C9orf72 Amyotrophic Lateral Sclerosis Research Articles

C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD

AbstractHexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are debilitating neurodegenerative conditions affecting either motor neurons (ALS) in the brain and spinal cord or neurons in the frontal and/or temporal cortical lobes (FTD). HREs undergo repeat-associated non-ATG (RAN) translation on both sense and anti-sense strands, generating five distinct dipeptide repeat proteins (DPRs), poly-GA, -GR, -GP, -PA and -PR. Perturbed proteostasis is well-recognised in ALS pathogenesis, including processes affecting the endoplasmic reticulum (ER) and Golgi compartments. However, these mechanisms have not been well characterised for C9orf72-mediated ALS/FTD. In this study we demonstrate that C9orf72 DPRs polyGA, polyGR and polyGP (× 40 repeats) disrupt secretory protein transport from the ER to the Golgi apparatus in neuronal cells. Consistent with this finding, these DPRs also induce fragmentation of the Golgi apparatus, activate ER stress, and inhibit the formation of the omegasome, the precursor of the autophagosome that originates from ER membranes. We also demonstrate Golgi fragmentation in cells undergoing RAN translation that express polyGP. Furthermore, dysregulated ER-Golgi transport was confirmed in C9orf72 patient dermal fibroblasts. Evidence of aberrant ER-derived vesicles in spinal cord motor neurons from C9orf72 ALS patients compared to controls was also obtained. These data thus confirm that ER proteostasis and ER-Golgi transport is perturbed in C9orf72-ALS in the absence of protein over-expression. Hence this study identifies novel molecular mechanisms associated with the ER and Golgi compartments induced by the C9orf72 HRE.

Apilimod dimesylate in C9orf72 amyotrophic lateral sclerosis: a randomized phase 2a clinical trial.

Apilimod dimesylate is a first-in-class phosphoinositide kinase, FYVE-type zinc finger containing (PIKfyve) inhibitor with favourable clinical safety profile and has demonstrated activity in preclinical C9orf72 and TDP-43 amyotrophic lateral sclerosis models. In this amyotrophic lateral sclerosis clinical trial, the safety, tolerability, CNS penetrance, and modulation of pharmacodynamic target engagement biomarkers were evaluated. This Phase 2a, randomized, double-blind, placebo-controlled, biomarker-endpoint clinical trial was conducted in four USA centres (ClinicalTrials.gov NCT05163886). Participants with C9orf72 repeat expansion were randomly assigned (2:1) to receive twice-daily oral treatment of 125 mg apilimod dimesylate capsules or matching placebo for 12 weeks, followed by a 12-week open-label extension. Safety was measured as the occurrence of treatment-emergent adverse or serious adverse events attributable to study drug, and tolerability as trial completion on treatment over 12 weeks. Changes from baseline in plasma and CSF and concentrations of apilimod and its active metabolites and of pharmacodynamic biomarkers of PIKfyve inhibition (soluble glycoprotein nonmetastatic melanoma protein B [sGPNMB] upregulation) and disease-specific CNS target engagement (poly[GP]). Between Dec 16, 2021, and Jul 7, 2022, 15 eligible participants were enrolled. There were no drug-related serious adverse events reported in the trial. Fourteen (93%) participants completed the double-blind period with 99% dose compliance (N=9 [90%] apilimod dimesylate; N=5 [100%] placebo). At Week 12, apilimod dimesylate was measurable in CSF at 1.63 ng/mL (SD: 0.937). At Week 12, apilimod dimesylate increased plasma sGPNMB by > 2.5-fold (p < 0.001) indicating PIKfyve inhibition and lowered CSF poly(GP) protein levels by 73% (p < 0.001) indicating CNS tissue-level proof of mechanism. Apilimod dimesylate met prespecified key safety and biomarker endpoints in this Phase 2a trial and demonstrated CNS penetrance and pharmacodynamic target engagement. Apilimod dimesylate was observed to have the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.

Distinct neural signatures of pulvinar in C9orf72 amyotrophic lateral sclerosis mutation carriers and noncarriers.

Thalamic alterations have been reported as a major feature in presymptomatic and symptomatic patients carrying the C9orf72 mutation across the frontotemporal dementia-amyotrophic lateral sclerosis (ALS) spectrum. Specifically, the pulvinar, a high-order thalamic nucleus and timekeeper for large-scale cortical networks, has been hypothesized to be involved in C9orf72-related neurodegenerative diseases. We investigated whether pulvinar volume can be useful for differential diagnosis in ALS C9orf72 mutation carriers and noncarriers and how underlying functional connectivity changes affect this region. We studied 19 ALS C9orf72 mutation carriers (ALSC9+) accurately matched with wild-type ALS (ALSC9-) and ALS mimic (ALSmimic) patients using structural and resting-state functional magnetic resonance imaging data. Pulvinar volume was computed using automatic segmentation. Seed-to-voxel functional connectivity analyses were performed using seeds from a pulvinar functional parcellation. Pulvinar structural integrity had high discriminative values for ALSC9+ patients compared to ALSmimic (area under the curve [AUC] = 0.86) and ALSC9- (AUC = 0.77) patients, yielding a volume cutpoint of approximately 0.23%. Compared to ALSmimic, ALSC9- showed increased anterior, inferior, and lateral pulvinar connections with bilateral occipital-temporal-parietal regions, whereas ALSC9+ showed no differences. ALSC9+ patients when compared to ALSC9- patients showed reduced pulvinar-occipital connectivity for anterior and inferior pulvinar seeds. Pulvinar volume could be a differential biomarker closely related to the C9orf72 mutation. A pulvinar-cortical circuit dysfunction might play a critical role in disease progression and development, in both the genetic phenotype and ALS wild-type patients.

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity

Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA–protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2)58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2)58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.

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

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

Causal ALS genes impact the MHC class II antigen presentation pathway

Mutations in RNA/DNA-binding proteins cause amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain unclear. Here, we report that a set of ALS-associated proteins, namely FUS, EWSR1, TAF15, and MATR3, impact the expression of genes encoding the major histocompatibility complex II (MHC II) antigen presentation pathway. Both subunits of the MHC II heterodimer, HLA-DR, are down-regulated in ALS gene knockouts/knockdown in HeLa and human microglial cells, due to loss of the MHC II transcription factor CIITA. Importantly, hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells bearing the FUSR495X mutation and HPCs derived from C9ORF72 ALS patient induced pluripotent stem cells also exhibit disrupted MHC II expression. Given that HPCs give rise to numerous immune cells, our data raise the possibility that loss of the MHC II pathway results in global failure of the immune system to protect motor neurons from damage that leads to ALS.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity.

Intronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored. To study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs). We found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs. This study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

C9orf72 ALS mutation carriers show extensive cortical and subcortical damage compared to matched wild-type ALS patients

ObjectiveC9orf72 mutation carriers with different neurological phenotypes show cortical and subcortical atrophy in multiple different brain regions, even in pre-symptomatic phases. Despite there is a substantial amount of knowledge, small sample sizes, clinical heterogeneity, as well as different choices of image analysis may hide anatomical abnormalities that are unique to amyotrophic lateral sclerosis (ALS) patients with this genotype or that are indicative of the C9orf72-specific trait overlain in fronto-temporal dementia patients. MethodsBrain structural and resting state functional magnetic imaging was obtained in 24 C9orf72 positive (ALSC9+) ALS patients paired for burden disease with 24 C9orf72 negative (ALSC9-) ALS patients. A comprehensive structural evaluation of cortical thickness and subcortical volumes between ALSC9+ and ALSC9- patients was performed while a region of interest (ROI)-ROI analysis of functional connectivity was implemented to assess functional alterations among abnormal cortical and subcortical regions. Results were corrected for multiple comparisons. ResultsCompared to ALSC9- patients, ALSC9+ patients exhibited extensive disease-specific patterns of thalamo-cortico-striatal atrophy, supported by functional alterations of the identified abnormal regions. Cortical thinning was most pronounced in posterior areas and extended to frontal regions. Bilateral atrophy of the mediodorsal and pulvinar nuclei was observed, emphasizing a focal rather than global thalamus atrophy. Volume loss in a large portion of bilateral caudate and left putamen was reported. The marked reduction of functional connectivity observed between the left posterior thalamus and almost all the atrophic cortical regions support the central role of the thalamus in the pathogenic mechanism of C9orf72-mediated disease. ConclusionsThese findings constitute a coherent and robust picture of ALS patients with C9orf72-mediated disease, unveiling a specific structural and functional characterization of thalamo-cortico-striatal circuit alteration. Our study introduces new evidence in the characterization of the pathogenic mechanisms of C9orf72 mutation.

Dipeptide Repeat Pathology in C9orf72-ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance.

The hexanucleotide expansion of the C9orf72 gene is found in 40% of familial amyotrophic lateral sclerosis (ALS) patients. This genetic alteration has been connected with impaired management of reactive oxygen species. In this study, we conducted targeted transcriptional profiling in leukocytes from C9orf72 patients and control subjects by examining the mRNA levels of 84 redox-related genes. The expression of ten redox genes was altered in samples from C9orf72 ALS patients compared to healthy controls. Considering that Nuclear factor erythroid 2-Related Factor 2 (NRF2) modulates the expression of a wide range of redox genes, we further investigated its status on an in vitro model of dipeptide repeat (DPR) toxicity. This model mimics the gain of function, toxic mechanisms attributed to C9orf72 pathology. We found that exposure to DPRs increased superoxide levels and reduced mitochondrial potential as well as cell survival. Importantly, cells overexpressing DPRs exhibited reduced protein levels of NRF2 and its target genes upon inhibition of the proteasome or its canonical repressor, the E3 ligase adapter KEAP1. However, NRF2 activation was sufficient to recover cell viability and redox homeostasis. This study identifies NRF2 as a putative target in precision medicine for the therapy of ALS patients harboring C9orf72 expansion repeats.

HNRNPK alleviates RNA toxicity by counteracting DNA damage in C9orf72 ALS

A ‘GGGGCC’ repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.

A mouse model with widespread expression of the C9orf72-linked glycine\u2013arginine dipeptide displays non-lethal ALS/FTD-like phenotypes

Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.

Poly-dipeptides produced from C9orf72 hexanucleotide repeats cause selective motor neuron hyperexcitability in ALS

SignificanceThe GGGGCC hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS). Despite myriad studies on the toxic effects of poly-dipeptides produced from the C9orf72 repeats, the mechanisms underlying the selective hyperexcitability of motor cortex that characterizes the early stages of C9orf72 ALS patients remain elusive. Here, we show that the proline-arginine poly-dipeptides cause hyperexcitability in cortical motor neurons by increasing persistent sodium currents conducted by the Nav1.2/β4 sodium channel complex, which is highly expressed in the motor cortex. These findings provide the basis for understanding how the C9orf72 mutation causes motor neuron hyperactivation that can lead to the motor neuron death in C9orf72 ALS.

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

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

Emerging Connections between Nuclear Pore Complex Homeostasis and ALS.

Developing effective treatments for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) requires understanding of the underlying pathomechanisms that contribute to the motor neuron loss that defines the disease. As it causes the largest fraction of familial ALS cases, considerable effort has focused on hexanucleotide repeat expansions in the C9ORF72 gene, which encode toxic repeat RNA and dipeptide repeat (DPR) proteins. Both the repeat RNA and DPRs interact with and perturb multiple elements of the nuclear transport machinery, including shuttling nuclear transport receptors, the Ran GTPase and the nucleoporin proteins (nups) that build the nuclear pore complex (NPC). Here, we consider recent work that describes changes to the molecular composition of the NPC in C9ORF72 model and patient neurons in the context of quality control mechanisms that function at the nuclear envelope (NE). For example, changes to NPC structure may be caused by the dysregulation of a conserved NE surveillance pathway mediated by the endosomal sorting complexes required for the transport protein, CHMP7. Thus, these studies are introducing NE and NPC quality control pathways as key elements in a pathological cascade that leads to C9ORF72 ALS, opening entirely new experimental avenues and possibilities for targeted therapeutic intervention.

Single-cell transcriptomics identifies master regulators of neurodegeneration in SOD1 ALS iPSC-derived motor neurons.

SummaryAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by the loss of motor neurons. We utilized single-cell transcriptomics to uncover dysfunctional pathways in degenerating motor neurons differentiated from SOD1 E100G ALS patient-derived induced pluripotent stem cells (iPSCs) and respective isogenic controls. Differential gene expression and network analysis identified activation of developmental pathways and core transcriptional factors driving the ALS motor neuron gene dysregulation. Specifically, we identified activation of SMAD2, a downstream mediator of the transforming growth factor β (TGF-β) signaling pathway as a key driver of SOD1 iPSC-derived motor neuron degeneration. Importantly, our analysis indicates that activation of TGFβ signaling may be a common mechanism shared between SOD1, FUS, C9ORF72, VCP, and sporadic ALS motor neurons. Our results demonstrate the utility of single-cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We find that ALS-associated mutant SOD1 targets transcriptional networks that perturb motor neuron homeostasis.

Human spinal cord-like organoids to model C9ORF72 ALS and test new therapies in vitro

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. Underlying genetic pathomechanisms include the C9orf72 repeat expansion, the most frequent genetic cause of ALS (C9ALS) in Western countries. Despite recent progress in unraveling C9ALS pathogenesis, reliable disease models and disease-modifying therapies still lack. Here, we aim to model C9ALS in vitro using 3D human spinal cord organoids (SCOs).

Viral vector gene delivery of the novel chaperone protein SRCP1 to modify insoluble protein in in vitro and in vivo models of ALS

Protein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington’s disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1-G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

Targeting S100A4 with niclosamide attenuates inflammatory and profibrotic pathways in models of amyotrophic lateral sclerosis

BackgroundAn increasing number of studies evidences that amyotrophic lateral sclerosis (ALS) is characterized by extensive alterations in different cell types and in different regions besides the CNS. We previously reported the upregulation in ALS models of a gene called fibroblast-specific protein-1 or S100A4, recognized as a pro-inflammatory and profibrotic factor. Since inflammation and fibrosis are often mutual-sustaining events that contribute to establish a hostile environment for organ functions, the comprehension of the elements responsible for these interconnected pathways is crucial to disclose novel aspects involved in ALS pathology.MethodsHere, we employed fibroblasts derived from ALS patients harboring the C9orf72 hexanucleotide repeat expansion and ALS patients with no mutations in known ALS-associated genes and we downregulated S100A4 using siRNA or the S100A4 transcriptional inhibitor niclosamide. Mice overexpressing human FUS were adopted to assess the effects of niclosamide in vivo on ALS pathology.ResultsWe demonstrated that S100A4 underlies impaired autophagy and a profibrotic phenotype, which characterize ALS fibroblasts. Indeed, its inhibition reduces inflammatory, autophagic, and profibrotic pathways in ALS fibroblasts, and interferes with different markers known as pathogenic in the disease, such as mTOR, SQSTM1/p62, STAT3, α-SMA, and NF-κB. Importantly, niclosamide in vivo treatment of ALS-FUS mice reduces the expression of S100A4, α-SMA, and PDGFRβ in the spinal cord, as well as gliosis in central and peripheral nervous tissues, together with axonal impairment and displays beneficial effects on muscle atrophy, by promoting muscle regeneration and reducing fibrosis.ConclusionOur findings show that S100A4 has a role in ALS-related mechanisms, and that drugs such as niclosamide which are able to target inflammatory and fibrotic pathways could represent promising pharmacological tools for ALS.