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Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia (FTD) are now known as parts of a disease spectrum with common pathological features and genetic causes. However, as both conditions are clinically heterogeneous, patient groups may be phenotypically similar but pathogenically and genetically variable. Despite numerous clinical trials, there remains no effective therapy for these conditions, which in part, may be due to challenges of therapy development in a heterogeneous patient population. Disruption to protein homeostasis is a key feature of different forms of ALS and FTD. Targeting the endogenous protein chaperone system, the heat shock response (HSR), may therefore be a potential therapeutic approach. We conducted a pre-clinical study of a known pharmacological amplifier of the HSR called arimoclomol, in mice with a mutation in valosin containing protein (VCP) which causes both ALS and FTD in patients. We demonstrate that amplification of the HSR ameliorates the ALS/FTD-like phenotype in the spinal cord and brain of mutant VCP mice and prevents neuronal loss, replicating our earlier findings in the SOD1 mouse model of ALS. Moreover, in human cell models, we demonstrate improvements in pathology upon arimoclomol treatment in mutant VCP patient fibroblasts and iPSC-derived motor neurons. Our findings suggest that targeting of the heat shock response may have therapeutic potential, not only in non-SOD1 ALS, but also for the treatment of FTD. Arimoclomol has recently been tested in a Phase 3 clinical trial for ALS for which we await post hoc analysis of the effects of this drug on patient sub-populations.

Accumulating evidence suggests that neurodegenerative diseases are not merely neuronal in nature but comprise multicellular involvement, with astrocytes emerging as key players. The pathomechanisms of several neurodegenerative diseases involve the deposition of misfolded protein aggregates in neurons that have characteristic prion-like behaviours such as template-directed seeding, intercellular propagation, distinct conformational strains and protein-mediated toxicity. The role of astrocytes in dealing with these pathological prion-like protein aggregates and whether their responses either protect from or conspire with the disease process is currently unclear. Here we review the existing literature implicating astrocytes in multiple neurodegenerative proteinopathies with a focus on prion-like behaviour in this context.

Mutations in theSNCAgene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons in the substantia nigra, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from theSNCAmutation during development, to its end stage pathology is unknown. Utilising human induced pluripotent stem cells (hiPSCs) withSNCAmutations, we resolved the temporal sequence of pathophysiological events that occur during neuronal differentiation in order to discover the early, and likely causative, events in synucleinopathies. We adapted a small molecule-based protocol that generates highly enriched midbrain dopaminergic (mDA) neurons (>80%). We characterised their molecular identity using single-cell RNA sequencing and their functional identity through the synthesis and secretion of dopamine, the ability to generate action potentials, and form functional synapses and networks. RNA velocity analyses confirmed the developmental transcriptomic trajectory of midbrain neural precursors into mDA neurons using our approach, and identified key driver genes in mDA neuronal development. To characterise the synucleinopathy, we adopted super-resolution methods to determine the number, size and structure of aggregates inSNCA-mutant mDA neurons. At one week of differentiation, prior to maturation to mDA neurons of molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, inSNCA-mutant midbrain immature neurons. The aggregation progresses over time to accumulate phosphorylated aggregates, and later fibrillar aggregates. When the midbrain neurons were functional, we observed evidence of impaired physiological calcium signalling, with raised basal calcium, and impairments in cytosolic and mitochondrial calcium efflux. Once midbrain identity fully developed,SNCA-mutant neurons exhibited bioenergetic impairments, mitochondrial dysfunction and oxidative stress. During the maturation of mDA neurons, upregulation of mitophagy and autophagy occured, and ultimately these multiple cellular stresses lead to an increase in cell death by six weeks post-differentiation. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

An intronic GGGGCC repeat expansion inC9orf72is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR) and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a genome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites.In vitroexperiments showed that polyGAAGA RNA binds poly(PR) with higher affinity than control RNA and induces phase-separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to polyGAAGA-containing RNAs, which may have physiological consequences.

Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development; however, the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. Here we show that IR is a physiological process that is spatiotemporally regulated during MN lineage restriction and that IRTs in the cytoplasm are detected in as many as 13% (n= 2297) of the genes expressed during this process. We identify a major class of cytoplasmic IRTs that are not associated with reduced expression of their own genes but instead show a high capacity for RNA-binding protein and miRNA occupancy. Finally, we show that ALS-causingVCPmutations lead to a selective increase in cytoplasmic abundance of this particular class of IRTs, which in turn temporally coincides with an increase in the nuclear expression level of predicted miRNA target genes. Altogether, our study identifies a previously unrecognized class of cytoplasmic intronic sequences with potential regulatory function beyond gene expression.

Amyotrophic Lateral Sclerosis (ALS) is characterised by progressive motor neuron degeneration, but there is marked genetic and clinical heterogeneity1. It has been challenging to identify common ALS mechanisms among this diversity; however, a systematic framework examining motor neurons across the ALS spectrum may reveal unifying insights. Here, we present the most comprehensive compendium of ALS human induced pluripotent stem cell-derived motor neurons (iPSNs) from 429 donors, spanning 10 ALS mutations and sporadic ALS, from 15 datasets, including Answer ALS and NeuroLINCS. Using gold-standard reproducible bioinformatic workflows, we identified that ALS iPSNs show a common increase in the DNA damage response, which is characterised by activation of p53 signalling. The strongest p53 activation was observed in C9orf72 repeat expansions but was also found in TARDBP, FUS and sporadic subgroups. p53 activation was replicated in an ALS postmortem spinal cord cohort of 203 samples, indicating that the p53-dependent DNA damage response in ALS begins early and persists into the later stages of the disease. ALS iPSNs showed extensive genomic instability, as evidenced by enrichment of splicing alterations, single nucleotide variants, insertions, deletions and gene fusions, which may contribute to their p53 signature. In summary, by integrating the global landscape of ALS iPSNs, we reveal genome instability and p53 activation as common hallmarks of ALS motor neurons and provide a resource for identifying future ALS drug targets.

Amyotrophic Lateral Sclerosis (ALS) is characterised by progressive motor neuron degeneration but there is marked genetic and clinical heterogeneity1. Identifying common mechanisms of ALS amongst this diversity has been challenging, however, a systematic framework examining motor neurons across the ALS spectrum may reveal unifying insights. Here, we present the most comprehensive compendium of ALS human-induced pluripotent stem cell-derived motor neurons (iPSNs) from 429 donors across 15 datasets including Answer ALS and NeuroLINCS, spanning 10 ALS mutations and sporadic ALS. Using gold-standard reproducible bioinformatic workflows, we identify that ALS iPSNs show common activation of the DNA damage response and p53 signalling, which was replicated in the NYGC ALS postmortem cohort of 203 spinal cord samples. The strongest p53 activation was observed in C9orf72 repeat expansions but was also independently increased in TARDBP, FUS and sporadic subgroups. ALS iPSNs showed extensive splicing alterations and enrichment of SNVs, indels and gene fusions, which may contribute to their damage-induced mutation signature. Our results integrate the global landscape of motor neuron alterations in ALS, revealing that genome instability is a common hallmark of ALS motor neurons and provides a resource to identify future ALS drug targets.

We recently described aberrantly increased cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as new hallmarks of amyotrophic lateral sclerosis (ALS). However, the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep sequencing of nuclear and cytoplasmic fractions of human induced pluripotent stem cells undergoing motor neurogenesis, we reveal that ALS-causing VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify >100 IRTs with increased cytoplasmic abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins. Remarkably, TDP-43, SFPQ and FUS—RNA binding proteins known for nuclear-to-cytoplasmic mislocalization in ALS—abundantly and specifically bind to this aberrant cytoplasmic pool of IRTs. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets., Aberrant cytoplasmic SFPQ intron-retaining transcripts (IRTs) have recently been described in ALS, but their roles remained unclear. Tyzack, Neeves et al. now identify >100 cytoplasmic IRTs and show that they are ‘blueprints’ for ALS-related RNA binding protein mislocalization, as well as potential therapeutic targets.

Clarke and Patani review the involvement of microglia in ALS, focusing specifically on evidence for spatiotemporally regulated neurotoxic and neuroprotective roles. Understanding how microglia contribute to the ‘cellular phase’ of ALS promises to advance the development of therapies with a mechanistic rationale., Microglia are the primary immune cells of the CNS, carrying out key homeostatic roles and undergoing context-dependent and temporally regulated changes in response to injury and neurodegenerative diseases. Microglia have been implicated in playing a role in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by extensive motor neuron loss leading to paralysis and premature death. However, as the pathomechansims of ALS are increasingly recognized to involve a multitude of different cell types, it has been difficult to delineate the specific contribution of microglia to disease. Here, we review the literature of microglial involvement in ALS and discuss the evidence for the neurotoxic and neuroprotective pathways that have been attributed to microglia in this disease. We also discuss accumulating evidence for spatiotemporal regulation of microglial activation in this context. A deeper understanding of the role of microglia in the ‘cellular phase’ of ALS is crucial in the development of mechanistically rationalized therapies.

Traumatic brain injury leads to cellular damage which in turn results in the rapid release of damage-associated molecular patterns (DAMPs) that prompt resident cells to release cytokines and chemokines. These in turn rapidly recruit neutrophils, which assist in limiting the spread of injury and removing cellular debris. Microglia continuously survey the CNS (central nervous system) compartment and identify structural abnormalities in neurons contributing to the response. After some days, when neutrophil numbers start to decline, activated microglia and astrocytes assemble at the injury site—segregating injured tissue from healthy tissue and facilitating restorative processes. Monocytes infiltrate the injury site to produce chemokines that recruit astrocytes which successively extend their processes towards monocytes during the recovery phase. In this fashion, monocytes infiltration serves to help repair the injured brain. Neurons and astrocytes also moderate brain inflammation via downregulation of cytotoxic inflammation. Depending on the severity of the brain injury, T and B cells can also be recruited to the brain pathology sites at later time points.

Amyotrophic lateral sclerosis (ALS) is a devastating disease with a lifetime risk of approximately 1:400. It is incurable and invariably fatal. Average survival is between 3 and 5 years and patients become increasingly paralyzed, losing the ability to speak, eat, and breathe. Therapies in development either (i) target specific familial forms of ALS (comprising a minority of around 10% of cases) or ii) emanate from (over)reliance on animal models or non-human/non-neuronal cell models. There is a desperate and unmet clinical need for effective treatments. Deciphering the primacy and relative contributions of defective protein homeostasis and RNA metabolism in ALS across different model systems will facilitate the identification of putative therapeutic targets.This review examines the putative common primary molecular events that lead to ALS pathogenesis. We focus on deregulated RNA metabolism, protein mislocalization/pathological aggregation and the role of glia in ALS-related motor neuron degeneration. Finally, we describe promising targets for therapeutic evaluation.Moving forward, an effective strategy could be achieved by a poly-therapeutic approach which targets both deregulated RNA metabolism and protein dyshomeostasis in the relevant cell types, at the appropriate phase of disease.

Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis.

Summary Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These “binding-region condensates” are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution., Graphical abstract, Highlights • TDP-43 mutants affect condensation properties to a similar extent at multiple scales • Binding-region condensates form on long RNA regions with dispersed UG-rich motifs • RBPchimera-CLIP indicates homomeric interactions promote molecular-scale condensates • Condensation selectively tunes the regulatory capacity of TDP-43; e.g., autoregulation, The condensation propensity of an RNA-binding protein tunes its binding to specific RNA regions across the transcriptome and affects its RNA processing functions. Formation of these “binding-region condensates,” promoted by specific motif types that are dispersed across long RNA regions, expands the ways in which RNA binding can be selectively controlled beyond canonical RNA-binding domains.

The microtubule-associated protein tau gene (MAPT) 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers which, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau transcript and protein ratios. These elevated levels of 4R-tau in astrocytes implicate glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of pre-clinical tauopathies.

AIMS Although morphological attributes of cells and their substructures are recognised readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. METHODS In this study, we integrate multichannel fluorescence high-content microscopy data with deep learning imaging methods to reveal-directly from unsegmented images-novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). RESULTS Surprisingly, we reveal that previously unrecognised disease-relevant information is withheld in broadly used and often considered 'generic' biological markers of nuclei (DAPI) and neurons ( β III-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analysing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. CONCLUSIONS Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers and establishes the use of image-based deep learning methods for rapid, automated and unbiased identification of biological hypotheses.

RNA binding proteins have been shown to play a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in valosin-containing protein (VCP/p97) cause ALS and exhibit the hallmark nuclear-to-cytoplasmic mislocalization of RNA binding proteins (RBPs). However, the mechanism by which mutations in VCP lead to this mislocalization of RBPs remains incompletely resolved. To address this, we used human-induced pluripotent stem cell-derived motor neurons carrying VCP mutations. We first demonstrate reduced nuclear-to-cytoplasmic ratios of transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS) and splicing factor proline and glutamine rich (SFPQ) in VCP mutant motor neurons. Upon closer analysis, we also find these RBPs are mislocalized to motor neuron neurites themselves. To address the hypothesis that altered function of the D2 ATPase domain of VCP causes RBP mislocalization, we used pharmacological inhibition of this domain in control motor neurons and found this does not recapitulate RBP mislocalization phenotypes. However, D2 domain inhibition in VCP mutant motor neurons was able to robustly reverse mislocalization of both TDP-43 and FUS, in addition to partially relocalizing SFPQ from the neurites. Together these results argue for a gain-of-function of D2 ATPase in VCP mutant human motor neurons driving the mislocalization of TDP-43 and FUS. Our data raise the intriguing possibility of harnessing VCP D2 ATPase inhibitors in the treatment of VCP-related ALS., Graphical Abstract Graphical Abstract, Harley et al. report that mutations in the VCP gene that cause amyotrophic lateral sclerosis result in the mislocalization of RNA binding proteins TDP-43, FUS and SFPQ in human motor neurons. Treatment with a VCP D2-ATPase inhibitor reverses the mislocalization of TDP-43 and FUS, suggesting a gain-of-function of D2-ATPase is driving mislocalization.

See Vidal and Atkin (doi:10.1093/brain/awz256) for a scientific commentary on this article. ALS remains incurable due to an incomplete understanding of its molecular pathogenesis. Tyzack, Luisier et al. report that FUS protein, previously thought to mislocalise only in patients with FUS mutations, is also mislocalised in many cases of sporadic ALS. They further propose an underlying molecular mechanism for the mislocalisation., Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

Astrocytes contribute to motor neuron death in amyotrophic lateral sclerosis (ALS), but whether they adopt deleterious features consistent with inflammatory reactive states remains incompletely resolved. To identify inflammatory reactive features in ALS human induced pluripotent stem cell (hiPSC)–derived astrocytes, we examined transcriptomics, proteomics, and glutamate uptake in VCP-mutant astrocytes. We complemented this by examining other ALS mutations and models using a systematic meta-analysis of all publicly-available ALS astrocyte sequencing data, which included hiPSC-derived astrocytes carrying SOD1, C9orf72, and FUS gene mutations as well as mouse ALS astrocyte models with SOD1G93A mutation, Tardbp deletion, and Tmem259 (also known as membralin) deletion. ALS astrocytes were characterized by up-regulation of genes involved in the extracellular matrix, endoplasmic reticulum stress, and the immune response and down-regulation of synaptic integrity, glutamate uptake, and other neuronal support processes. We identify activation of the TGFB, Wnt, and hypoxia signaling pathways in both hiPSC and mouse ALS astrocytes. ALS changes positively correlate with TNF, IL1A, and complement pathway component C1q-treated inflammatory reactive astrocytes, with significant overlap of differentially expressed genes. By contrasting ALS changes with models of protective reactive astrocytes, including middle cerebral artery occlusion and spinal cord injury, we uncover a cluster of genes changing in opposing directions, which may represent down-regulated homeostatic genes and up-regulated deleterious genes in ALS astrocytes. These observations indicate that ALS astrocytes augment inflammatory processes while concomitantly suppressing neuronal supporting mechanisms, thus resembling inflammatory reactive states and offering potential therapeutic targets.

The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.

Neurodegenerative diseases are a group of largely incurable disorders characterised by the progressive loss of neurons and for which often the molecular mechanisms are poorly understood. To bridge this gap, researchers employ a range of techniques. A very prominent and useful technique adopted across many different fields is imaging and the analysis of histopathological and fluorescent label tissue samples. Although image acquisition has been efficiently automated recently, automated analysis still presents a bottleneck. Although various methods have been developed to automate this task, they tend to make use of single-purpose machine learning models that require extensive training, imposing a significant workload on the experts and introducing variability in the analysis. Moreover, these methods are impractical to audit and adapt, as their internal parameters are difficult to interpret and change. Here, we present a novel unsupervised automated schema for object segmentation of images, exemplified on a dataset of tissue images. Our schema does not require training data, can be fully audited and is based on a series of understandable biological decisions. In order to evaluate and validate our schema, we compared it with a state-of-the-art automated segmentation method for post-mortem tissues of ALS patients.

SUMMARYAlthough morphological attributes of cells and their substructures are recognized readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. In this study we integrate multichannel fluorescence high-content microscopy data with deep-learning imaging methods to reveal - directly from unsegmented images - novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). Surprisingly, we reveal that previously unrecognized disease-relevant information is withheld in broadly used and often considered ‘generic’ biological markers of nuclei (DAPI) and neurons (βIII-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analyzing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers, and establishes the use of image-based deep learning methods for rapid, automated and unbiased testing of biological hypotheses.

RNA binding proteins fulfil a wide number of roles in gene expression. Multiple mechanisms of RNA binding protein dysregulation have been implicated in the pathomechanisms of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Oxidative stress and mitochondrial dysfunction also play important roles in these diseases. In this review, we highlight the mechanistic interplay between RNA binding protein dysregulation, oxidative stress and mitochondrial dysfunction in ALS. We also discuss different potential therapeutic strategies targeting these pathways.

Histopathological analysis of tissue sections is invaluable in neurodegeneration research. However, cell‐to‐cell variation in both the presence and severity of a given phenotype is a key limitation of this approach, reducing the signal to noise ratio and leaving unresolved the potential of single‐cell scoring for a given disease attribute. Here, we tested different machine learning methods to analyse high‐content microscopy measurements of hundreds of motor neurons (MNs) from amyotrophic lateral sclerosis (ALS) post‐mortem tissue sections. Furthermore, we automated the identification of phenotypically distinct MN subpopulations in VCP‐ and SOD1‐mutant transgenic mice, revealing common morphological cellular phenotypes. Additionally we established scoring metrics to rank cells and tissue samples for both disease probability and severity. By adapting this paradigm to human post‐mortem tissue, we validated our core finding that morphological descriptors robustly discriminate ALS from control healthy tissue at single cell resolution. Determining disease presence, severity and unbiased phenotypes at single cell resolution might prove transformational in our understanding of ALS and neurodegeneration more broadly., With their novel pipeline for automated segmentation, profiling and identification of phenotypically distinct motor neuron subpopulations in ALS pathological tissue sections, Hageman et al. report that morphological descriptors strongly discriminate ALS from control healthy tissue at the single‐cell level.

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes., Graphical Abstract Graphical abstractHealthy astrocytes (left, blue) have prevalent nuclear intron retention in reactivity transcripts, promoting their nuclear confinement (introns shown as red rectangles). Conversely, ALS astrocytes (right, red) exhibit decreased intron retention in reactivity transcripts, enabling cytoplasmic translocation and translation upon engaging ribosomes, underlying their reactive transformation. A small number of intron retaining transcripts escape to the cytoplasm where they are degraded by nonsense mediated decay (NMD), which is enhanced in ALS astrocytes.

SUMMARYIntron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development, however the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. By resolving the spatiotemporal dynamics of IR underlying distinct stages of MN lineage restriction, we identify a cytoplasmic group of IRTs that is not associated with reduced expression of their own genes but instead with an upregulation of predicted target genes of specific miRNAs, the motifs of which are enriched within the intronic sequences of this group. Next, we show that ALS-causing VCP mutations lead to a selective increase in IR of this particular class of introns. This in turn temporally coincides with an increase in the expression level of predicted target genes of these miRNAs, providing a potential mechanistic insight into ALS pathogenesis. Altogether, we propose a novel role for the cytoplasmic intronic sequences in regulating miRNA activity through miRNA sequestration, which potentially contributes to ALS pathogenesis.