Amyotrophic Lateral Sclerosis Mutations Research Articles

Genotype-phenotype association and functional analysis of hnRNPA1 mutations in amyotrophic lateral sclerosis

Background Pathogenic variants in hnRNPA1 have been reported in amyotrophic lateral sclerosis (ALS) patients. However, studies on hnRNPA1 mutant spectrum and pathogenicity of variants were rare. Methods We performed whole exome sequencing of ALS-associated genes and subsequent verification of rare variants in hnRNPA1 in our ALS patients. The hnRNPA1 mutations reported in literature were reviewed and combined with our results to determine the genotype-phenotype relationship. Functional analysis of the novel variant p.G195A was performed in vitro by transfection of mutant hnRNPA1 into 293T cell. Results Among 207 ALS patients recruited, 3 rare hnRNPA1 variants were identified (mutant frequency 1.45%), including two recurrent mutations (p.P340S and p.G283R), and a novel rare variant p.G195A. In combination with previous reports, there are 27 ALS patients with 15 hnRNPA1 mutations identified. Disease onset age was 47.90 ± 1.52 years with predominant limb onset. The p.P340S mutation caused flail arm syndrome (FAS) in two independent families with extended life expectancy. The newly identified p.G195A mutation, lying at the start of the PrLD (“prion-like” domain)/LCD (low-complexity domain), causes local structural changes in 3D protein prediction. Upon sodium arsenite exposure, mutant hnRNPA1 retained in the nucleus but deficit of cytoplasmic G3BP1-positive stress granule clearance was observed. This is different from the p.P340S mutation which caused both cytoplasmic translocation and stress granule formation. No cytoplasmic TDP-43 translocation was observed. Conclusion Mutations in hnRNPA1 are overall minor in ALS patients. The p.P340S mutation is associated with manifestation of FAS. Mutations in LCD of hnRNPA1 cause stress granule misprocessing.

ALS-associated VRK1 R321C mutation causes proteostatic imbalance and mitochondrial defects in iPSC-derived motor neurons

Vaccinia-related kinase 1 (VRK1) is a gene which has been implicated in the pathological process of a broad range of neurodevelopmental disorders as well as neuropathies, such as Amyotrophic Lateral Sclerosis (ALS). Here we report a family presenting ALS in an autosomal recessive mode of inheritance, segregating with a homozygous missense mutation located in VRK1 gene (p.R321C; Arg321Cys). Proteomic analyses from iPSC-derived motor neurons identified 720 proteins eligible for subsequent investigation, and our exploration of protein profiles revealed significant enrichments in pathways such as mTOR signaling, E2F, MYC targets, DNA repair response, cell proliferation and energetic metabolism. Functional studies further validated such alterations, showing that affected motor neurons presented decreased levels of global protein output, ER stress and downregulation of mTOR signaling. Mitochondrial alterations also pointed to decreased reserve capacity and increased non-mitochondrial oxygen consumption. Taken together, our results present the main pathological alterations associated with VRK1 mutation in ALS.

Dissecting the effect of ALS mutation S375G on the conformational properties and aggregation dynamics of TDP-43370-375 fragment

The aggregation of transactive response deoxyribonucleic acid (DNA) binding protein of 43 kDa (TDP-43) into ubiquitin-positive inclusions is closely associated with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and chronic traumatic encephalopathy. The 370–375 fragment of TDP-43 (370GNNSYS375, TDP-43370-375), the amyloidogenic hexapeptides, can be prone to forming pathogenic amyloid fibrils with the characteristic of steric zippers. Previous experiments reported the ALS-associated mutation, serine 375 substituted by glycine (S375G) is linked to early onset disease and protein aggregation of TDP-43. Based on this, it is necessary to explore the underlying molecular mechanisms. By utilizing all-atom molecular dynamics (MD) simulations of 102 μs in total, we investigated the impact of S375G mutation on the conformational ensembles and oligomerization dynamics of TDP-43370-375 peptides. Our replica exchange MD simulations show that S375G mutation could promote the unstructured conformation formation and induce peptides to form a loose packed oligomer, thus inhibiting the aggregation of TDP-43370-375. Further analyses suggest that S375G mutation displays a reduction effect on the number of total hydrogen bonds and contacts among TDP-43370-375 peptides. Hydrogen bonding and polar interactions among TDP-43370-375 peptides, as well as Y374-Y374 π-π stacking interaction, are attenuated by S375G mutation. Additional microsecond MD simulations demonstrate that S375G mutation could prohibit the conformational conversion to β-structure-rich aggregates and possess an inhibitory effect on the oligomerization dynamics of TDP-43370-375. This study offers for the first time of molecular insights into the S375G mutation affecting the aggregation of TDP-43370-375 at the atomic level, and may open new avenues in the development of future site-specific mutation therapeutics.

FUS reads histone H3K36me3 to regulate alternative polyadenylation.

Complex organisms generate differential gene expression through the same set of DNA sequences in distinct cells. The communication between chromatin and RNA regulates cellular behavior in tissues. However, little is known about how chromatin, especially histone modifications, regulates RNA polyadenylation. In this study, we found that FUS was recruited to chromatin by H3K36me3 at gene bodies. The H3K36me3 recognition of FUS was mediated by the proline residues in the ZNF domain. After these proline residues were mutated or H3K36me3 was abolished, FUS dissociated from chromatin and bound more to RNA, resulting in an increase in polyadenylation sites far from stop codons genome-wide. A proline mutation corresponding to a mutation in amyotrophic lateral sclerosis contributed to the hyperactivation of mitochondria and hyperdifferentiation in mouse embryonic stem cells. These findings reveal that FUS is an H3K36me3 reader protein that links chromatin-mediated alternative polyadenylation to human disease.

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.

RNP granules in ALS and neurodegeneration: From multifunctional membraneless organelles to therapeutic opportunities.

Amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases are characterised by dysfunction of a host of RNA-binding proteins (RBPs) and a severely disrupted RNA metabolism. Recently, RBP-harbouring phase-separated complexes, ribonucleoprotein (RNP) granules, have come into the limelight as "crucibles" of neuronal pathology in ALS. RNP granules are indispensable for the multitude of regulatory processes underlying cellular RNA metabolism and serve as critical organisers of cellular biochemistry. Neurons, highly specialised cells, heavily rely on RNP granules for efficient trafficking, signalling and stress responses. Multiple RNP granule components, primarily RBPs such as TDP-43 and FUS, are affected by ALS mutations. However, even in the absence of mutations, RBP proteinopathies represent pathophysiological hallmarks of ALS. Given the high local concentrations of RBPs and RNAs, their weakened or enhanced interactions within RNP granules disrupt their homeostasis. Thus, the physiological process of phase separation and RNP granule formation, vital for maintaining the high-functioning state of neuronal cells, becomes their Achilles heel. Here, we will review the recent literature on the causes and consequences of abnormal RNP granule functioning in ALS and related disorders. In particular, we will summarise the evidence for the network-level dysfunction of RNP granules in these conditions and discuss considerations for therapeutic interventions to target RBPs, RNP granules and their network as a whole.

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

Glycine-Alanine Dipeptide Repeat Protein from C9-ALS Interacts with Sulfide Quinone Oxidoreductase (SQOR) to Induce the Activity of the NLRP3 Inflammasome in HMC3 Microglia: Irisflorentin Reverses This Interaction.

Amyotrophic lateral sclerosis (ALS) is a fatal rare disease of progressive degeneration of motor neurons. The most common genetic mutation in ALS is the hexanucleotide repeat expansion (HRE) located in the first intron of the C9orf72 gene (C9-ALS). HRE can produce dipeptide repeat proteins (DPRs) such as poly glycine-alanine (GA) in a repeat-associated non-ATG (RAN) translation. GA-DPR has been shown to be toxic to motor neurons in various biological models. However, its effects on microglia involved in C9-ALS have not been reported. Here, we show that GA-DPR (GA50) activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome in a human HMC3 microglia model. MCC950 (specific inhibitor of the NLRP3) treatment can abrogate this activity. Next, using yeast two-hybrid screening, we identified sulfide quinone oxidoreductase (SQOR) as a GA50 interacting protein. SQOR knockdown in HMC3 cells can significantly induce the activity of the NLRP3 inflammasome by upregulating the level of intracellular reactive oxygen species and the cytoplasmic escape of mitochondrial DNA. Furthermore, we obtained irisflorentin as an effective blocker of the interaction between SQOR and GA50, thus inhibiting NLRP3 inflammasome activity in GA50-expressing HMC3 cells. These results imply the association of GA-DPR, SQOR, and NLRP3 inflammasomes in microglia and establish a treatment strategy for C9-ALS with irisflorentin.

Identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence

Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

IPSC motor neurons, but not other derived cell types, capture gene expression changes in postmortem sporadic ALS motor neurons

Motor neuron degeneration, the defining feature of amyotrophic lateral sclerosis (ALS), is a primary example of cell-type specificity in neurodegenerative diseases. Using isogenic pairs of induced pluripotent stem cells (iPSCs) harboring different familial ALS mutations, we assess the capacity of iPSC-derived lower motor neurons, sensory neurons, astrocytes, and superficial cortical neurons to capture disease features including transcriptional and splicing dysregulation observed in human postmortem neurons. At early time points, differentially regulated genes in iPSC-derived lower motor neurons, but not other cell types, overlap with one-third of the differentially regulated genes in laser-dissected motor neurons from ALS compared with control postmortem spinal cords. For genes altered in both the iPSC model and bona fide human lower motor neurons, expression changes correlate between the two populations. In iPSC-derived lower motor neurons, but not other derived cell types, we detect the downregulation of genes affected by TDP-43-dependent splicing. This reduction takes place exclusively within genotypes known to involve TDP-43 pathology.

Impaired ATF3 signaling involves SNAP25 in SOD1 mutant ALS patients

Epigenetic remodeling is emerging as a critical process for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Genetics alone fails to explain the etiology of ALS, the investigation of the epigenome might therefore provide novel insights into the molecular mechanisms of the disease. In this study, we interrogated the epigenetic landscape in peripheral blood mononuclear cells (PBMCs) of familial ALS (fALS) patients with either chromosome 9 open reading frame 72 (C9orf72) or superoxide dismutase 1 (SOD1) mutation and aimed to identify key epigenetic footprints of the disease. To this end, we used an integrative approach that combines chromatin immunoprecipitation targeting H3K27me3 (ChIP-Seq) with the matching gene expression data to gain new insights into the likely impact of blood-specific chromatin remodeling on ALS-related molecular mechanisms. We demonstrated that one of the hub molecules that modulates changes in PBMC transcriptome in SOD1-mutant ALS patients is ATF3, which has been previously reported in an SOD1G93A mouse model. We also identified potential suppression of SNAP25, with impaired ATF3 signaling in SOD1-mutant ALS blood. Together, our study shed light on the mechanistic underpinnings of SOD1 mutations in ALS.

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalization in ALS motor neurons and is restored by VCP ATPase inhibition

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition.

Automated Fast-Flow Synthesis of Chromosome 9 Open Reading Frame 72 Dipeptide Repeat Proteins.

An expansion of the hexanucleotide (GGGGCC) repeat sequence in chromosome 9 open frame 72 (c9orf72) is the most common genetic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutation leads to the production of toxic dipeptide repeat proteins (DPRs) that induce neurodegeneration. However, the fundamental physicochemical properties of DPRs remain largely unknown due to their limited availability. Here, we synthesized the c9orf72 DPRs poly-glycine-arginine (poly-GR), poly-proline-arginine (poly-PR), poly-glycine-proline (poly-GP), poly-proline-alanine (poly-PA), and poly-glycine-alanine (poly-GA) using automated fast-flow peptide synthesis (AFPS) and achieved single-domain chemical synthesis of proteins with up to 200 amino acids. Circular dichroism spectroscopy of the synthetic DPRs revealed that proline-containing poly-PR, poly-GP, and poly-PA could adopt polyproline II-like helical secondary structures. In addition, structural analysis by size-exclusion chromatography indicated that longer poly-GP and poly-PA might aggregate. Furthermore, cell viability assays showed that human neuroblastoma cells cultured with poly-GR and poly-PR with longer repeat lengths resulted in reduced cell viability, while poly-GP and poly-PA did not, thereby reproducing the cytotoxic property of endogenous DPRs. This research demonstrates the potential of AFPS to synthesize low-complexity peptides and proteins necessary for studying their pathogenic mechanisms and constructing disease models.

Identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1(SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence.

Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins.

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

The Neuroprotective Activities of the Novel Multi-Target Iron-Chelators in Models of Alzheimer's Disease, Amyotrophic Lateral Sclerosis and Aging.

The concept of chelation therapy as a valuable therapeutic approach in neurological disorders led us to develop multi-target, non-toxic, lipophilic, brain-permeable compounds with iron chelation and anti-apoptotic properties for neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), age-related dementia and amyotrophic lateral sclerosis (ALS). Herein, we reviewed our two most effective such compounds, M30 and HLA20, based on a multimodal drug design paradigm. The compounds have been tested for their mechanisms of action using animal and cellular models such as APP/PS1 AD transgenic (Tg) mice, G93A-SOD1 mutant ALS Tg mice, C57BL/6 mice, Neuroblastoma × Spinal Cord-34 (NSC-34) hybrid cells, a battery of behavior tests, and various immunohistochemical and biochemical techniques. These novel iron chelators exhibit neuroprotective activities by attenuating relevant neurodegenerative pathology, promoting positive behavior changes, and up-regulating neuroprotective signaling pathways. Taken together, these results suggest that our multifunctional iron-chelating compounds can upregulate several neuroprotective-adaptive mechanisms and pro-survival signaling pathways in the brain and might function as ideal drugs for neurodegenerative disorders, such as PD, AD, ALS, and aging-related cognitive decline, in which oxidative stress and iron-mediated toxicity and dysregulation of iron homeostasis have been implicated.

A double point mutation of SOD1 targeting net charge promotes aggregation under destabilizing conditions: Correlation of charge distribution and ALS-provoking mutation

A progressive neurodegenerative illness such as amyotrophic lateral sclerosis (ALS) is characterized by the misfolding and aggregation of human CuZn superoxide dismutase (hSOD1) into amyloid aggregates. Thus, designing strategies for the choice of WT-SOD1 and double mutant (G12D/G138E) with an increased net negative charge can be a good idea to elucidate the pathological mechanism of SOD1 in ALS under some destabilizing conditions. Consequently, we show evidence that protein charge, together with other destabilizing conditions, plays an important role in ALS disease. To achieve this purpose, we use methods, such as spectroscopy and transmission electron microscopy (TEM) to monitor the formation of amyloid aggregation. The specific activity of WT-SOD1 was approximately 1.72 times higher than that of the double mutant. Under amyloidogenic circumstances, structural properties such as local, secondary, oligomeric, and fibrillar structures were explored. The double mutant's far-UV CD spectra displayed a broad minimum peak in the region 213 to 218 nm, suggesting the production of β-rich amyloid fibrils. FTIR spectra of the double mutant samples at different incubation times showed a low-frequency peak around 1630–1640 cm-1, attributed to a parallel β-sheet. Moreover, CR-binding assay and TEM analysis revealed and confirmed that mutation with an increased repulsive charge promotes the formation of fibrous aggregates. Consequently, ALS mutations with a higher repulsive charge are the apparent exceptions that validate the rule. This findings revealed that the double mutant increases protein aggregation through a novel mechanism, likely involving destabilization of structure and a change in the net negative charge.

Involvement of Lipids in the Pathogenesis of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons. To study its underlying mechanisms, a variety of models are currently used at the cellular level and in animals with mutations in multiple ALS associated genes, including SOD1, C9ORF72, TDP-43, and FUS. Key mechanisms involved in the disease include excitotoxicity, oxidative stress, mitochondrial dysfunction, neuroinflammatory, and immune reactions. In addition, significant metabolism alterations of various lipids classes, including phospholipids, fatty acids, sphingolipids, and others have been increasingly recognized. Recently, the mechanisms of programmed cell death (apoptosis), which may be responsible for the degeneration of motor neurons observed in the disease, have been intensively studied. In this context, sphingolipids, which are the most important sources of secondary messengers transmitting signals for cell proliferation, differentiation, and apoptosis, are gaining increasing attention in the context of ALS pathogenesis given their role in the development of neuroinflammatory and immune responses. This review describes changes in lipids content and activity of enzymes involved in their metabolism in ALS, both summarizing current evidence from animal models and clinical studies and discussing the potential of new drugs among modulators of lipid metabolism enzymes.

In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration

Amyotrophic lateral sclerosis (ALS) is an inexorably progressive and degenerative disorder of motor neurons with no currently-known cure. Studies to determine the mechanism of neurotoxicity and the impact of ALS-linked mutations (SOD1, FUS, TARDP, C9ORF72, PFN1, TUBA4A and others) have greatly expanded our knowledge of ALS disease mechanisms and have helped to identify potential targets for ALS therapy. Cellular pathologies (e.g., aggregation of mutant forms of SOD1, TDP43, FUS, Ubiqulin2, PFN1, and C9ORF72), mitochondrial dysfunction, neuroinflammation, and oxidative damage are major pathways implicated in ALS. Nevertheless, the selective vulnerability of motor neurons remains unexplained. The importance of tubulins for long-axon infrastructure, and the special morphology and function of motor neurons, underscore the central role of the cytoskeleton. The recent linkage of mutations to the tubulin α chain, TUBA4A, to familial and sporadic cases of ALS provides a new investigative opportunity to shed light on both mechanisms of ALS and the vulnerability of motor neurons. In the current study we investigate TUBA4A, a structural microtubule protein with mutations causal to familial ALS, using molecular-dynamic (MD) modeling of protein structure to predict the effects of each mutation and its overall impact on GTP binding, chain stability, tubulin assembly, and aggregation propensity. These studies predict that each of the reported mutations will cause notable structural changes to the TUBA4A (α chain) tertiary protein structure, adversely affecting its physical properties and functions. Molecular docking and MD simulations indicate certain α chain mutations (e.g. K430N, R215C, and W407X) may cause structural deviations that impair GTP binding, and plausibly prevent or destabilize tubulin polymerization. Furthermore, several mutations (including R320C and K430N) confer a significant increase in predicted aggregation propensity of TUBA4A mutants relative to wild-type. Taken together, these in silico modeling studies predict structural perturbations and disruption of GTP binding, culminating in failure to form a stable tubulin heterocomplex, which may furnish an important pathogenic mechanism to trigger motor neuron degeneration in ALS.

Cofactor-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

Impetus leading to the toxic gain of function of Cu/Zn superoxide dismutase (SOD1) whose aggregation forms the hallmark of amyotrophic lateral sclerosis (ALS) is still a controversial area. The formation of membraneless organelles such as stress granules (SGs) is believed to occur through the process of liquid-liquid phase separation (LLPS) and involves numerous proteins containing intrinsically disordered regions. Whether SOD1, which is also associated with SGs and whose aggregation is associated with Amyotrophic lateral sclerosis (ALS), can independently undergo LLPS, is not known. Using a combination of experiments and computational methods, we report here that local structural disorder in two loop regions of SOD1 induced by the absence of metal cofactor - Zn, triggers its LLPS. The liquid condensates give rise to aggregates which eventually form toxic amyloids in a longer timescale. The addition of exogenous Zn to immature, metal-free SOD1 and the disease-relate ALS mutant - I113T, stabilized the loops and restored the folded structure, thereby inhibiting LLPS and subsequent aggregation. On the contrary, the Zn-induced inhibition of phase separation and aggregation was found to be partial in the case of another severe ALS-associated mutant - G85R, which exhibits reduced Zn-binding. Moreover, a less-severe ALS mutant - G37R with perturbed Cu binding does not undergo LLPS. In this work, we want to highlight the modulation of SOD1 LLPS propensity in a Zn-dependent manner due to underlying conformational transitions between folded and partially disordered states. Our work establishes a link between SOD1 LLPS and aggregation, which is relevant to ALS pathogenesis.