oligonucleotides

2025-12-22 | SOD1 mutations in Taiwanese ALS patients: Clinical characteristics, frequency, and a p.T138R founder effect

Objective Mutations in SOD1 are a well-established genetic cause of amyotrophic lateral sclerosis (ALS), exerting toxic gain-of-function effects that promote protein misfolding and aggregation in motor neurons and glial cells. The emergence of SOD1-targeted antisense oligonucleotide therapy underscores the clinical importance of precise genetic diagnosis. This study aimed to determine the frequency, clinical characteristics, and potential founder effect of SOD1 mutations in a large Taiwanese ALS cohort, and to evaluate their aggregation propensity in vitro.Methods All coding exons of SOD1 were analyzed by Sanger sequencing in 650 unrelated Taiwanese patients with ALS. Haplotype analysis using single nucleotide polymorphism markers flanking SOD1 was conducted to assess a potential founder effect. Protein cross-linking assays were performed to assess the aggregation propensity of 11 SOD1 variants.Results Seventeen pathogenic SOD1 variants were identified in 26 probands and 12 affected relatives. Mean age at onset was 48.9 ± 14.9 years, and 8% had bulbar-onset ALS. The most frequent variant was p.T138R (8 probands), followed by p.G11A (3 probands). The other 15 variants each occurred in a single family. A shared ancestral haplotype was observed among p.T138R carriers. Cross-linking experiments demonstrated oligomer formation in all tested mutant SOD1 proteins compared to the wild-type protein, supporting their pathogenicity.Conclusions SOD1 mutations account for approximately 4% of ALS cases in Taiwan, are associated with earlier onset and predominantly spinal-onset ALS, and include a p.T138R founder variant. These findings highlight the importance of genetic screening in ALS, particularly in guiding eligibility for emerging targeted therapies.

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2025-11-06 | RNA-Focused DNA-Encoded Library Construction, Screening, and Integration of Docking Identify Bioactive Ligands of Pathogenic r(G 4 C 2 ) exp RNA

Abstract Disease-associated RNAs are increasingly recognized as promising therapeutic targets for small-molecule intervention. While DNA-encoded libraries (DELs) have long been established for protein ligand discovery, recent studies have demonstrated their feasibility for identifying RNA-binding small molecules. To further advance RNA-targeted ligand discovery, a diverse, solid-phase DEL enriched in privileged RNA-binding scaffolds was constructed and applied to identify ligands of r(G 4 C 2 ) exp , a toxic RNA repeat expansion implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). DEL selection outcomes were analyzed through large-scale molecular docking integrated with physicochemical and structure-activity relationship (SAR) analyses. Strong correlations were observed between docking predictions and experimental enrichment trends, supporting lead identification. The lead compound was subsequently optimized based on its docked pose to an NMR structure, resulting in analogs with enhanced binding affinity and bioactivity. These findings demonstrate that RNA ligand identification can be effectively achieved by combining DNA-encoded library technology with computational approaches for rational design and analysis, and highlight a broadly adaptable platform for RNA-targeted small molecule discovery. TOC graphic

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2025-06-19 | An Advancements In Treatment Of Amyotrophic Lateral Sclerosis (ALS)

Introduction and Objective: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the loss of motor neurons. Each year, amyotrophic lateral sclerosis is diagnosed in 4-5 people per 100,000 population. The disease is commonly known as Lou-Gehrig's disease after the famous American baseball player who died two years after receiving the diagnosis at the age of 39. The etiology of the disease is not fully understood, but gene mutations that contribute to the development of ALS have been identified. Such mutations include the gene encoding superoxide dismutase type 1 (SOD1), C9orf72, TBK1 and FUS125, among others. Since the first SOD1 mutation was identified as the genetic cause of SLA, there has been a significant milestone in clinical research aimed at finding a cure for this terminal and dreaded disease. In this article, we will discuss the early symptoms of ALS, factors that increase the risk of developing the disease and, most important, the knowledge and latest treatments, such as the use of antisense oligonucleotides (ASOs). Aim of Study: This article presents up-to-date treatments for the neurodegenerative disease amyotrophic lateral sclerosis, focusing on recent clinical trials and their potential therapeutic targets. We will also review interdisciplinary treatments such as mechanical ventilation and pain management. We will also discuss factors that increase the risk of developing this incurable disease. Materials and methods: For this review article, databases such as PubMed and Google Scholar were searched. The search terms used to find relevant scientific articles included: Amyotrophic Lateral Sclerosis, ALS treatment, Tofersen, Riluzole. Ultimately, 35 research articles were cited.

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2024-03-01 | Abstract 2166 RNA Aptamer Selection Against SOD1 to Create a Therapeutic for ALS Patients

Diagnoses are on the rise for the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) and current treatments do little to extend life, creating a dire forecast that punctuates the need for effective ALS treatments. ALS affects approximately 200,000 people globally (ALS Society of Canada) and cases are predicted to increase by 69% between 2015 and 2040 (Nature Communications). There are only 7 drugs that treat ALS, most of which only improve life expectancy by a few months (ALS Association). One of these drugs, Toferson, attacks the mutated form of a protein whose overexpression in the brain is linked to ALS: Superoxide Dismutase 1 (SOD1). In the disease state, mutant SOD1 leads to accumulations or inclusion bodies of proteins in the brain. Although originally only aggregates of mutated SOD1 were associated with ALS, it is now understood that aggregates of wild-type SOD1 can also accelerate its progression. This work seeks to develop an ALS treatment that dispels overexpressed wild-type SOD1, dismantling its aggregates, by introducing anti-SOD1 aptamers. Aptamers are short, single-stranded RNA molecules that can selectively bind to a specific target, such as SOD1, and are identified through a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). SELEX involves iterative rounds of selection, amplification, and purification. These steps narrow a large pool of random RNA sequences to those that bind SOD1. An aptamer that binds wild-type SOD1 protein will be used to create an oligonucleotide complex which will act as a delivery mechanism for the enzyme ubiquitin. The complex will form a bridge between the aptamer and ubiquitin. Because the complex connects them, when the aptamer binds to an aggregate of wild-type SOD1 protein, ubiquitin will be delivered to that aggregate. The ubiquitin coming into contact with the aggregates will initiate aggrephagy, a type of autophagy that degrades protein aggregates. Because ubiquitin tags proteins in the body for degradation, the use of this complex will result in the body degrading the aggregates of wild-type SOD1 protein in the brain. In order to prevent the aptamer from binding to SOD1 monomers, additional rounds of SELEX will be performed to increase the aptamers' binding affinity specifically to SOD1 aggregates as opposed to SOD1 monomers. Currently, 3 full rounds of SELEX have been completed, as well as the early phases of round 4. The results of selection will be cloned and sequenced after the fifth or sixth round of selection in order to determine if a viable aptamer against SOD1 has been found. If an appropriately stringent aptamer against SOD1 is found, progress can begin on building the oligonucleotide complex to create the proposed therapeutic for ALS. The main objective of this research is to utilize SELEX to find an aptamer against SOD1 for the purpose of creating a ubiquitin-delivery system to combat the negative effects of excess wild-type SOD1 in the brains of ALS patients. Because this method would target aggregates of wild-type SOD1, when used in combination with a drug like Toferson, which limits the harmful effects of aggregates of mutated SOD1, it could decrease the rate of progression of ALS by attacking ALS symptoms from two fronts. Finding this anti-SOD1 aptamer and creating an oligonucleotide complex to use in a ubiquitin delivery-system may advance ALS treatment significantly and improve the health outcomes of the hundreds of thousands of patients afflicted with this disease.

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2023-08-14 | Genetic Downregulation of the Metabotropic Glutamate Receptor Type 5 Dampens the Reactive and Neurotoxic Phenotype of Adult ALS Astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.

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small molecules

2026-07-28 | R-loops: Biological functions, regulatory mechanisms, and therapeutic implications in brain diseases-A review.

R-loops are three-stranded nucleic acid structures formed by a DNA-RNA hybrid and a displaced single-stranded DNA. They regulate transcription, replication, and DNA repair, but their dysregulation causes genomic instability and inflammation, contributing to brain diseases. The nervous system exhibits selective vulnerability to R-loop stress due to ultra-long gene transcription, post-mitotic longevity, and high metabolic demands. This review synthesizes current literature from PubMed, Scopus, Web of Science, and Embase (2010-2026) on R-loop biology, with a focus on brain-specific mechanisms, regulatory factors (SETX, ZPR1, METTL3, TDP-43/FUS), and disease models. In neurodegeneration, R-loop accumulation drives repeat expansion disorders (Fragile X, Huntington's disease) and loss-of-function SETX mutations (AOA2), whereas gain-of-function SETX (L389S) causes pathological R-loop depletion in ALS4, disrupting TGF-β signaling. TDP-43/FUS and SMN are integral to R-loop resolution, unifying ALS/FTD and SMA. In brain cancers, METTL3-mediated m6A modification of TERRA stabilizes telomeric R-loops in ALT-positive neuroblastoma, creating a therapeutic vulnerability to METTL3 inhibitors (STM2457, STC-15). Glioma stem cells rely on m6A-modified circPOLR2B to regulate R-loop formation and malignancy. Clinical-stage agents (EP102, TUG1ASO, ATX-559) and R-loop-derived prognostic signatures (RLPI) are emerging, but translation is hindered by a lack of non-invasive biomarkers and the dual physiological/pathological roles of R-loops. R-loops are central to brain disease pathogenesis, offering promising therapeutic targets. Future research should prioritize precision R-loop modulators, non-invasive biomarkers, and combinatorial strategies.

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2026-07-01 | RNA dysregulation and compromised neuronal identity drive pathogenesis in Senataxin-associated ALS

ABSTRACT RNA dysregulation is a recognized contributor to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the most common motor neuron (MN) disease. However, the molecular mechanisms linking defects in RNA metabolism to selective neuronal vulnerability remain poorly understood. Alterations in the cellular levels of R-loops –structures forming by reannealing of the nascent RNA with the template DNA during transcription- have been observed in neurodegeneration, but it is unclear how perturbations in R-loop homeostasis contribute to neuronal dysfunction. Here we investigate the molecular basis of a juvenile form of ALS dubbed ALS4 that is caused by mutations in the helicase SETX, which plays important roles in the resolution of R-loops and transcription termination. Using isogenic human induced pluripotent stem cell-derived MNs, we show that ALS4-associated SETX mutations induce progressive axonal defects and widespread transcriptomic alterations, including reduced expression or altered splicing of transcripts critical for neuronal function. ALS4 MNs exhibit a transcriptional signature marked by cellular stress, aberrant cell cycle re-entry, and compromised neuronal identity that is partially shared by other forms of ALS. Mechanistically, these defects are partly driven by downstream aberrant activation of the TGF-β signaling pathway, whose pharmacological inhibition ameliorates axonal defects. Finally, our analyses support a link between mutant SETX ectopic activity at R-loops and the observed alterations in RNA expression and splicing, providing new insights into how RNA dysregulation can drive neuronal dysfunction Altogether, our work reveals how perturbations at the interface of transcription and R-loop metabolism can reshape neuronal identity and drive disease. Teaser Deregulation of TGF-β signaling drives axonal defects and compromised motor neuron identity in senataxin-mediated ALS

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2025-10-07 | Glial cell-intrinsic and non-cell autonomous toxicity in a Drosophila C9orf72 neurodegeneration model

The most common genetic cause of both familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G4C2 repeat in the first intron of the gene C9orf72. The C9orf72 repeat expansion is bidirectionally transcribed into sense and anti-sense RNA foci, and also produces dipeptide repeats (DPRs) via a non-canonical translation mechanism known as repeat-associated (RAN) translation. Each of these components of the G4C2 repeat expansion cause neurodegenerative effects in animal models when expressed in neurons, but impacts from glial expression are more poorly understood. Here, we use glial cell type-specific expression of individual DPRs, of RNA repeat-only, or of the G4C2 repeat that is capable of producing both DPRs and RNA repeats to systematically investigate both the glial cell-intrinsic and non-cell autonomous toxicity of each of these components. Our results show that as with neurons, the GR and G4C2 transgenes, produce the highest degree of cell-intrinsic toxicity when expressed in glia. Both of these transgenes are capable of producing the GR DPR, which is also typically found to be the most toxic factor in neurons. We demonstrate that both the GR and G4C2 transgenes cause activation of mdg4, an endogenous retrovirus (ERV). Such ERV expression is a hallmark of TDP-43 dysfunction that is commonly observed in C9orf72 patients and contributes to both cell intrinsic and non-cell autonomous toxicity. We find that only the G4C2 transgene produces measurable non-cell autonomous effects that result in loss of nearby neurons. But manipulations of apoptosis reveal non-cell autonomous or systemic effects from either GR or G4C2 expressing glia. Blocking apoptotic cell death of either GR or G4C2 expressing glia via the p35 caspase inhibitor further exacerbates effects on lifespan and ablating such glia via expression of the proapoptotic reaper gene partially ameliorates these effects.

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2025-07-31 | Repurposing FDA-approved drugs for treatment of amyotrophic lateral sclerosis using machine learning

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons. Current medications are largely ineffective, associated with side effects, and hindered by a lack of agreement over treatment pathways. The time-intensive process and high costs further limit the development of therapeutics. Therefore, this research aimed to identify FDA-approved drugs that inhibit three proteins (Casein kinase 1, Protein tyrosine kinase 2, Ephrin type-A receptor 4) associated with ALS. A machine learning (ML) model was trained for each protein to identify an inputted compound as an active inhibitor of that protein. The FDA-approved drugs were then screened through these models, and 18 drugs were identified as likely inhibitors for all three proteins. The results were validated through protein-ligand docking of each drug to its respective protein(s). Risperidone was the most active drug, with an average ML score of 1 and binding affinity of -8.9. The ML scores and binding affinities had a strong correlation, indicating reliability. This research predicted multiple drugs that can simultaneously target many proteins involved in ALS, creating more effective treatment options at a lower cost. This procedure can be applied to efficiently discover drugs for other diseases in the future.

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2025-03-01 | Monoaminergic Alterations at the Subregional Cervical and Thoracic Spinal Cord Level of Patients Within the FTD ‐ ALS Continuum and Early‐Onset AD : Low Thoracic Dopaminergic Activity in ALS

ABSTRACT Early‐onset neurodegeneration leads to cognitive and behavioral symptoms in frontotemporal dementia (FTD) and motor disturbances in amyotrophic lateral sclerosis (ALS). Despite distinct clinical profiles, more than half of FTD patients experience ALS‐related symptoms and vice versa. Spinal cord monoamine neurotransmitter alterations were reported in ALS, but not yet in FTD. Therefore, we compared monoaminergic turnover across the FTD–ALS continuum. Reversed‐phase, ultra‐high‐performance liquid chromatography with electrochemical detection was used to measure levels of the monoamines (nor)adrenaline ((N)A), dopamine (DA) and serotonin (5‐hydroxytryptamine, 5‐HT) and their metabolites 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA) in five cervical and thoracic spinal cord regions in 10 FTD, 14 ALS, 6 mixed FTD–ALS, 14 early‐onset Alzheimer's disease (EOAD), and 7 control (CONTR) individuals. At the cervical level, NA levels were lower in FTD‐ALS versus CONTR, whereas the HVA/5‐HIAA ratio was higher in ALS versus EOAD in the lateral funiculus. In the dorsal horn–intermediate gray matter, DA levels were decreased in FTD‐ALS compared to FTD. At the thoracic level, DOPAC was lower in ALS than in FTD‐ALS patients in the ventral and lateral funiculus, ventral horn, and dorsal horn–intermediate gray matter, as was the DOPAC/DA ratio in the lateral funiculus and dorsal horn–intermediate gray matter. Contrarily, HVA/DA turnover was lower in FTD‐ALS than in FTD in the dorsal and ventral funiculus. We observed lower NA levels in FTD‐ALS than in FTD in the ventral funiculus, and lower MHPG/NA turnover in the dorsal horn–intermediate gray matter. A levels were lower in ALS versus FTD. This study indicates differences in monoaminergic turnover across the FTD‐ALS continuum, at the cervical and thoracic levels, with primarily a decrease in dopaminergic activity in ALS. Characterizing disease‐specific neurochemical profiles for FTD, ALS, or FTD‐ALS could contribute to the identification of novel interesting pharmacological targets. image

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proteins

2026-03-03 | Disruption of the angiopoietin-like system connects lipid homeostasis and hypothalamic dysfunction in ALS

BACKGROUND: Alterations in lipid metabolism are manifestations of amyotrophic lateral sclerosis (ALS) that contribute to the risk and rate of progression. Blood levels of triglycerides and cholesterol are altered in ALS patients and pre-symptomatic gene carriers, but mechanistic insights into these changes are lacking. METHODS: murine model was studied at pre-symptomatic (P50), early symptomatic (P90), and fully symptomatic (P110) stages, along with their wild-type (WT) littermates for ANGPTLs. Untargeted lipidomics on serum was performed using high-resolution liquid chromatography-mass spectrometry. Further, the involvement of the hypothalamus was studied using hypothalamic volumetry in patients and an antibody array spanning 308 proteins in mice. RESULTS: We show that mutation-specific patterns of systemic lipid abnormalities appear in ALS and that they correlate with reduced levels of angiopoietin-like proteins 3 and 4. ANGPTL-3/4, in turn, correlates with hypothalamic atrophy but not with corticospinal involvement, as determined by MRI volumetry and diffusion tensor imaging. Lipid phenotype and decreased ANGPTL in humans are recapitulated in two SOD1 murine ALS models, in which ANGPTL-3, -4, and -8 expression patterns are consistent with the repartitioning of lipid utilisation from muscles to the brown adipose tissue; systemic levels of ANGPTL-3 correlate with hypothalamic neuroinflammation and vascular permeability and with hypothalamic levels of agouti-related protein and neuropeptide Y. CONCLUSIONS: These data provide a molecular mechanism linking peripheral lipid metabolism to the dysfunction of a specific hypothalamic circuit through the mediation of systemic ANGPTL-3 and -4. This finding constitutes a molecularly defined entry point to manipulate lipid metabolism in ALS.

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2026-01-08 | Immune Modulation Through Directed Th2 Polarization: Programming Naïve T Cells to Suppress Autoimmune Pathways in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron loss, neuroinflammation, and immune dysregulation. Increasing evidence demonstrates that a peripheral and central shift toward proinflammatory Th1 and Th17 immune responses correlate with disease severity and progression. In contrast, T helper type-2 (Th2)–associated immune responses exert neuroprotective and anti-inflammatory effects through cytokine-mediated suppression of pathogenic autoimmunity and modulation of microglial activity. This review positions immune modulation as a programmable therapeutic strategy, in which naïve CD4⁺ T cells (Th0) are actively biased toward a Th2 phenotype through cytokine conditioning and controlled induction of atopic immune signaling. Forced atopy establishes a Type 2–dominant cytokine microenvironment rich in IL-4, IL-10, IL-13, and thymic stromal lymphopoietin (TSLP), thereby suppressing Th1 differentiation, inhibiting IFN-γ–driven neurotoxicity, and reshaping immune–glial crosstalk. Alleamit’s patented hyperallergenic platform provides a mechanistically grounded method for inducing sustained Th2 polarization while artificial intelligence–assisted immune monitoring can enhance safety, personalization, and therapeutic precision. Collectively, this framework supports immune reprogramming as a viable disease-modifying strategy in ALS and establishes a foundation for translational development.

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2025-07-01 | Botulinum toxin type A for amyotrophic lateral sclerosis lower limb spasm: two case reports

Patients with amyotrophic lateral sclerosis (ALS) often experience spasticity, which can severely affect their ability to perform basic activities like standing and walking, potentially diminishing their already compromised quality of life. Botulinum toxin type A (BTX-A) is a first-line drug for spastic management. However, there are limited reports on its effectiveness in reducing muscle tone among ALS patients, with scarcely any related research conducted in China. We conducted the clinical observation and follow-up study through the relevant ethical post (ChiCTR2200061794). Clinical registration was on July 2, 2022. All participants provided written informed consent. We report two cases of middle-aged male patients, both diagnosed with ALS, who presented with symptoms such as limb stiffness and walking limitation due to increased muscle tone in the lower limbs. Based on the spasticity of the patient's lower limbs, the corresponding target muscles were selected for BTX-A treatment under ultrasound guidance, and the patients were evaluated on relevant functional scales before injection (baseline, T0) and at three follow-up visits (T1: 2 weeks, T2: 4 weeks, T3: 8 weeks). Appropriate BTX-A injected into the target muscles could effectively depress the spasticity of ALS patients without apparent side effects.

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2025-06-01 | Overexpression of DnaJ chaperones in ameliorating toxicities associated with FUS and TDP-43

Abstract Amyotrophic Lateral Sclerosis (ALS) is an adult-onset neurodegenerative disease. It primarily affects the motor neurons, leading to muscle weakness and eventually death of the patients. Various factors, such as genetics, environment, age, etc, are involved in the etiopathogenesis of ALS. As ALS is a highly challenging and complex disease involving various pathogeneses linked with progressive motor neuron degeneration, it is difficult to have a single therapeutic target against this disease. ALS mutants of TDP-43 and FUS are showing slow growth as well as protein aggregation in the cytoplasm in yeast. Our study involves cloning of 38 Drosophila DnaJ domain chaperones and overexpressing them in a yeast ALS model. After the interaction of mutants TDP-43 and FUS with the DnaJ domain chaperones, they were categorized into suppressors and enhancers. The major hits on our screen were CG7872, Hsc70-4, and Mrj, which rescued TDP-43 toxicity by reducing protein aggregation and slow growth in yeast cells. In FUS, slow growth and protein aggregation are reduced with the chaperones CG32641 and CG6693, resulting in a lowering of FUS toxicity. On the other hand, chaperones CG2911, CG2790, and CG8476 in presence of TDP-43 overexpression, grows slower with an increase in the number of aggregates in yeast cells. These chaperones increase the toxicity of TDP-43. Similarly, in FUS, Hsc20, CG7130, CG2911, and JdpC also showed slow growth phenotype and an increase in protein aggregation in yeast cells. These chaperones enhance the toxicity of FUS. This screening will enhance our understanding of the ALS pathology, and the chaperones identified in this study can serve as neuroprotective agents for ALS. Graphical abstract

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2024-12-19 | Neuroprotective effects of activated fibroblast growth factor receptor 1 via the suppression of p53 accumulation against poly-PR-mediated toxicity.

A GGGGCC hexanucleotide repeat expansion (HRE) within the C9orf72 gene is a major causative factor in amyotrophic lateral sclerosis (ALS). This aberrant HRE results in the generation of five distinct dipeptide repeat proteins (DPRs). Among the DPRs, poly-PR accumulates in the nucleus and exhibits particularly strong toxicity to motor and cortical neurons. Fibroblast growth factor receptor 1 (FGFR1) is known to promote neurogenesis and inhibit apoptosis in neurons. Nevertheless, there has been no previous report of its neuroprotective effects against poly-PR toxicity. The objective of this study was to investigate the neuroprotective effects of FGFR1 activation in poly-PR-expressing NSC34 motor neuron-like cells. RT-qPCR analysis in NSC34 cells showed that Fgfr1 was the most highly expressed member of the Fgfr family in NSC34 cells. The activation of FGFR1 by FGF2, a common ligand for all FGFRs, exerted neuroprotective effects against the toxicity of poly-PR. Additionally, FGFR1 activation was observed to enhance cell viability through the PI3K-AKT pathway, while the contribution of the MEK-ERK pathway was found to be limited. Furthermore, FGFR1 activation suppressed the accumulation of p53 protein and promoted its degradation through increased murine double minute 2 (MDM2), an E3 ubiquitin ligase that targets p53. The neuroprotective effects were attenuated by PD173074, a selective FGFR1 inhibitor or Nutlin-3a, an inhibitor of the p53-MDM2 interaction. Overall, these findings suggest that FGFR1 activation provides neuroprotection against poly-PR toxicity. Consequently, this study suggests the potential utility of FGFR1 activation as a therapeutic strategy for ALS.

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cell therapies

2025-12-01 | Immediate Effects of Adipose‐Derived Mesenchymal Stem Cell Administration on Cholinesterase Levels and Grip Strength

BACKGROUND: Immediate improvements in subjective and objective symptoms can sometimes be observed following the administration of adipose-derived mesenchymal stem cells (ADSCs). This rapid improvement was surprising, as we had assumed that regenerative medicine would require several days or more to show effects. Therefore, we measured grip strength and cholinesterase values before and after treatment. ADSCs have gained attention in regenerative medicine due to their multipotent nature and potential therapeutic effects. METHOD: We conducted a prospective observational study on 21 patients who received ADSC treatment. Informed consent was obtained from all participants. Serum cholinesterase levels and grip strength were measured immediately before and after ADSC administration. Grip strength was measured on the non-infusion side, maintaining the same posture for both pre- and post-treatment measurements. RESULT: The study included patients with various conditions: Parkinson's disease (PD, n=9), Amyotrophic Lateral Sclerosis (ALS, n=4), Alzheimer's disease (n=2), Chronic Obstructive Pulmonary Disease (COPD, n=4), and Progressive Supranuclear Palsy (PSP, n=1). The mean age was 67.5 years (range: 53-81), with 14 males and 7 females. A paired samples t-test revealed a statistically significant decrease in cholinesterase levels after ADSC administration (mean difference: -21.905 U/L, p < 0.001). Although not statistically significant, there was a trend towards improvement in grip strength (mean difference: 1.0619 kg, p = 0.119). CONCLUSION: Our findings demonstrate a significant immediate decrease in serum cholinesterase levels following ADSC administration. The observed decrease in cholinesterase levels might indicate an enhancement of acetylcholine activity, potentially explaining the trend towards improved grip strength. The immediate effects observed in this study are intriguing, as they suggest that ADSCs may have rapid neuromodulatory effects beyond their known regenerative properties. Previous studies have shown that ADSCs can differentiate into various cell types, including neural cells, and secrete neurotrophic factors. The trend towards improved grip strength, although not statistically significant, is noteworthy. Grip strength is a reliable indicator of overall muscle strength and has been associated with various health outcomes. The potential improvement in grip strength immediately after ADSC administration warrants further investigation, as it could have implications for patients with neuromuscular disorders.

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2025-11-13 | CRISPR/Cas9-mediated generation of a de novo C9ORF72 knock-in isogenic iPSC cell bank to model ALS and FTD

Abstract Background The pathogenic G 4 C 2 repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies focused on delineating the underlying perturbed mechanisms resulting from this genetic mutation are often confounded by the heterogeneity present in current disease models, such as patient-derived iPSC lines, with estimations of up to 50% of the variation in iPSC cell phenotypes resulting from inter-individual differences. Isogenic models, in which the pathogenic mutation is introduced into a defined genetic background, offer a powerful approach to isolating mutation-specific effects and enable high-resolution comparison across distinct ALS/FTD-associated mutations. Such models are essential for uncovering convergent disease mechanisms and improving reproducibility in ALS/FTD research. Methods A two-step scarless CRISPR/Cas9 genome editing strategy was used to generate isogenic human iPSC lines carrying a de novo knock-in of a disease-length G 4 C 2 repeat expansion in the C9ORF72 locus. The resulting lines underwent thorough quality control and were differentiated into lower motor neurons and assessed for the presence of key ALS/FTD pathologies, including changes to C9ORF72 mRNA and protein expression, RNA foci and dipeptide repeat proteins. Results Two C9ORF72 knock-in iPSC lines were generated with 631 and 600 G 4 C 2 repeats, alongside an isogenic genome editing control line. The C9ORF72 G 4 C 2 repeat expansion knock-in iPSC lines exhibit both loss-of-function and gain-of-function pathological features characteristic of ALS/FTD. Compared to the parental wild-type KOLF2.1J line and isogenic (wild-type) CRISPR control line, these exhibit a significant reduction in C9ORF72 mRNA and protein levels, the presence of RNA foci accumulation, and a marked increase in poly(GA) and poly(GP) dipeptide repeat protein levels in iPSCs and motor neurons. Conclusions This is one of the first reports of a successful knock-in of the pathogenic C9ORF72 G 4 C 2 repeat expansion into a human iPSC line, establishing a genetically defined and physiologically relevant model of ALS/FTD. These isogenic lines recapitulate both key loss- and gain-of-function disease pathologies, providing a crucial complement to existing patient-derived iPSC banks. By eliminating confounding genetic background variability, these cell lines will enable more precise interrogation of C9ORF72 -linked pathomechanisms and offer a robust platform for comparative studies across the ALS and FTD spectrum, mechanistic investigations, and future therapeutic targeting with enhanced translational relevance.

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2024-07-23 | The Current Landscape of Hypotheses Describing the Contribution of CD4+ Heterogeneous Populations to ALS

Amyotrophic Lateral Sclerosis (ALS) is a poorly understood and fatal disease. It has a low prevalence and a 2–4 year survival period. Various theories and hypotheses relating to its development process have been proposed, albeit with no breakthrough in its treatment. Recently, the role of the adaptive immune system in ALS, particularly CD4+ T cells, has begun to be investigated. CD4+ T cells are a heterogeneous group of immune cells. They include highly pro-inflammatory types such as Th1 and Th17, as well as highly anti-inflammatory cells such as Tregs. However, the landscape of the role of CD4+ T cells in ALS is still not clearly understood. This review covers current hypotheses that elucidate how various CD4+ T cells can contribute to ALS development. These hypotheses include the SWITCH model, which suggests that, in the early stages of the disease, Tregs are highly capable of regulating the immune response. However, in the later stages of the disease, it seems that pro-inflammatory cells such as Th1 and Th17 are capable of overwhelming Treg function. The reason why this occurs is not known. Several research groups have proposed that CD4+ T cells as a whole might experience aging. Others have proposed that gamma delta T cells might directly target Tregs. Additionally, other research groups have argued that less well-known CD4+ T cells, such as Emoes+ CD4+ T cells, may be directly responsible for neuron death by producing granzyme B. We propose that the ALS landscape is highly complicated and that there is more than one feasible hypothesis. However, it is critical to take into consideration the differences in the ability of different populations of CD4+ T cells to infiltrate the blood–brain barrier, taking into account the brain region and the time of infiltration. Shedding more light on these still obscure factors can help to create a personalized therapy capable of regaining the balance of power in the battle between the anti-inflammatory and pro-inflammatory cells in the central nervous system of ALS patients.

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2024-07-11 | Preservation of Vocal Function in Amyotrophic Lateral Sclerosis (ALS) Patients Following Percutaneous Dilatational Tracheostomy (PDT) and Adjuvant Therapies

To evaluate the efficacy of percutaneous dilatational tracheostomy (PDT) combined with adjuvant therapies in preserving vocal function in amyotrophic lateral sclerosis (ALS) patients. Methods: We performed a retrospective analysis of 47 ALS patients who underwent PDT at the Rodem Hospital from 2021 to 2023. Post-operatively, these patients were provided with a comprehensive treatment plan that included regenerative injection therapy, low-frequency electrical stimulation, respiratory rehabilitation, and swallowing rehabilitation therapy. Additionally, a balloon reduction program was implemented for effective Tracheostomy tube (T-tube) management. The preservation of vocal functions was evaluated 4 weeks following the procedure. Results: While some patients maintained or slightly improved their ALSFRS-R speech scores, the overall trend indicated a de-crease in speech scores post-PDT. This suggests that PDT combined with adjuvant therapies may not universally improve vocal function, but can help maintain it in certain cases. Conclusion: Our findings indicate that PDT combined with mesotherapy, low-frequency electrical stimulation, and swallowing rehabilitation therapy may play a role in maintaining vocal function in limb type ALS patients, though further research is needed to optimize patient management and validate these results.

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2018-10-03 | Identification of a resilient mouse facial motoneuron population following target disconnection by injury or disease.

When nerve transection is performed on adult rodents, a substantial population of neurons survives short-term disconnection from target, and the immune system supports this neuronal survival, however long-term survival remains unknown. Understanding the effects of permanent axotomy on cell body survival is important as target disconnection is the first pathological occurrence in fatal motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). The goal of this study was to determine if facial motoneurons (FMN) could survive permanent target disconnection up to 26 weeks post-operation (wpo) after facial nerve axotomy (FNA). In addition, the potentially additive effects of immunodeficiency and motoneuron disease on post-axotomy FMN survival were examined. This study included three wild type (WT) mouse strains (C57BL/6J, B6SJL, and FVB/NJ) and three experimental models (RAG-2-/-: immunodeficiency; mSOD1: ALS; Smn-/-/SMN2+/+: SMA). All animals received a unilateral FNA, and FMN survival was quantified at early and extended post-operative timepoints. In the C57BL/6J WT group, FMN survival significantly decreased at 10 wpo (55±6%), and then remained stable out to 26 wpo (47±6%). In the RAG-2-/- and mSOD1 groups, FMN death occurred much earlier at 4 wpo, and survival plateaued at approximately 50% at 10 wpo. The SMA model and other WT strains also exhibited approximately 50% FMN survival after FNA. These results indicate that immunodeficiency and motoneuron disease accelerate axotomy-induced neuron death, but do not increase total neuron death in the context of permanent target disconnection. This consistent finding of a target disconnection-resilient motoneuron population is prevalent in other peripheral nerve injury models and in neurodegenerative disease models as well. Characterization of the distinct populations of vulnerable and resilient motoneurons may reveal new therapeutic approaches for injury and disease.

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2026-05-25 | Mammalian lysophagy: mechanisms and pathophysiological implications

Lysophagy is a form of selective macroautophagy/autophagy that preserves lysosomal integrity by eliminating damaged lysosomes. Lysosomal membrane permeabilization can arise from diverse physiological and pathological insults, including proteotoxic stress, crystalline particles, pathogens and chemical perturbations, and occurs along a continuum ranging from transient nanoscale lesions to catastrophic rupture. Cells respond to lysosomal injury through a hierarchical quality-control network in which membrane repair, lysophagic removal and lysosomal regeneration operate in a coordinated manner. Damage recognition involves sensing of exposed lumenal glycans and membrane lipids, followed by ubiquitin-dependent tagging that recruits selective autophagy receptors and activates the core autophagy machinery to form lysophagosomes. Lysophagy is closely integrated with membrane repair pathways, metabolic signaling and innate immune responses that together determine lysosomal fate. Dysregulated lysosomal quality control has been implicated in diverse diseases, including neurodegeneration, infection, cancer and chronic inflammatory disorders. In this review, we summarize current mechanistic insights and emerging experimental approaches for studying lysosomal quality control and lysophagy in mammalian cells.Abbreviations: ALR, autophagic lysosome reformation; ALS, amyotrophic lateral sclerosis; ATG8, mammalian Atg8-family protein; ER, endoplasmic reticulum; ESCRT, endosomal sorting complexes required for transport; LAMPs, lysosome associated membrane proteins; LIR, LC3-interacting region; LLOMe, L-leucyl-L-leucine methyl ester; LMP, lysosomal membrane permeabilization; PITT, phosphoinositide-initiated membrane tethering and lipid transport; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns4P, phosphatidylinositol-4-phosphate; ROS, reactive oxygen species; V-ATPase, vacuolar-type H+ -ATPase

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2025-05-01 | Brightening the Future of ALS using Optogenetics

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease that degenerates predominantly motor neurons in the brain, brainstem, and spinal cord, leading to complete paralysis of muscles essential for daily motor activities. Difficulty in swallowing (dysphagia) is one of the most severe symptoms, contributing to poor quality of life and an eightfold increase in mortality rates due to malnutrition, dehydration, and aspiration pneumonia. Dysphagia onset in ALS coincides with tongue weakness, due to the degeneration of hypoglossal motor units (XII MUs) providing the sole motor innervation of the tongue. Current treatments do not specifically target XII MU degeneration. To address this unmet clinical need, we propose optogenetics, a neuromodulation method that uses light to precisely control neural activity with high temporal and spatial resolution. We hypothesize that optogenetics will act as a targeted gene therapy approach to slow or halt the degeneration of XII MUs by therapeutically exciting them to drive neuronal repair and nerve terminal proliferation. In our previous study, we used a translational mouse model of ALS ( SOD1-G93A ) with dysphagia and injected adeno associated virus (AAV) mediated excitatory opsin Channelrhodopsin2 (ChR2) into the hypoglossal nucleus (HGN) to directly stimulate and record neural activity via an implanted optrode. Currently, we are developing a minimally invasive method using intralingual injections to retrogradely transfect XII MUs as a more translationally feasible approach. In our pilot study, AAV mediated ChR2 was injected into the tongue to transfect neurons and muscle cells in transgenic (n=4, at disease onset) and wild-type (n=7) mice. Results 3-4 weeks post-injection showed that midline injections into the tongue base and blade were sufficient to evoke visible tongue contractions via optogenetic stimulation (opto-stim) of the tongue surface under anesthesia, and to drive opsin expression in the HGN (tongue tissue not yet analyzed). However, both results were suboptimal compared to our direct HGN injection method. That this peripheral-based optogenetics approach worked at all is exciting because it was applied after clinical disease onset (to mirror intervention timing for human ALS), when mice presumably had axonal transport deficits (a hallmark early feature of ALS). We are now exploring methods to improve opsin expression in XII MUs to likewise improve opto-stim evoked tongue contractions. Following optimization, we will investigate the therapeutic effects of XII MU-targeted opto-stim in awake mice (while drinking from a light-emitting spout) vs. at rest (via a light-emitting patch implanted subcutaneously under the tongue). This essential pre-clinical work will demonstrate if this innovative optogenetics approach can safely and effectively slow or halt XII MU degeneration to preserve swallowing function and extend lifespan, setting the stage for translation to ALS clinical trials. This project is funded by NIH R21 NS123761 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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2024-12-04 | Aldh3a1-mediated detoxification of reactive aldehydes contributes to distinct muscle responses to Amyotrophic Lateral Sclerosis progression

Different muscles exhibit varied susceptibility to degeneration in Amyotrophic Lateral Sclerosis (ALS), a fatal neuromuscular disorder. Extraocular muscles (EOMs) are particularly resistant to ALS progression and exploring the underlying molecular nature may deliver great therapeutic value. Reactive aldehyde 4-hydroxynonenal (HNE) is implicated in ALS pathogenesis and ALDH3A1 is an inactivation-resistant intracellular detoxifier of 4-HNE protecting eyes against UV-induced oxidative stress. Here we detected prominently higher levels of ALDH3A1 in mouse EOMs than other muscles under normal physiological conditions. In an ALS mouse model (hSOD1G93A) reaching end-stage, ALDH3A1 expression was sustained at high level in EOMs, whereas substantial upregulation of ALDH3A1 occurred in soleus and diaphragm. The upregulation was less pronounced in extensor digitorum longus (EDL) muscle, which endured the most severe pathological remodeling as demonstrated by unparalleled upregulation of a denervation marker ANKRD1 expression. Interestingly, sciatic nerve transection in wildtype mice induced ALDH3A1 and ANKRD1 expression in an inverse manner over muscle type and time. Adeno-associated virus enforced overexpression of ALDH3A1 protected myotubes from 4-HNE-induced DNA fragmentation, plasma membrane leakage and restored MG53-mediated membrane repair. Our data indicate that ALDH3A1 may contribute to distinct muscle resistance to ALS through detoxifying reactive aldehydes.

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2024-10-31 | Safety and tolerability of tegoprubart in patients with amyotrophic lateral sclerosis: A Phase 2A clinical trial

Background The interaction of CD40L and its receptor CD40 on activated T cells and B cells respectively control pro-inflammatory activation in the pathophysiology of autoimmunity and transplant rejection. Previous studies have implicated signaling pathways involving CD40L (interchangeably referred to as CD154), as well as adaptive and innate immune cell activation, in the induction of neuroinflammation in neurodegenerative diseases. This study aimed to assess the safety, tolerability, and impact on pro-inflammatory biomarker profiles of an anti CD40L antibody, tegoprubart, in individuals with amyotrophic lateral sclerosis (ALS). Methods and findings In this multicenter dose-escalating open-label Phase 2A study, 54 participants with a diagnosis of ALS received 6 infusions of tegoprubart administered intravenously every 2 weeks. The study was comprised of 4 dose cohorts: 1 mg/kg, 2 mg/kg, 4 mg/kg, and 8 mg/kg. The primary endpoint of the study was safety and tolerability. Exploratory endpoints assessed the pharmacokinetics of tegoprubart as well as anti-drug antibody (ADA) responses, changes in disease progression utilizing the Revised ALS Functional Rating Scale (ALSFRS-R), CD154 target engagement, changes in pro-inflammatory biomarkers, and neurofilament light chain (NFL). Seventy subjects were screened, and 54 subjects were enrolled in the study. Forty-nine of 54 subjects completed the study (90.7%) receiving all 6 infusions of tegoprubart and completing their final follow-up visit. The most common treatment emergent adverse events (TEAEs) overall (>10%) were fatigue (25.9%), falls (22.2%), headaches (20.4%), and muscle spasms (11.1%). Mean tegoprubart plasma concentrations increased proportionally with increasing dose with a half-life of approximately 24 days. ADA titers were low and circulating levels of tegoprubart were as predicted for all cohorts. Tegoprubart demonstrated dose dependent target engagement associated and a reduction in 18 pro-inflammatory biomarkers in circulation. Conclusions Tegoprubart appeared to be safe and well tolerated in adults with ALS demonstrating dose-dependent reduction in pro-inflammatory chemokines and cytokines associated with ALS. These results warrant further clinical studies with sufficient power and duration to assess clinical outcomes as a potential treatment for adults with ALS. Trial registration Clintrials.gov ID: NCT04322149 .

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2023-10-18 | T cell biology in neuromuscular disorders: a focus on Duchenne Muscular Dystrophy and Amyotrophic Lateral Sclerosis

Growing evidence demonstrates a continuous interaction between the immune system, the nerve and the muscle in neuromuscular disorders of different pathogenetic origins, such as Duchenne Muscular Dystrophy (DMD) and Amyotrophic Lateral Sclerosis (ALS), the focus of this review. Herein we highlight the complexity of the cellular and molecular interactions involving the immune system in neuromuscular disorders, as exemplified by DMD and ALS. We describe the distinct types of cell-mediated interactions, such as cytokine/chemokine production as well as cell-matrix and cell-cell interactions between T lymphocytes and other immune cells, which target cells of the muscular or nervous tissues. Most of these interactions occur independently of exogenous pathogens, through ligand-receptor binding and subsequent signal transduction cascades, at distinct levels of specificity. Although this issue reveals the complexity of the system, it can also be envisioned as a window of opportunity to design therapeutic strategies (including synthetic moieties, cell and gene therapy, as well as immunotherapy) by acting upon one or more targets. In this respect, we discuss ongoing clinical trials using VLA-4 inhibition in DMD, and in ALS, with a focus on regulatory T cells, both revealing promising results.

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