of CSF TDP-43 as a biomarker for ALS.

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease. Electronic supplementary material The online version of this article (10.1186/s40478-017-0481-1) contains supplementary material, which is available to authorized users.

Almost all cases of sporadic amyotrophic lateral sclerosis (ALS), and some cases of the familial form, are characterised by the deposition of TDP-43, a member of a family of heteronuclear ribonucleoproteins (hnRNP). Although protein misfolding and deposition is thought to be a causative feature of many of the most prevalent neurodegenerative diseases, a link between TDP-43 aggregation and the dysfunction of motor neurons has yet to be established, despite many correlative neuropathological studies. We have investigated this relationship in the present study by probing the effect of altering TDP-43 aggregation behaviour in vivo by modulating the levels of molecular chaperones in a Drosophila model. More specifically, we quantify the effect of either pharmacological upregulation of the heat shock response or specific genetic upregulation of a small heat shock protein, CG14207, on the neurotoxicity of both TDP-43 and of its disease associated 25 kDa fragment (TDP-25) in a Drosophila model. Inhibition of the aggregation of TDP-43 by either method results in a partial reduction of its neurotoxic effects on both photoreceptor and motor neurons, whereas inhibition of the aggregation of TDP-25 results not only in a complete suppression of its toxicity but also its clearance from the brain in both neuronal subtypes studied. The results demonstrate, therefore, that aggregation plays a crucial role in mediating the neurotoxic effects of both full length and truncated TDP-43, and furthermore reveal that the in vivo propensity of these two proteins to aggregate and their susceptibility to molecular chaperone mediated clearance are quite distinct.