技术资料

AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV,mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation,neuronal dysfunction,and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia,we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1,ISG15,ISG20,IFI27,IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART,consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However,S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells,mature and immature ChP cells,neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons,accompanied by increased expression of genes promoting cellular senescence and cell death. Together,these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons. View Publication

AbstractA GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD),while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF,LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence,rather than amino acid content,is central to the impact of RQC factor depletion on RAN translation—suggesting a role for RNA secondary structure in these processes. Together,these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders. Graphical Abstract Graphical Abstract View Publication

BackgroundAmyloid-beta (A?) plaques and their associated glial responses are hallmark features of Alzheimer’s disease (AD),yet their interactions within the human brain remain poorly defined.MethodsWe applied spatial transcriptomics (ST) and immunohistochemistry (IHC) to 78 postmortem brain sections from 21 individuals in the Religious Orders Study and Memory and Aging Project (ROSMAP). We paired ST with histological data and stratified spots into major categories of plaque-glia niches based on A?,GFAP,and IBA1 intensity. Leveraging published ROSMAP single-nucleus RNA-seq data,we examined differences in gene expression,cellular composition,and intercellular communication across these niches. Neuronal and glial changes were validated by IHC and quantitative analyses. We further characterized glial responses using gene set enrichment analysis (GSEA) with known mouse glial signatures and human AD-associated microglial states. Finally,we used iPSC-derived multicellular cultures and single-cell RNA sequencing (scRNA-seq) to identify cell types that,upon short-term A? exposure,recapitulate the glial responses observed in the human spatial data.ResultsLow-A? regions,enriched for diffuse plaques,exhibited transcriptomic profiles consistent with greater neuronal loss than high-A? regions. High-glia regions showed increased expression of inflammatory and neurodegenerative pathways. Spatial glial responses aligned with established gene modules,including plaque-induced genes (PIGs),oligodendrocyte (OLIG) responses,disease-associated microglia (DAM),disease-associated astrocytes (DAA),and human AD-associated microglial states,indicating that diverse glial phenotypes emerge around plaques and shape the local immune environment. IHC confirmed elevated neuronal apoptosis near low-A? plaques and greater CD68 abundance and synaptic loss near glia-high plaques. In vitro,iPSC-derived microglia—but not astrocytes—exposed to A? displayed transcriptomic changes that closely mirrored the glial states identified in our ST dataset.ConclusionsOur study provides a comprehensive spatial transcriptomic dataset from human AD brain tissue and bridges spatial gene expression with traditional neuropathology. By integrating ST,snRNA-seq,and human multicellular models,we map cellular states and molecular events within plaque-glia niches. This work offers a spatially resolved framework for dissecting plaque-glia interactions and reveals new insights into the cellular and molecular heterogeneity underlying neurodegenerative pathology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s44477-025-00002-z. View Publication

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. View Publication

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. View Publication

ABSTRACTPatched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling,an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development,molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here,we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling,and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models. Summary: Cerebellar organoids recapitulate in vivo processes of regionalisation and SHH signalling,and offer new insight into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models. View Publication

SummaryThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS),a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits,ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes,little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/? induced pluripotent stem cells to model CNCC specification,we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT),a process necessary for CNCC delamination and migration from the neural tube. Furthermore,wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level,these EMT enhancers contain binding motifs for ZIC2,and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers,ZIC2 relocates to neuronal enhancers,triggering aberrant neuronal gene activation. In mice,deletion of Zic2 impairs NCC delamination,while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. Graphical abstract ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition,a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers,resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. View Publication

N6-methyladenosine (m6A) is an abundant internal RNA modification that can impact gene expression at both post-transcriptional and transcriptional levels. However,the landscapes and functions of m6A in human brains and neurodegenerative diseases,including Alzheimer’s disease (AD),are under-explored. Here,we examined RNA m6A methylome using total RNA-seq and meRIP-seq in middle frontal cortex of post-mortem brains from individuals with or without AD,which revealed m6A alteration on both mRNAs and various noncoding RNAs. Notably,many promoter-antisense RNAs (paRNAs) displayed cell-type-specific expression and changes in AD,including one produced adjacent to MAPT that encodes the Tau protein. MAPT-paRNA is highly expressed in neurons,and m6A positively controls its expression. In iPSC-derived human excitatory neurons,MAPT-paRNA does not impact the nearby MAPT mRNA,but instead promotes expression of hundreds of neuronal and synaptic genes,and is protective against excitotoxicity. Analysis of single nuclei RNA-DNA interactome in human brains supports that brain paRNAs interact with both cis- and trans-chromosomal target genes to impact their transcription. These data reveal landscapes and functions of noncoding RNAs and m6A in brain gene regulation and AD pathogenesis. This study characterised the landscapes and changes of RNA m6A in brains of individuals with or without Alzheimer’s disease,and revealed roles of a promoter antisense RNA next to MAPT in neuronal gene regulation that promote neuronal survival. View Publication

Fellows et al. report that individual dynein motors can move the entire axon length during retrograde transport. They find factors LIS1 and NDEL1,needed for transport initiation,also move with cargos. In the anterograde direction,dynein and its cofactor dynactin are transported separately,keeping them apart until required. Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein,which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level,it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here,we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction,we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore,LIS1 and NDEL1 also undergo long-distance movement,despite being mainly implicated with the initiation of dynein transport. Intriguingly,in the anterograde direction,dynein/LIS1 moves faster than dynactin/NDEL1,consistent with transport on different cargos. Therefore,neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required. View Publication

Vascular dementia is the second most common form of dementia. Yet,the mechanisms by which cerebrovascular damage progresses are insufficiently understood. Here,we create bilateral common carotid artery stenosis in mice,which effectively impairs blood flow to the brain,a major cause of the disease. Through imaging and single-cell transcriptomics of the mouse cortex,we uncover that blood vessel venous cells undergo maladaptive structural changes associated with increased Epas1 expression and activation of developmental angiogenic pathways. In a human cell model comparing arterial and venous cells,we observe that low-oxygen condition leads to sustained EPAS1 signaling specifically in venous cells. EPAS1 inhibition reduces cerebrovascular abnormalities,microglial activation,and improves markers of cerebral perfusion in vivo. In human subjects,levels of damaged endothelial cells from venous vessels are correlated with white matter injury in the brain and poorer cognitive functions. Together,these findings indicate EPAS1 as a potential therapeutic target to restore cerebrovascular integrity and mitigate neuroinflammation. How changes in brain blood vessels lead to a chronic reduction in blood flow and,consequently,to vascular dementia is poorly understood. Here,the authors show that venous endothelial dysfunction driven by EPAS1 promotes abnormal vascular remodeling and contributes to cognitive decline. View Publication

BackgroundThe causal relationship between the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the preservation of SERCA2a function in mitigating myocardial ischemia–reperfusion (mI/R) injury,along with the associated regulatory mechanisms,remains incompletely understood. This study aims to unravel how NRF2 directly or indirectly influences SERCA2a function and its regulators,phospholamban (PLN) and Dwarf Open Reading Frame (DWORF),by testing the pharmacological repositioning of AEOL-10150 (AEOL) in the context of mI/R injury.MethodsC57BL6/J,Nrf2 knockout (Nrf2?/?),and wild-type (Nrf2+/+) mice,as well as human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) were subjected to I/R injury. Gain/loss of function techniques,RT-qPCR,western blotting,LC/MS/MS,and fluorescence spectroscopy were utilized. Cardiac dimensions and function were assessed by echocardiography.ResultsIn the early stages of mI/R injury,AEOL administration reduced mitochondrial ROS production,decreased myocardial infarct size,and improved cardiac function. These effects were due to NRF2 activation,leading to the overexpression of the micro-peptide DWORF,consequently enhancing SERCA2a activity. The cardioprotective effect induced by AEOL was diminished in Nrf2?/? mice and in Nrf2/Dworf knockdown models in hiPSCMs subjected to simulated I/R injury. Our data show that AEOL-induced NRF2-mediated upregulation of DWORF disrupts the phospholamban-SERCA2a interaction,leading to enhanced SERCA2a activation and improved cardiac function.ConclusionsTaken together,our study reveals that AEOL-induced NRF2-mediated overexpression of DWORF enhances myocardial function through the activation of the SERCA2a offering promising therapeutic avenues for mI/R injury.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01242-1. Highlights• Novel AEOL-10150 therapeutic potential. AEOL-10150 demonstrates promise in activating NRF2 and mitigating myocardial ischemia-reperfusion injury.• DWORF overexpression breakthrough. Overexpression of DWORF significantly contributes to preserving cardiac function and reducing myocardial injury through the NRF2-DWORF pathway.• Enhanced cardiac protection mechanisms. The study highlights the dual role of AEOL-10150 and DWORF in enhancing cardiac protection and preventing heart failure.• Future research directions. Additional studies are required to validate the long-term efficacy of AEOL-10150 and the regulatory effects of NRF2-DWORF axis in clinical applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01242-1. View Publication

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart,with several clinical trials now in planning or recruitment stages. However,because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs,it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here,we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically,we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures,signal regulatory protein ? (SIRPA)+CD90?CD200+ and SIRPA+CD90?CD200?,identify arrhythmogenic and non-arrhythmogenic cardiomyocytes,respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias. Selvakumar,Clayton et al. use a porcine model of myocardial infarction and PSC-CM transplantation and identify atrial and pacemaker-like cardiomyocytes as the cause of engraftment arrhythmias and surface marker signatures to distinguish between arrhythmogenic and non-arrhythmogenic cardiomyocytes. View Publication