Chitinases are hydrolytic enzymes responsible for degrading chitin and have been evolutionarily conserved across various species. Although their signaling pathways are not fully understood, the chitinases are considered active immunomodulators across several cell types. Specific isoforms, including Chitotriosidase-1 (CHIT1), Chitinase-3-like protein 1 (CHI3L1), and human-specific Chitinase-3-like protein 2 (CHI3L2), have emerged as markers of inflammation across the neurodegenerative spectrum, including amyotrophic lateral sclerosis (ALS). ALS is a fatal neuromuscular condition, and therapeutic development has been severely hindered by phenotypic heterogeneity and an incomplete understanding of etiology. Although several overlapping disease mechanisms can contribute to neuronal death, inflammation can exacerbate pathology. Prior studies have reported that CHIT1, CHI3L1, and CHI3L2 levels are elevated in the cerebrospinal fluid (CSF) of ALS patients and associated with disease aggressiveness. Nevertheless, several open questions critical to our understanding of the chitinases' role in ALS disease burden remain: namely, 1) which cell types in the central nervous system (CNS) are chitinase sources under physiological conditions, 2) which of these display chitinase upregulation in ALS, and 3) what is the diagnostic utility of the chitinases relative to established biomarkers. Here, we utilize pre-clinical models and post-mortem human tissue to demonstrate at both the transcriptomic and protein level that neurons are a primary source of chitinases; furthermore, neuronal chitinase expression is conserved across species. Under physiological conditions, CHI3L1 is more abundant and widely expressed across various cell types, whereas CHIT1 is predominantly expressed in neurons. Additionally, utilizing symptomatic mice from three familial ALS models, we demonstrate isoform-specific expression profiles, with astroglial and microglial upregulation of CHI3L1, and neuronal and microglial upregulation of CHIT1. Differing expression dynamics and diagnostic utility were also noted in our clinical cohort: CSF CHIT1 and CHI3L2 levels had more discriminatory power when distinguishing between ALS vs. non-ALS controls, while CHI3L1 was more closely associated with inflammation and aging across the neurodegenerative spectrum. Although the chitinases did not diagnostically outperform the neurofilament proteins as biomarkers, we propose that appreciating their expression patterns can aid in optimizing biomarker-guided trial design. Taken together, we demonstrate that chitinase upregulation in ALS is evident in various CNS cell types and that its neuronal expression may provide new insights into its role in disease activity.

BACKGROUND:Membrane contact sites are crucial for the exchange of ions or lipids and thus are critical for the function and maintenance of organelles. VPS13A is a membrane-residing, bridge-like protein connecting two membranes to enable bulk lipid transfer. Loss-of-function mutations in the VPS13A gene cause VPS13A disease. Previous studies showed alterations of lipid transfer and impaired calcium homeostasis. OBJECTIVE:Although membrane contact sites are becoming increasingly important in neurodegenerative disease research, their contribution to cellular homeostasis is still unclear. We attempted to investigate the consequences of loss of VPS13A function on membrane contact sites and related mechanisms in the context of VPS13A disease. METHODS:VPS13A-deficient patient-derived fibroblasts were compared with fibroblasts from healthy donors. Specific dyes, labeled fatty acids, and a specific marker for mitochondrial-endoplasmic reticulum contact sites were used to investigate lipid transfer and distribution in involved organelles. Mitochondrial calcium handling was investigated using the calcium indicator Rhod-2, AM. Images were obtained by super-resolution microscopy using Airyscan2 technology. RESULTS:We observed a general disturbance of membrane contact sites in VPS13A disease, accompanied by a reduction in lipid droplet formation, diminished lipid transfer into mitochondria, and unusual mitochondrial calcium uptake behavior in VPS13A disease fibroblasts. CONCLUSIONS:Loss of VPS13A causes alterations beyond an impairment of lipid shuttling, which includes a dysregulation of membrane contact sites as well as impaired mitochondrial calcium handling. Accordingly, our findings contribute significantly to the understanding of mechanisms directly or indirectly linked to the function of VPS13A. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

OBJECTIVE:Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron degeneration. Fused in sarcoma (FUS)-associated juvenile ALS (jALS) represents a distinct and aggressive subgroup with rapid deterioration and poor prognosis. Certain FUS mutations have been associated with comorbid intellectual disability, suggesting neurodevelopmental involvement. We compared FUS-jALS with adult-onset FUS-ALS cases (aALS) to evaluate the association between premorbid cognitive impairment, genetic and clinical features incorporating neuroimaging data. METHODS:Patients with genetically confirmed FUS-ALS were classified as jALS (onset < 25 years) or aALS (onset ≥ 25 years). Neuropsychological assessment used Mehrfachwahl-Wortschatz-Test (MWT) for verbal IQ, and the Edinburgh Cognitive and Behavioral ALS Screen (ECAS), with cognitive impairment classified according to Strong criteria. Volumetric analysis was conducted on structural MRI and FDG-PET data. RESULTS:All three jALS (P525L [n = 2], H517_Q519del [n = 1]) showed rapid progression with early severe clinical events. Neuropsychological assessment revealed global cognitive deficits (ALS-ci) with widespread dysfunction beyond typical ALS-specific patterns and reduced verbal IQ, pointing towards premorbid cognitive impairment. aALS demonstrated slower progression and were predominantly cognitively unimpaired (ALS-ni) or showed an ALS-specific impairment. Neuroimaging revealed distinct patterns: jALS cases demonstrated posterior cortical atrophy and hypometabolism on FDG-PET, while aALS showed largely preserved brain volumes and limbic-subcortical hypometabolism. INTERPRETATION:Specific FUS mutations (P525L, H517_Q519del) predispose to jALS with severe progression and premorbid cognitive impairments, supporting a genotype-phenotype association. Posterior cortical findings suggest neurodevelopmental delay rather than disease-related neurodegeneration. Genetic FUS screening may be warranted in patients with intellectual disability and motor signs, given emerging targeted therapies.

The pathogenesis of Parkinson's disease is multifactorial, but disruption of calcium and iron is a common feature. The mitochondrial Rho GTPase Miro1 is a component of the mitochondrial-endoplasmic reticulum contact sites and a key regulator of calcium homeostasis. Heterozygous variants in the Miro1-encoding gene RHOT1 were identified in Parkinson's disease patients. Neurons harboring Parkinson's disease-associated variants show defects in mitochondrial calcium regulation and mitochondria-ER contact sites organization which we hypothesize to contribute to neuronal vulnerability. However, the exact mechanism is not fully understood. We systematically assessed the role of Miro1 and its different domains by using a set of isogenic lines with gene edited mutations S156A and K572R in PINK1/Parkin regulatory elements and the Parkinson's disease-associated mutation R272Q. This showed us a general role of Miro1 in the regulation of cellular calcium homeostasis and the regulation of mitochondrial-ER contact sites, but more importantly, a domain-specific involvement of local calcium distribution, impaired store operated calcium entry and vulnerability to ferroptosis. These findings indicate that Miro1-mutant specific impairments in cellular calcium handling contributes to neuronal vulnerability via mitochondria-ER contact sites and provides further insights in the mechanism how impaired regulation of Miro1 impacts neurons in the context of Parkinson's disease.

(Acta Neuropathologica commun) During the progression of amyotrophic lateral sclerosis (ALS), only specific motor neurons (MNs) preferentially deteriorate, while others are spared until the disease reaches its end stage. Resilient MNs possess several protective factors, yet the precise molecular mechanism(s) underlying selective neuronal vulnerability remains poorly understood. Vesicle-associated membrane protein (VAMP)-binding protein B (VAPB) is an endoplasmic reticulum (ER) protein involved in protein quality control (PQC) mechanisms, including unfolded protein response (UPR) as well as autophagy. A dominantly inherited P56S mutation in the VAPB gene has been linked to ALS8, atypical ALS, and late-onset spinal muscular atrophy (SMA). The P56S VAPB mutation causes ER-associated inclusions, disorganization, and ER stress, contributing to MN degeneration through toxic gain and loss of function. Over-expression of VAPB protein confers neuroprotection in a mouse model of ALS, and increased levels of neuronal VAPB inversely correlate with the absence of pathological aggregates. We hypothesize that VAPB is crucial for motor neuron survival by promoting autophagic degradation of ALS-associated aggregates, while lack of VAPB confers neuronal vulnerability. We analyzed the brain and spinal cord from sporadic (s) and familial (f) ALS patients, comparing patterns of VAPB immunoreactivity using immunohistochemistry, complemented by Western and dot blot analysis. Pathophysiological insights from these studies were further explored using cell culture models, including MNs derived from induced pluripotent stem cells (iPSCs). Consistent with our hypothesis we observed that MNs/neurons resistant to ALS exhibited elevated levels of VAPB and were devoid of pathogenic aggregates. Similarly, ALS-resistant oculomotor neurons showed increased VAPB immunoreactivity compared to normal controls. VAPB was often found to be sequestered within toxic aggregates alongside autophagy-related proteins in the lumbar spinal cord MNs. Notably, a compensatory increase in VAPB immunoreactivity was observed at the C-bouton synapse, suggesting a potential alternative mechanism of neuroprotection. Supporting these findings, in vitro experiments indicated that VAPB overexpression promoted autophagy and assisted in clearing ALS-associated RNA-binding protein aggregates. In summary, VAPB promotes selective neuronal survival by facilitating the autophagic clearance of toxic aggregates. Abnormal VAPB accumulations likely disrupt these neuroprotective processes.

OBJECTIVES:Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) share neuropathological features, including tau, amyloid, and TDP-43 pathology. This study investigated whether AD-related pathological changes are associated with cognitive impairment ALS. METHODS:Cerebrospinal fluid (CSF total-tau, phosphorylated-tau, beta-amyloid) and plasma biomarkers (TDP-43; neurofilament light chain [NfL]) were analyzed in 192 individuals with ALS or ALS with frontotemporal dementia (ALS-FTD) and 100 healthy controls. Cognitive performance was assessed using the Edinburgh Cognitive and Behavioral ALS Screen (ECAS). Group comparisons and regression analyses examined associations between biomarker profiles and cognitive status. Autopsy data were available for a subset of participants. RESULTS:Compared with healthy controls, patients with ALS - particularly those with cognitive impairment (ALSci) or ALS-FTD - showed elevated AD-related biomarkers. Significant differences in beta-amyloid levels were observed between healthy controls (HCs) and patients with ALSci, but not between controls and cognitively unimpaired patients. CSF p-tau and total-tau levels were strongly associated with domain-specific cognitive performance. In contrast, plasma extracellular vesicle TDP-43 and NfL showed weak or no association with cognition. In vivo biomarkers alone reliably distinguished cognitive impairment only in ALSci and ALS-FTD. Postmortem analyses showed no strong association between ABC scores or overall TDP-43 burden and cognitive state; however, temporal and hippocampal TDP-43 burden was associated with cognitive dysfunction. INTERPRETATION:Our findings suggest that tau-related CSF biomarkers, particularly p-tau and total-tau, are associated with cognitive deficits in ALS, indicating that AD-related pathology might be associated to cognitive decline in ALS. However, postmortem data showed even stronger relation of TDP43 pathology to cognitive deficits in ALS. ANN NEUROL 2026;100:123-138.

Abstract The erythrocyte sedimentation rate (ESR) is one of the most widely used laboratory diagnostic parameters in the preliminary assessment of inflammation; indeed, every reader of this work has likely received an ESR assessment in their lifetime. A rapid ESR is a non-specific parameter that provides information about the inflammatory process. Although the origins of this methodology date back to antiquity, the prevailing view that ESR simply reflected particle settling of erythrocytes has recently undergone a paradigm shift: once cell aggregates form a system-spanning network, gravitational collapse of a weak and percolating gel reveals a more complex process reflecting the failure. The apparent non-specific nature of the cells and proteins involved also called into question the medical utility of ESR, at least in well-resourced environments. Here we show a new experimentally derived and physically modelled approach (“supraESR”) that enhances the value and accuracy of ESR for a variety of conditions that exhibit abnormally slow ESR (e.g., sickle cell disease, neuroacanthocytosis syndromes, chronic mountain sickness). We introduce a completely new diagnostic parameter that is based on an established and easily automated measurement method that promises low-cost screening for neuroacanthocytosis syndrome, a group of rare neurodegenerative diseases that are currently detectable only through integration of complex multimodal findings.

Stimulation of the innate immune system has been implicated in ALS and particularly in distinct monogenic forms of ALS. To address whether this is of diagnostic value, we performed a proof-of concept study using qPCR to assess the Interferon score in blood samples of genetic ALS. 56.5% of genetic ALS patients showed significant IFN activation, highest in C9orf72HRE patients (77.3%). About half of FUS-ALS (52.2%), but none of SOD1-ALS patients demonstrated pathological IFN scores. The IFN score significantly correlated with the ALSFRS-R slope and inversely with the time to severe event as a survival surrogate in this genetic ALS cohort. IFN + patients were more likely to be male, showed more rapid disease progression and higher neurofilament levels. The IFN score might have the potential as a stratification and readout tool for biomarker-guided individualized therapy in ALS.

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, leading to an inexorable decline in voluntary muscle function, and finally to death within 2–4 years. The provision of professional ALS care is a multifaceted and continually evolving challenge, including the management of symptoms, advanced care planning, and the provision of psychosocial support. The core objective is to minimize suffering by optimizing symptom management and preserving quality of life. European and German guidelines recommend a specialized, multidisciplinary, and multiprofessional team, including collaborations with palliative care providers. While this is a validated approach to ensure optimal care and patient satisfaction, real-world experience suggests that the German healthcare system may not fully meet these requirements. Methods This study assessed resources of specialized ALS centres in Germany, focusing on the structural prerequisites for the provision of multidimensional care and collaboration with specialised palliative care (SPC) providers. A mixed methods design was used, comprising remote video interviews with neurologists specialized in ALS, including standardised questions and an open section. Results Sixteen neurologists representing their ALS centres were interviewed. The findings indicated a substantial discrepancy in the allocation of time and personnel resources among the centres. The majority of interviewees regarded resources to be inadequate and reported deficiencies in multidisciplinarity and networking. Consequently, certain components of ALS care - particularly psychosocial concerns - have been documented as being occasionally disregarded due to limitations in time or human resources. A number of interviewees expressed criticism regarding the inadequate access to and suboptimal collaboration with SPC providers. The compensation for patient care and interprofessional communication was not perceived as cost-effective. Conclusions Our results suggest that limited resources may prevent the provision of guideline-based care for people living with ALS and their families, even in specialized outpatient clinics. To facilitate the delivery of comprehensive care by ALS centers throughout the entire disease course, the establishment of operational standards concerning their multi-professional staffing and adequate compensation is imperative. Further research is needed to develop feasible concepts of how specialized neurological palliative care can be made reliably accessible to all patients in need.

ABSTRACT Phosphorylated tau181 (p‐tau181), an Alzheimer's disease biomarker, was recently evaluated in amyotrophic lateral sclerosis (ALS). We investigated plasma p‐tau181 in 202 ALS/ALS‐FTD patients and 94 healthy controls, assessing cognitive performance, motor function, and longitudinal dynamics. Plasma p‐tau181 and NfL were significantly elevated in ALS, with p‐tau181 increasing over 1 year while NfL remained stable. Neither marker correlated with cognitive performance, and only NfL was associated with disease severity and progression. Plasma p‐tau181 was higher in patients with predominant lower motor neuron involvement. The results indicate that p‐tau181 reflects peripheral processes in ALS, providing a complementary, mechanistically distinct biomarker from NfL.

Recent research demonstrated activation of the innate immune system in ALS models. This pathway can be activated by cGAS-STING sensing of cytosolic DNA that accumulates as a result of chronic DNA damage and defective mitochondria, both of which was identified as pathology in FUS-ALS. Therefore, we analyzed innate immune pathways in FUS-ALS, which revealed upregulation of interferon-stimulated genes (ISGs) and activation of the TBK1-IRF3 pathway in FUSmut iPSC-derived spinal motor neurons (sMNs). Accumulation of cytosolic dsRNA and its sensor RIG-I, but not MDA5, was found to be significantly upregulated in FUSmut sMNs, which was abolished upon siRNA-mediated knockdown of RIG-I. RIG-I was highly expressed in FUS-ALS post-mortem α-MNs. IFN treatment of FUSwt sMNs phenocopied the axonal degeneration of FUSmut sMNs. Mitochondrial transcription, a known source of dsRNA, was found to be upregulated in compartmental axonal RNAseq analysis and its inhibition reduced ISGs in FUS-ALS sMNs. The JAK-STAT inhibitor ruxolitinib alleviated the upregulated ISG expression and reversed the axonal degeneration of sMNs. Finally, we analyzed ISG expression in peripheral blood from 18 FUS-ALS patients, eight of whom had a significantly elevated interferon signature. RIG-I-mediated innate immune activation in sMNs may be an interesting novel individualized biomarker-driven therapeutic target in (FUS-) ALS. A one-sentence summary of your paper: RIG-I-mediated innate immune activation is found in FUS-ALS spinal motor neurons caused by cytosolic dsRNA accumulation due to mitochondrial transcriptional activation and is amenable to JAK-STAT inhibition and might thus be an interesting novel individualized biomarker-driven therapeutic approach in (FUS-) ALS.

Mutations in FUSED IN SARCOMA (FUS) cause juvenile-onset amyotrophic lateral sclerosis (ALS). Early pathogenesis of FUS-ALS involves impaired transcription and splicing, DNA damage response, and axonal degeneration. However, the molecular pathophysiology and the link between somatic and axonal phenotypes are still poorly understood. We evaluated whether compartment-specific transcriptome differences could distinguish and drive early axonal degeneration. We used iPSC-derived motor neurons (MNs) coupled with microfluidic approaches to generate RNA-sequencing profiles from axonal and somatodendritic compartments. We demonstrate that the axonal transcriptome is unique and distinct, with RNA metabolism, extracellular secretion, and matrix disassembly pathways particularly enriched in distal axonal compartments.FUS mutation leads to changes in distinct pathways that were clustered in only a few distinct protein-protein interaction (PPI) networks. Somatodendritic changes upon FUS mutation include WNT signaling, mitochondrial, extracellular matrix (ECM)-, and synapse-related functions. In contrast, analysis of the axonal transcriptome in mutant MNs centers on the PLK1 pathway, mitochondrial gene expression, and regulation of inflammation. Comparison to CLIP-seq data revealed a significant enrichment for PLK1 and DNA replication pathways in axons. PLK1 upregulation did not activate cell-cycle re-entry but contributed to mutant MNs survival, and its inhibition increased neuronal cell death. We propose that upregulation of PLK1 represents an early event in the pathogenesis of ALS and could act in response to DNA damage, mitochondrial damage, and immune response activation in the affected cells. Additionally, downregulation of ECM pathways in the somatodendritic compartment and axons could explain strongly compromised dynamics of axonal outgrowth. Overall, we provide a novel valuable resource of the potential targets and affected processes changed in the specific compartments of FUS-ALS motor neurons.

INTRODUCTION:The alpha-synuclein seed amplification assay (SAA) has shown excellent performance in the detection of Lewy body pathology in cerebrospinal fluid (CSF). Lewy body pathology is prognostically relevant in patients at risk for dementia. Current assays only provide binary results, so there is a need to quantify the extent of pathology in living patients. METHODS:In addition to the "standard" SAA, we developed a quantitative SAA (qnSAA) and measured 432 CSF samples (216 baseline-follow-up pairs). RESULTS:qnSAA results correlated with cognitive performance. Seventy-five percent of participants with fast qnSAA kinetics converted to dementia in the observed interval. Overall, participants with fast qnSAA kinetics accounted for 27.3% of dementia converters in the entire cohort. DISCUSSION:Findings demonstrate promising properties of qnSAA measurements in a cohort of patients at risk for dementia. qnSAA results showed improved prognostic relevance and have potential to measure target engagement of therapies against Lewy body pathology.

The common single nucleotide polymorphism (SNP) rs12608932 located at a cryptic splice in the UNC13A gene has been reported to modify the clinical phenotype of ALS, but it is unclear whether homozygosity for the C-allele at UNC13A rs12608932 modifies specific domains of cognition in ALS. We analyzed retrospective data from a German cohort and found that the proportion of cognitively or behaviorally impaired patients was higher in the high-risk group of homozygous C-allele carriers. Patients with C/C alleles had lower scores than controls on verbal fluency, executive functioning, and delayed memory recall, but did not differ significantly from other ALS genotypes. Furthermore, informant ratings suggested higher disinhibition in the C/C carriers. These findings indicate that the C/C risk variant of UNC13A rs12608932 may contribute to general cognitive vulnerability rather than domain-specific deficit.

During the progression of amyotrophic lateral sclerosis (ALS), only specific motor neurons (MNs) preferentially deteriorate, while others are spared until the disease reaches its end stage. Resilient MNs possess several protective factors, yet the precise molecular mechanism(s) underlying selective neuronal vulnerability remains poorly understood. Vesicle-associated membrane protein (VAMP)-binding protein B (VAPB) is an endoplasmic reticulum (ER) protein involved in protein quality control (PQC) mechanisms, including unfolded protein response (UPR) as well as autophagy. A dominantly inherited P56S mutation in the VAPB gene has been linked to ALS8, atypical ALS, and late-onset spinal muscular atrophy (SMA). The P56S VAPB mutation causes ER-associated inclusions, disorganization, and ER stress, contributing to MN degeneration through toxic gain and loss of function. Over-expression of VAPB protein confers neuroprotection in a mouse model of ALS, and increased levels of neuronal VAPB inversely correlate with the absence of pathological aggregates. We hypothesize that VAPB is crucial for motor neuron survival by promoting autophagic degradation of ALS-associated aggregates, while lack of VAPB confers neuronal vulnerability. We analyzed the brain and spinal cord from sporadic (s) and familial (f) ALS patients, comparing patterns of VAPB immunoreactivity using immunohistochemistry, complemented by Western and dot blot analysis. Pathophysiological insights from these studies were further explored using cell culture models, including MNs derived from induced pluripotent stem cells (iPSCs). Consistent with our hypothesis we observed that MNs/neurons resistant to ALS exhibited elevated levels of VAPB and were devoid of pathogenic aggregates. Similarly, ALS-resistant oculomotor neurons showed increased VAPB immunoreactivity compared to normal controls. VAPB was often found to be sequestered within toxic aggregates alongside autophagy-related proteins in the lumbar spinal cord MNs. Notably, a compensatory increase in VAPB immunoreactivity was observed at the C-bouton synapse, suggesting a potential alternative mechanism of neuroprotection. Supporting these findings, in vitro experiments indicated that VAPB overexpression promoted autophagy and assisted in clearing ALS-associated RNA-binding protein aggregates. In summary, VAPB promotes selective neuronal survival by facilitating the autophagic clearance of toxic aggregates. Abnormal VAPB accumulations likely disrupt these neuroprotective processes.

VPS13A disease (chorea-acanthocytosis), is an ultra-rare autosomal recessive neurodegenerative disorder caused by mutations of the VPS13A gene encoding Vps13A. Increased serum levels of the muscle isoform of creatine kinase associated with often asymptomatic muscle pathology are among the poorly understood early clinical manifestations of VPS13A disease. Here, we carried out an integrated analysis of skeletal muscle from Vps13a-/- mice and from VPS13A disease patient muscle biopsies. The absence of Vps13A impaired autophagy, resulting in pathologic metabolic remodeling characterized by cellular energy depletion, increased protein/lipid oxidation and a hyperactivated unfolded protein response. This was associated with defects in myofibril stability and the myofibrillar regulatory proteome, with accumulation of the myocyte senescence marker, NCAM1. In Vps13a-/- mice, the impairment of autophagy was further supported by the lacking effect of starvation alone or in combination with colchicine on autophagy markers. As a proof of concept, we showed that rapamycin treatment rescued the accumulation of terminal phase autophagy markers LAMP1 and p62 as well as NCAM1, supporting a connection between impaired autophagy and accelerated aging in the absence of VPS13A. The premature senescence was also corroborated by local activation of pro-inflammatory NF-kB-related pathways in both Vps13a-/- mice and patients with VPS13A disease. Our data link for the first time impaired autophagy and inflammaging with muscle dysfunction in the absence of VPS13A. The biological relevance of our mouse findings, supported by human muscle biopsy data, shed new light on the role of VPS13A in muscle homeostasis.

Amyotrophic lateral sclerosis (ALS) is a fatal type of neurodegenerative disease marked by progressive and selective degeneration of motor neurons (MNs) present in the spinal cord, brain stem and motor cortex. However, the intricate molecular mechanisms underlying primary cell death pathways, including ferroptosis-related genes (FRGs) mediating MN dysfunction in ALS, remain elusive. Ferroptosis, a novel type of iron-dependent cell death with the accumulation of lipid peroxidation products, stands distinct from apoptotic-related stress and other cell death mechanisms. Although growing advances have highlighted the role of iron deposition, apoptosis and alteration of antioxidant systems in ALS pathogenesis, there is little data at the systems biology level. Therefore, we performed a comprehensive bioinformatic analysis of bulk RNA-sequencing (RNA-seq) data by systematically comparing the gene expression profiles from iPSC-derived MNs of ALS patients and healthy controls using our datasets as well as from the GEO database to reveal the role of ferroptosis-related gene alterations in ALS, especially in selective MN vulnerability of FUSED IN SARCOMA (FUS) mutations. In this study, we first identified differentially expressed genes (DEGs) between FUS mutant and healthy controls. Subsequently, the crossover genes between DEGs and FRGs were selected as differentially expressed ferroptosis-related genes (DEFRGs). Functional enrichment and protein-protein interaction (PPI) analysis of DEFRGs identified that DNA damage, stress response and extra cellular matrix (ECM) were the most significantly dysregulated functions/pathways in FUS-ALS causing mutations compared to healthy controls. While GSEA analysis showed enrichment of genes associated with apoptosis, the degree of ferroptosis and iron ion homeostasis/response to iron of FUS MNs was lower. Altogether, our findings may contribute to a better understanding of the relevant role of cell death pathways underlying selective vulnerability of MNs to neurodegeneration in FUS-ALS pathophysiology.

Fatty-acid-hydroxylase-associated neurodegeneration (FAHN) is a rare neurodegenerative disorder caused by loss-of-function mutations in the FA2H gene, leading to impaired enzymatic activity and resulting in myelin sheath instability, demyelination, and axonal degeneration. In this study, we established a human in vitro model using neurons and oligodendrocytes derived from induced pluripotent stem cells (hiPSCs) of a FAHN patient. This coculture system enabled the investigation of myelination processes and myelin integrity in a disease-relevant context. Analyses using immunofluorescence and Western blot revealed impaired expression and localisation of key myelin proteins in oligodendrocytes and cocultures. FA2H-deficient cells showed reduced myelination, shortened internodes, and disrupted formation of the nodes of Ranvier. Additionally, we identified autophagy defects—a hallmark of many neurodegenerative diseases—including reduced p62 expression, elevated LC3B levels, and impaired fusion of autophagosomes with lysosomes. This study presents a robust hiPSC-based model to study FAHN, offering new insights into the molecular pathology of the disease. Our findings suggest that FA2H mutations compromise both the structural integrity of myelin and the efficiency of the autophagic machinery, highlighting potential targets for future therapeutic interventions.

Extracellular vesicles (EVs) are cell-secreted membranous particles contributing to intercellular communication. Coding and noncoding RNAs can be detected as EV cargo, and RNA-binding proteins (RBPs), such as hnRNPA2B1, have been circumstantially implicated in EV-RNA sorting mechanisms. However, the contribution of competitive RBP–RNA interactions responsible for RNA-sorting outcomes is still unclear, especially for predicting the EV-RNA content. We designed a reverse proteomic analysis exploiting the EV-RNA to identify intracellular protein binders in vitro. Using cells expressing a recombinant hnRNPA2B1 to normalize competitive interactions, we prioritized a network of heterogeneous nuclear ribonucleoproteins and purine-rich RNA sequences subsequently validated in secreted EV-RNA through short fluorescent RNA oligos. Then, we designed a GGGAG-enriched RNA probe that efficiently interacted with a full-length human hnRNPA2B1 protein. We exploited the interaction to conduct a pharmacological screening and identify inhibitors of the protein–RNA binding. Small molecules were orthogonally validated through biochemical and cell-based approaches. Selected drugs remarkably impacted secreted EV-RNAs and reduced an RNA-dependent, EV-mediated paracrine activation of NF-kB in recipient cells. These results demonstrate the relevance of post-transcriptional mechanisms for EV-RNA sorting and the possibility of predicting the EV-RNA quality for developing innovative strategies targeting discrete paracrine functions.