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A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease, evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues, and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations, we performed whole-transcriptome profiling of motor neurons (MNs) differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database, we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation, the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity, highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12147-8.

Neurotropic viruses are the most common cause of infectious encephalitis and highly target neurons for infection. Our understanding of the intrinsic capacity of neuronal innate immune responses to mediate protective antiviral responses remains incomplete. Here, we evaluated the role of intercellular crosstalk in mediating intrinsic neuronal immunity and its contribution to limiting viral infection. We found that in the absence of viral antagonism, neurons transcriptionally induce robust interferon signaling and can effectively signal to uninfected bystander neurons. Yet, in two-dimensional cultures, this dynamic response did not restrict viral spread. Interestingly, this differed in the context of viral infection in three-dimensional forebrain organoids with complex neuronal subtypes and cellular organization, where we observed protective capacity. We showed antiviral crosstalk between infected neurons and bystander neural progenitors is mediated by type I interferon signaling. Using spatial transcriptomics, we then uncovered regions containing bystander neural progenitors that expressed distinct antiviral genes, revealing critical underpinnings of protective antiviral responses among neuronal subtypes. These findings underscore the importance of interneuronal communication in protective antiviral immunity in the brain and implicate key contributions to protective antiviral signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03381-y.

Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia.

IntroductionGlucose Transporter 1-Deficiency Syndrome (GLUT1-DS) is a rare genetic disorder caused by mutations in the gene encoding for GLUT1 and characterized by impaired glucose uptake in the brain. This leads to brain hypometabolism and the development of symptoms that include epilepsy, motor dysfunctions and cognitive impairment. The development of patient-specific in vitro models is a valuable tool for understanding the pathophysiology of rare genetic disorders and testing new therapeutic interventions.MethodsIn this study, we generated brain organoids from induced pluripotent stem cells (iPSCs) derived either from a GLUT1-DS patient or a healthy individual. The functional organoids were analyzed for cellular composition, maturity, and electrophysiological activity using a custom-made microelectrode array (MEA) platform, which allowed for the detection of spikes, burst patterns, and epileptiform discharges.ResultsImmunostaining revealed a similar distribution of neurons and astrocytes in both healthy and GLUT1-DS brain organoids, though GLUT1-DS brain organoids exhibited reduced cellular density and smaller overall size. Electrophysiological recordings demonstrated functional spike profiles in both organoid types. Notably, our study demonstrates that brain organoids derived from a GLUT1-DS patient exhibit distinct epileptiform activity and heightened sensitivity to glucose deprivation, reflecting key features of the disorder.DiscussionThese findings validate the use of brain organoids as a model for studying GLUT1-DS and highlight their potential for testing novel therapeutic strategies aimed at improving glucose metabolism and managing epilepsy in patients.

Recent studies demonstrate that brain infiltration of peripheral immune cells and their interaction with brain-resident cells contribute to Parkinson’s disease (PD). However, mechanisms of T cell-brain cell communication are not fully elucidated and models allowing investigation of interaction between T cells and brain-resident cells are required. In this study, we developed a three-dimensional (3D) model composed of stem cell-derived human midbrain organoids (hMO) and peripheral blood T cells. We demonstrated that organoids consist of multiple midbrain-specific cell types, allowing to study T cell motility and interactions with midbrain tissue in a spatially organized microenvironment. We optimized co-culture conditions and demonstrated that T cells infiltrate hMO tissue, leading to neural cell loss. Our work establishes a novel 3D cell co-culture model as a promising tool to investigate the effect of the adaptive immune system on the midbrain and can be used in future studies to address these processes in the context of PD.