Adipose mesenchymal stem cells-derived extracellular vesicles exert their preferential action in damaged central sites of SOD1 mice rather than peripherally.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.

Decoding genetic and pathophysiological mechanisms in amyotrophic lateral sclerosis and primary lateral sclerosis: A comparative study of differentially expressed genes and implicated pathways in motor neuron disorders.

Motor Neuron Disorders (MNDs), characterized by the degradation and loss of function of motor neurons, are recognized as fatal conditions with limited treatment options and no known cure. The present study aimed to identify the pathophysiological functions and affected genes in patients with MNDs, specifically Amyotrophic Lateral Sclerosis (ALS) and Primary Lateral Sclerosis (PLS). The GSE56808 dataset comprised three sample groups: six patients diagnosed with ALS (GSM1369650, GSM1369652, GSM1369654, GSM1369656, GSM1369657, GSM1369658), five patients diagnosed with PLS (GSM1369648, GSM1369649, GSM1369653, GSM1369655, GSM1369659), and six normal controls (GSM1369642, GSM1369643, GSM1369644, GSM1369645, GSM1369646, and GSM1369647). The application of computational analysis of microarray gene expression profiles enabled us to identify 346 significantly differentially expressed genes (DEGs), 169 genes for the ALS sample study, and 177 genes for the PLS sample study. Enrichment was carried out using MCODE, a Cytoscape plugin. Functional annotation of DEGs was carried out via ClueGO/CluePedia (v2.5.9) and further validated via the DAVID database. NRP2, SEMA3D, ROBO3 and, CACNB1, CACNG2 genes were identified as the gene of interest for ALS and PLS sample groups, respectively. Axonal guidance (GO:0007411) and calcium ion transmembrane transport (GO:0070588) were identified to be some of the significantly dysregulated gene ontology (GO) terms, with arrhythmogenic right ventricular cardiomyopathy (KEGG:05412) to be the top relevant KEGG pathway which is affected in MND patients. ROBO3 gene was observed to have distinctive roles in ALS and PLS-affected patients, hinting towards the differential progression of ALS from PLS. The insights derived from our comprehensive analysis accentuate the distinct variances in the underlying molecular pathogenesis of ALS and PLS. Further research should investigate the mechanistic roles of the identified DEGs and molecular pathways, leading to potential targeted therapies for ALS and PLS.

Annexin A11 aggregation in FTLD-TDP type C and related neurodegenerative disease proteinopathies.

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 protein forms inclusions in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and limbic predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Annexin A11 is also known to form aggregates in ALS cases with pathogenic variants in ANXA11. Annexin A11 aggregation has not been described in sporadic ALS, FTLD-TDP or LATE-NC cases. To explore the relationship between TDP-43 and annexin A11, genetic analysis of 822 autopsy cases was performed to identify rare ANXA11 variants. In addition, an immunohistochemical study of 368 autopsy cases was performed to identify annexin A11 aggregates. Insoluble annexin A11 aggregates which colocalize with TDP-43 inclusions were present in all FTLD-TDP Type C cases. Annexin A11 inclusions were also seen in a small proportion (3-6%) of sporadic and genetic forms of FTLD-TDP types A and B, ALS, and LATE-NC. In addition, we confirm the comingling of annexin A11 and TDP-43 aggregates in an ALS case with the pathogenic ANXA11 p.G38R variant. Finally, we found abundant annexin A11 inclusions as the primary pathologic finding in a case of progressive supranuclear palsy-like frontotemporal dementia with prominent striatal vacuolization due to a novel variant, ANXA11 p.P75S. By immunoblot, FTLD-TDP with annexinopathy and ANXA11 variant cases show accumulation of insoluble ANXA11 including a truncated fragment. These results indicate that annexin A11 forms a diverse and heterogeneous range of aggregates in both sporadic and genetic forms of TDP-43 proteinopathies. In addition, the finding of a primary vacuolar annexinopathy due to ANXA11 p.P75S suggests that annexin A11 aggregation is sufficient to cause neurodegeneration.

Comprehensive assessment of TDP-43 neuropathology data in the National Alzheimer's Coordinating Center database.

TDP-43 proteinopathy is a salient neuropathologic feature in a subset of frontotemporal lobar degeneration (FTLD-TDP), in amyotrophic lateral sclerosis (ALS-TDP), and in limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and is associated with hippocampal sclerosis of aging (HS-A). We examined TDP-43-related pathology data in the National Alzheimer's Coordinating Center (NACC) in two parts: (I) availability of assessments, and (II) associations with clinical diagnoses and other neuropathologies in those with all TDP-43 measures available. Part I: Of 4326 participants with neuropathology data collected using forms that included TDP-43 assessments, data availability was highest for HS-A (97%) and ALS (94%), followed by FTLD-TDP (83%). Regional TDP-43 pathologic assessment was available for 77% of participants, with hippocampus the most common region. Availability for the TDP-43-related measures increased over time, and was higher in centers with high proportions of participants with clinical FTLD. Part II: In 2142 participants with all TDP-43-related assessments available, 27% of participants had LATE-NC, whereas ALS-TDP or FTLD-TDP (ALS/FTLD-TDP) was present in 9% of participants, and 2% of participants had TDP-43 related to other pathologies ("Other TDP-43"). HS-A was present in 14% of participants, of whom 55% had LATE-NC, 20% ASL/FTLD-TDP, 3% Other TDP-43, and 23% no TDP-43. LATE-NC, ALS/FTLD-TDP, and Other TDP-43, were each associated with higher odds of dementia, HS-A, and hippocampal atrophy, compared to those without TDP-43 pathology. LATE-NC was associated with higher odds for Alzheimer's disease (AD) clinical diagnosis, AD neuropathologic change (ADNC), Lewy bodies, arteriolosclerosis, and cortical atrophy. ALS/FTLD-TDP was associated with higher odds of clinical diagnoses of primary progressive aphasia and behavioral-variant frontotemporal dementia, and cortical/frontotemporal lobar atrophy. When using NACC data for TDP-43-related analyses, researchers should carefully consider the incomplete availability of the different regional TDP-43 assessments, the high frequency of participants with ALS/FTLD-TDP, and the presence of other forms of TDP-43 pathology.

Shotgun Proteomics Links Proteoglycan-4+ Extracellular Vesicles to Cognitive Protection in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder lacking reliable biomarkers for early diagnosis and disease progression monitoring. This study aimed to identify the novel biomarkers in plasmatic extracellular vesicles (EVs) isolated from ALS patients and healthy controls (HCs). A total of 61 ALS patients and 30 age-matched HCs were enrolled in the study and the protein content of circulating EVs was analyzed by shotgun proteomics. The study was divided into a discovery phase (involving 12 ALS and 12 HC patients) and a validation one (involving 49 ALS and 20 HC patients). In the discovery phase, more than 300 proteins were identified, with 32 proteins showing differential regulation in ALS patients compared to HCs. In the validation phase, over 400 proteins were identified, with 20 demonstrating differential regulation in ALS patients compared to HCs. Notably, seven proteins were found to be common to both phases, all of which were significantly upregulated in EVs from ALS patients. Most of them have previously been linked to ALS since they have been detected in the serum or cerebrospinal fluid of ALS patients. Among them, proteoglycan (PRG)-4, also known as lubricin, was of particular interest since it was significantly increased in ALS patients with normal cognitive and motor functions. This study highlights the significance of EVs as a promising avenue for biomarker discovery in ALS. Moreover, it sheds light on the unexpected role of PRG-4 in relation to cognitive status in ALS patients.

Extracellular Vesicles from NSC-34 MN-like Cells Transfected with Mutant SOD1 Modulate Inflammatory Status of Raw 264.7 Macrophages.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the disruption of motor neurons (MNs), neuromuscular junction dismantling and clinical signs of ALS. Understanding the modality and the effect of MNs-macrophage communication is pivotal. Here, we focus on extracellular vesicle (EVS)-mediated communication and, in particular, we analyze the response of macrophages. NSC-34 cells transfected with mutant SOD1 (G93A, A4V, G85R, G37R) and differentiated towards MN-like cells, and Raw 264.7 macrophages are the cellular models of the study. mSOD1 NSC-34 cells release a high number of vesicles, both large-lEVs (300 nm diameter) and small-sEVs (90 nm diameter), containing inflammation-modulating molecules, and are efficiently taken up by macrophages. RT-PCR analysis of inflammation mediators demonstrated that the conditioned medium of mSOD1 NSC-34 cells polarizes Raw 264.7 macrophages towards both pro-inflammatory and anti-inflammatory phenotypes. sEVs act on macrophages in a time-dependent manner: an anti-inflammatory response mediated by TGFß firstly starts (12 h); successively, the response shifts towards a pro-inflammation IL-1ß-mediated (48 h). The response of macrophages is strictly dependent on the SOD1 mutation type. The results suggest that EVs impact physiological and behavioral macrophage processes and are of potential relevance to MN degeneration.

Protein Disulfide Isomerase Endoplasmic Reticulum Protein 57 (ERp57) is Protective Against ALS-Associated Mutant TDP-43 in Neuronal Cells.

Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Pathological forms of Tar-DNA binding protein-43 (TDP-43), involving its mislocalisation to the cytoplasm and the formation of misfolded inclusions, are present in almost all ALS cases (97%), and ~ 50% cases of the related condition, frontotemporal dementia (FTD), highlighting its importance in neurodegeneration. Previous studies have shown that endoplasmic reticulum protein 57 (ERp57), a member of the protein disulphide isomerase (PDI) family of redox chaperones, is protective against ALS-linked mutant superoxide dismutase (SOD1) in neuronal cells and transgenic SOD1G93A mouse models. However, it remains unclear whether ERp57 is protective against pathological TDP-43 in ALS. Here, we demonstrate that ERp57 is protective against key features of TDP-43 pathology in neuronal cells. ERp57 inhibited the mislocalisation of TDP-43M337V from the nucleus to the cytoplasm. In addition, ERp57 inhibited the number of inclusions formed by ALS-associated variant TDP-43M337V and reduced the size of these inclusions. ERp57 was also protective against ER stress and induction of apoptosis. Furthermore, ERp57 modulated the steady-state expression levels of TDP-43. This study therefore demonstrates a novel mechanism of action of ERp57 in ALS. It also implies that ERp57 may have potential as a novel therapeutic target to prevent the TDP-43 pathology associated with neurodegeneration.

Association Between Hypothalamic Volume and Metabolism, Cognition, and Behavior in Patients With Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: Dysfunction of energy metabolism, cognition, and behavior are important nonmotor symptoms of amyotrophic lateral sclerosis (ALS), negatively affecting survival and quality of life, but poorly understood. Neuroimaging is ideally suited to studying nonmotor neurodegeneration in ALS, but few studies have focused on the hypothalamus, a key region for regulating energy homeostasis, cognition, and behavior. We evaluated, therefore, hypothalamic neurodegeneration in ALS and explored the relationship between hypothalamic volumes and dysregulation of energy metabolism, cognitive and behavioral changes, disease progression, and survival. METHODS: Patients with ALS and population-based controls were included for this cross-sectional and longitudinal MRI study. The hypothalamus was segmented into 5 subregions and their volumes were calculated. Linear (mixed) models, adjusted for age, sex, and total intracranial volume, were used to compare hypothalamic volumes between groups and to analyze associations with metabolism, cognition, behavior, and disease progression. Cox proportional hazard models were used to investigate the relationship of hypothalamic volumes with survival. Permutation-based corrections for multiple hypothesis testing were applied to all analyses to control the family-wise error rate. RESULTS: Data were available for 564 patients with ALS and 356 controls. The volume of the anterior superior subregion of the hypothalamus was smaller in patients with ALS than in controls (ß = -0.70 [-1.15 to -0.25], p = 0.013). Weight loss, memory impairments, and behavioral disinhibition were associated with a smaller posterior hypothalamus (ß = -4.79 [-8.39 to -2.49], p = 0.001, ß = -10.14 [-15.88 to -4.39], p = 0.004, and ß = -12.09 [-18.83 to -5.35], p = 0.003, respectively). Furthermore, the volume of this subregion decreased faster over time in patients than in controls (ß = -0.25 [0.42 to -0.09], p = 0.013), and a smaller volume of this structure was correlated with shorter survival (hazard ratio = 0.36 [0.21-0.61], p = 0.029). DISCUSSION: We obtained evidence for hypothalamic involvement in ALS, specifically marked by atrophy of the anterior superior subregion. Moreover, we found that atrophy of the posterior hypothalamus was associated with weight loss, memory dysfunction, behavioral disinhibition, and survival, and that this subregion deteriorated faster in patients with ALS than in controls. These findings improve our understanding of nonmotor involvement in ALS and could contribute to the identification of new treatment targets for this devastating disease.

The polyglutamine protein ATXN2: from its molecular functions to its involvement in disease.

A CAG repeat sequence in the ATXN2 gene encodes a polyglutamine (polyQ) tract within the ataxin-2 (ATXN2) protein, showcasing a complex landscape of functions that have been progressively unveiled over recent decades. Despite significant progresses in the field, a comprehensive overview of the mechanisms governed by ATXN2 remains elusive. This multifaceted protein emerges as a key player in RNA metabolism, stress granules dynamics, endocytosis, calcium signaling, and the regulation of the circadian rhythm. The CAG overexpansion within the ATXN2 gene produces a protein with an extended poly(Q) tract, inducing consequential alterations in conformational dynamics which confer a toxic gain and/or partial loss of function. Although overexpanded ATXN2 is predominantly linked to spinocerebellar ataxia type 2 (SCA2), intermediate expansions are also implicated in amyotrophic lateral sclerosis (ALS) and parkinsonism. While the molecular intricacies await full elucidation, SCA2 presents ATXN2-associated pathological features, encompassing autophagy impairment, RNA-mediated toxicity, heightened oxidative stress, and disruption of calcium homeostasis. Presently, SCA2 remains incurable, with patients reliant on symptomatic and supportive treatments. In the pursuit of therapeutic solutions, various studies have explored avenues ranging from pharmacological drugs to advanced therapies, including cell or gene-based approaches. These endeavours aim to address the root causes or counteract distinct pathological features of SCA2. This review is intended to provide an updated compendium of ATXN2 functions, delineate the associated pathological mechanisms, and present current perspectives on the development of innovative therapeutic strategies.

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS).

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

Multiomic ALS signatures highlight subclusters and sex differences suggesting the MAPK pathway as therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.

Chronological and Biological Aging in Amyotrophic Lateral Sclerosis and the Potential of Senolytic Therapies.

Amyotrophic Lateral Sclerosis (ALS) is a group of sporadic and genetic neurodegenerative disorders that result in losses of upper and lower motor neurons. Treatment of ALS is limited, and survival is 2-5 years after disease onset. While ALS can occur in younger individuals, the risk significantly increases with advancing age. Notably, both sporadic and genetic forms of ALS share pathophysiological features overlapping hallmarks of aging including genome instability/DNA damage, mitochondrial dysfunction, inflammation, proteostasis, and cellular senescence. This review explores chronological and biological aging in the context of ALS onset and progression. Age-related muscle weakness and motor unit loss mirror aspects of ALS pathology and coincide with peak ALS incidence, suggesting a potential link between aging and disease development. Hallmarks of biological aging, including DNA damage, mitochondrial dysfunction, and cellular senescence, are implicated in both aging and ALS, offering insights into shared mechanisms underlying disease pathogenesis. Furthermore, senescence-associated secretory phenotype and senolytic treatments emerge as promising avenues for ALS intervention, with the potential to mitigate neuroinflammation and modify disease progression.

From Environment to Gene Expression: Epigenetic Methylations and One-Carbon Metabolism in Amyotrophic Lateral Sclerosis.

The etiology of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) is complex and considered multifactorial. The majority of ALS cases are sporadic, but familial cases also exist. Estimates of heritability range from 8% to 61%, indicating that additional factors beyond genetics likely contribute to ALS. Numerous environmental factors are considered, which may add up and synergize throughout an individual's lifetime building its unique exposome. One level of integration between genetic and environmental factors is epigenetics, which results in alterations in gene expression without modification of the genome sequence. Methylation reactions, targeting DNA or histones, represent a large proportion of epigenetic regulations and strongly depend on the availability of methyl donors provided by the ubiquitous one-carbon (1C) metabolism. Thus, understanding the interplay between exposome, 1C metabolism, and epigenetic modifications will likely contribute to elucidating the mechanisms underlying altered gene expression related to ALS and to developing targeted therapeutic interventions. Here, we review evidence for 1C metabolism alterations and epigenetic methylation dysregulations in ALS, with a focus on the impairments reported in neural tissues, and discuss these environmentally driven mechanisms as the consequences of cumulative exposome or late environmental hits, but also as the possible result of early developmental defects.

M6A reduction relieves FUS-associated ALS granules.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m6A downregulation. Notably, cells expressing mutant FUS were characterized by higher m6A levels suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

Arctigenin derivative A-1 ameliorates motor dysfunction and pathological manifestations in SOD1G93A transgenic mice via the AMPK/SIRT1/PGC-1α and AMPK/SIRT1/IL-1ß/NF-κB pathways.

AIM: Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by progressive death of upper and lower motor neurons, leading to generalized muscle atrophy, paralysis, and even death. Mitochondrial damage and neuroinflammation play key roles in the pathogenesis of ALS. In the present study, the efficacy of A-1, a derivative of arctigenin with AMP-activated protein kinase (AMPK) and silent information regulator 1 (SIRT1) activation for ALS, was investigated. METHODS: A-1 at 33.3 mg/kg was administrated in SOD1G93A transgenic mice orally from the 13th week for a 6-week treatment period. Motor ability was assessed before terminal anesthesia. Muscle atrophy and fibrosis, motor neurons, astrocytes, and microglia in the spinal cord were evaluated by H&E, Masson, Sirius Red, Nissl, and immunohistochemistry staining. Protein expression was detected with proteomics analysis, Western blotting, and ELISA. Mitochondrial adenosine triphosphate (ATP) and malondialdehyde (MDA) levels were measured using an assay kit. RESULTS: A-1 administration in SOD1G93A mice enhanced mobility, decreased skeletal muscle atrophy and fibrosis, mitigated loss of spinal motor neurons, and reduced glial activation. Additionally, A-1 treatment improved mitochondrial function, evidenced by elevated ATP levels and increased expression of key mitochondrial-related proteins. The A-1 treatment group showed decreased levels of IL-1ß, pIκBα/IκBα, and pNF-κB/NF-κB. CONCLUSIONS: A-1 treatment reduced motor neuron loss, improved gastrocnemius atrophy, and delayed ALS progression through the AMPK/SIRT1/PGC-1α pathway, which promotes mitochondrial biogenesis. Furthermore, the AMPK/SIRT1/IL-1ß/NF-κB pathway exerted neuroprotective effects by reducing neuroinflammation. These findings suggest A-1 as a promising therapeutic approach for ALS.

Revisiting Glutamate Excitotoxicity in Amyotrophic Lateral Sclerosis and Age-Related Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. 'Primary pathways' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. 'Secondary pathways' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.

MATR3's Role beyond the Nuclear Matrix: From Gene Regulation to Its Implications in Amyotrophic Lateral Sclerosis and Other Diseases.

Matrin-3 (MATR3) was initially discovered as a component of the nuclear matrix about thirty years ago. Since then, accumulating studies have provided evidence that MATR3 not only plays a structural role in the nucleus, but that it is also an active protein involved in regulating gene expression at multiple levels, including chromatin organization, DNA transcription, RNA metabolism, and protein translation in the nucleus and cytoplasm. Furthermore, MATR3 may play a critical role in various cellular processes, including DNA damage response, cell proliferation, differentiation, and survival. In addition to the revelation of its biological role, recent studies have reported MATR3's involvement in the context of various diseases, including neurodegenerative and neurodevelopmental diseases, as well as cancer. Moreover, sequencing studies of patients revealed a handful of disease-associated mutations in MATR3 linked to amyotrophic lateral sclerosis (ALS), which further elevated the gene's importance as a topic of study. In this review, we synthesize the current knowledge regarding the diverse functions of MATR3 in DNA- and RNA-related processes, as well as its involvement in various diseases, with a particular emphasis on ALS.

MS785-MS27 Reactive Misfolded/Non-Native Zn-Deficient SOD1 Species Exhibit Cytotoxicity and Adopt Heterozygous Conformations in Motor Neurons.

Misfolding of superoxide dismutase-1 (SOD1) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) with SOD1 mutations. The development of antibodies specific for misfolded SOD1 deepens our understanding of how the protein participates in ALS pathogenesis. Since the term "misfolding" refers to various disordered conformers other than the natively folded one, which misfolded species are recognized by specific antibodies should be determined. Here, we molecularly characterized the recognition by MS785-MS27, an antibody cocktail experimentally confirmed to recognize over 100 ALS-linked SOD1 mutants. Indirect ELISA revealed that the antibody cocktail recognized Zn-deficient wild-type and mutated SOD1 species. It also recognized conformation-disordered wild-type and mutated SOD1 species, such as unfolded and oligomeric forms, but had less affinity for the aggregated form. Antibody-reactive SOD1 exhibited cytotoxicity to a motor neuron cell model, which was blocked by Zn treatment with Zn-deficient SOD1. Immunohistochemistry revealed antibody-reactive SOD1 mainly in spinal motor neurons of SOD1G93A mice throughout the disease course, and the distribution after symptomatic stages differed from that of other misfolded SOD1 species. This suggests that misfolded/non-native SOD1 species exist as heterogeneous populations. In conclusion, MS785-MS27 recognizes various conformation-disordered SOD1 species lacking the Zn ion.

A Cellular Model of Amyotrophic Lateral Sclerosis to Study the Therapeutic Effects of Extracellular Vesicles from Adipose Mesenchymal Stem Cells on Microglial Activation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to progressive paralysis and death. Increasing evidence indicates that neuroinflammation plays an important role in ALS's pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate/accelerate MN death. In particular, the role of the microglia in ALS remains unclear, partly due to the lack of experimental models that can fully recapitulate the complexity of ALS's pathology. In this study, we developed and characterized a microglial cell line, SIM-A9-expressing human mutant protein Cu+/Zn+ superoxide dismutase_1 (SIM-A9hSOD1(G93A)), as a suitable model in vitro mimicking the microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells induced a change in their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which is known to activate cell death processes and apoptosis. Afterward, we used our microglial model as an experimental set-up to investigate the therapeutic action of extracellular vesicles isolated from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represent a promising therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. Here, we demonstrated that treatment with ASC-EVs is able to modulate activated ALS microglia, reducing their metabolic activity and polarizing their phenotype toward an anti-inflammatory one through a mechanism of reduction of reactive oxygen species.

cAMP/PKA signaling regulates TDP-43 aggregation and mislocalization.

Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.