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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.

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.

Stem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). The axons of these neurons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. It follows that shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here we directly address this issue by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, permitting systematic investigation of the effects of length on human axonal biology for the first time. With this approach, we reveal a link between length and metabolism in human MNs in vitro, where axons above a “threshold” size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation and mitochondrial homeostasis. Our findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs in order to sustain long axons. Our findings have critical implications for in vitro modelling of several neurodegenerative disorders and reinforce the importance of modelling cell shape and biophysical constraints with fidelity and precision in vitro.