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Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by profound neuronal and cognitive decline, with increasing evidence implicating astrocyte dysfunction in disease pathology. While traditional therapeutic approaches have primarily targeted neurons, the crucial role of astrocytes in metabolism, neurotransmission, amyloid-beta clearance, and neuroinflammation underscores their potential as therapeutic targets. In this study, we employed a multiomic integrative analysis combining transcriptomic and kinomic profiling of human induced pluripotent stem cell (hiPSC)-derived astrocytes from patients with familial AD (fAD) compared to healthy controls (HCs). Our transcriptomic analysis identified 1249 significantly differentially expressed genes, highlighting a pronounced upregulation of inflammatory genes (SERPINA3, IL6R, IL1RAP, TNFRSF11A) and a concomitant downregulation of genes essential for synaptic support and ion channel function (STMN2, NMNAT2, SCN2A, GRIN1). Kinomic profiling revealed dysregulated kinase activities within DYRK, GSK, and MAPK families, further implicating altered kinase signaling pathways in astrocyte dysfunction. Integration of these datasets pinpointed critical molecular hubs, notably within the PI3K signaling and inflammatory pathways, highlighting targets such as JAK2, STAT3, and AKT1 as potential modulators of disease progression. Furthermore, leveraging the Library of Integrated Network-Based Cellular Signatures (LINCS) platform, we identified chemical perturbagens, including fluticasone propionate and Akt inhibitors, capable of reversing the transcriptomic signatures associated with fAD astrocytes. This integrative multiomic approach not only enhances our understanding of astrocyte-specific molecular mechanisms in AD but also provides novel targets for therapeutic intervention aimed at mitigating astrocyte-driven neurodegeneration. Highlights•Familial AD astrocytes display significant pro-inflammatory transcriptomic and kinomic dysregulation.•PI3K and inflammatory signaling pathways are highly dysregulated in familial AD astrocytes.•Expression of inflammatory markers such as SERPINA3, IL6R, and TNFRSF11A is increased in familial AD astrocytes.•Kinase activity analysis identifies DYRK, GSK, and MAPK pathways as key dysregulated axes in familial AD astrocytes.•Potential astrocyte-specific therapeutic approaches to AD include targeting PI3K, JAK, and STAT3.

SummaryLineage-specific differentiation of human induced pluripotent stem cells (hiPSCs) relies on complex interactions between biochemical and physical cues. Here we investigated the ability of hiPSCs to undergo lineage commitment in response to inductive signals and assessed how this competence is modulated by substrate stiffness. We showed that Activin A-induced hiPSC differentiation into mesendoderm and its derivative, definitive endoderm, is enhanced on gel-based substrates softer than glass. This correlated with changes in tight junction formation and extensive cytoskeletal remodeling. Further, live imaging and biophysical studies suggested changes in cell motility and interfacial contacts underlie hiPSC layer reshaping on soft substrates. Finally, we repurposed an ultra-soft silicone gel, which may provide a suitable substrate for culturing hiPSCs at physiological stiffnesses. Our results provide mechanistic insight into how epithelial mechanics dictate the hiPSC response to chemical signals and provide a tool for their efficient differentiation in emerging stem cell therapies. Graphical abstract Highlights•Tuning of substrate stiffness can enhance mesendoderm/endoderm hiPSC differentiation•Altered tight junction formation drives increased differentiation on soft substrates•Changes in cell motility and interfacial contacts underlie hiPSC layer remodeling Mechanobiology; Stem cells research; Biophysics

BackgroundGenome-wide association studies (GWAS) of Alzheimer’s disease (AD) have identified a plethora of risk loci. However, the disease variants/genes and the underlying mechanisms have not been extensively studied.MethodsBulk ATAC-seq was performed in induced pluripotent stem cells (iPSCs) differentiated various brain cell types to identify allele-specific open chromatin (ASoC) SNPs. CRISPR-Cas9 editing generated isogenic pairs, which were then differentiated into glutamatergic neurons (iGlut). Transcriptomic analysis and functional studies of iGlut co-cultured with mouse astrocytes assessed neuronal excitability and lipid droplet formation.ResultsWe identified a putative causal SNP of CLU that impacted neuronal chromatin accessibility to transcription-factor(s), with the AD protective allele upregulating neuronal CLU and promoting neuron excitability. And, neuronal CLU facilitated neuron-to-glia lipid transfer and astrocytic lipid droplet formation coupled with reactive oxygen species (ROS) accumulation. These changes caused astrocytes to uptake less glutamate thereby altering neuron excitability.ConclusionsFor a strong AD-associated locus near Clusterin (CLU), we connected an AD protective allele to a role of neuronal CLU in promoting neuron excitability through lipid-mediated neuron-glia communication. Our study provides insights into how CLU confers resilience to AD through neuron-glia interactions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00840-1.

BackgroundStem cell-derived secreted factors could protect neurons in neurodegenerative disease or after injury. The exact neuroprotective components in the secretome remain challenging to discover. Here we developed a cell-to-cell interaction model to identify a retinal ganglion cell (RGC)-protective factor derived from induced pluripotent stem cells (iPSCs).MethodsPrimary RGCs were co-cultured with iPSCs or treated with iPSC-conditioned media in vitro. Cell viability were assayed using live-cell staining, and culture supernatant were analyzed via multiplexed antibody-based assays and ELISA. In vivo tests were carried out under mouse optic nerve crush model and RGC transplantation study in rats. Paired t-tests were used for data analysis between two groups.ResultsRGC viability was significantly enhanced when iPSCs were first stimulated with RGC-derived supernatant before iPSC-conditioned medium was collected and added into RGC culture. A significant increase of stem cell growth factor-beta (SCGF-?) concentration was detected in the latter conditioned medium. SCGF-? enhanced RGC survival in vitro and in vivo, and RGC-derived interleukin-12(p70) (IL-12[p70]) promotes secretion of iPSC-derived SCGF-?. Downstream of this IL-12(p70)-to-SCGF-? axis, ngn2 was significantly upregulated, and was found both necessary and sufficient for RGC survival.ConclusionThis study addresses a longstanding question of how neurons and stem cells interact to promote neuroprotection, and define a novel molecular interaction pathway whereby RGC’s secretion of IL-12(p70) enhances iPSCs’ secretion of SCGF-?, and SCGF-? protects RGCs via upregulating ngn2, suggesting that neurons may call on stem cells for their own protection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04198-5.

AbstractDespite being one of the most prevalent RNA modifications, the role of N6?methyladenosine (m6A) in amyotrophic lateral sclerosis (ALS) remains ambiguous. In this investigation, we explore the contribution of genetic defects of m6A?related genes to ALS pathogenesis. We scrutinized the mutation landscape of m6A genes through a comprehensive analysis of whole?exome sequencing cohorts, encompassing 508 ALS patients and 1660 population?matched controls. Our findings reveal a noteworthy enrichment of RNA binding motif protein X?linked (RBMX) variants among ALS patients, with a significant correlation between pathogenic m6A variants and adverse clinical outcomes. Furthermore, Rbmx knockdown in NSC?34 cells overexpressing mutant TDP43Q331K results in cell death mediated by an augmented p53 response. Similarly, RBMX knockdown in ALS motor neurons derived from induced pluripotent stem cells (iPSCs) manifests morphological defects and activation of the p53 pathway. Transcriptional analysis using publicly available single?cell sequencing data from the primary motor cortex indicates that RBMX?regulated genes selectively influence excitatory neurons and exhibit enrichment in ALS?implicated pathways. Through integrated analyses, our study underscores the emerging roles played by RBMX in ALS, suggesting a potential nexus between the disease and dysregulated m6A?mediated mRNA metabolism. The dysregulation of m6A modification has gained recognition as a crucial factor in the development of amyotrophic lateral sclerosis (ALS). Among the m6A reader proteins, RNA binding motif protein X?linked (RBMX) stands out with a notable enrichment of variants in ALS patients, and the presence of pathogenic RBMX variants is associated with a faster disease progression. In vitro experiments have provided evidence that reducing RBMX levels can result in neuronal defects. Additionally, bioinformatic analyses have supported these findings by revealing that RBMX?associated genes specifically impact excitatory neurons. Furthermore, these genes are involved in the regulation of pathways and genes associated with neurodegeneration and RNA metabolism, underscoring the relevance of RBMX in ALS pathogenesis.

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants, as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast, larger SVs, which may significantly influence human phenotypes, have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study, we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines, which were not identified by long-read sequencing. Additionally, various exon deletions were confirmed by OGM in the PRKN gene of iPSCs, of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs, no gene fusions, no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary, OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques, which is supportive for OGM’s future use in gene discovery and iPSC line screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-024-10902-1.