Steven A. Sloan

Oligodendrocytes play essential roles in central nervous system development and homeostasis, and their dysfunction is a hallmark of numerous neurological disorders. However, human in vitro systems that support oligodendrocyte lineage progression while enabling the study of disease-relevant states remain limited. Here, we establish human spinal cord organoids (hSpO) and cortico-motor assembloids as platforms to model oligodendrocyte development, neuron-glia interactions, and cytokine-induced dysfunction. We show that hSpO generate oligodendrocyte lineage populations that transcriptionally resemble those found in the developing human spinal cord, and oligodendrocyte progenitor cells that exhibit physiologically-relevant functional properties, including migration and monosynaptic input from neurons. Exposure of assembloids to pro-inflammatory cytokines induces transcriptional changes across the oligodendrocyte lineage, characterized by altered lineage progression and acquisition of disease-associated gene expression programs that mirror signatures observed in multiple sclerosis patient tissue. Together, this work establishes hSpO and assembloids as in vitro systems for studying oligodendrocyte lineage development and disease-associated states in a human multi-cellular context.

Phenotyping cells at transcriptomic and proteomic levels is an essential step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcriptome level abundances modestly correlate, complementary profiling of both is needed. We report a method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously in vitro using BV2 microglial and HEK293 cell lines and in vivo using astrocytic and neuronal Cre driver mice crossed with Rosa26-TurboID knock-in mice. SPARO leverages TurboID to biotinylate RNA-interacting cytosolic proteins, enabling enrichment of proteins for proteomics and protein-associated RNA for transcriptomics. We validate SPARO first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the global proteomes and transcriptomes. The effect of neuroinflammatory activation by lipopolysaccharide is also faithfully captured. We apply SPARO to obtain native-state proteomes and transcriptomes from astrocytes and neurons, thereby validating the approach in vivo. We interrogate mRNA-protein concordance and discordance, providing insights into molecular processes that exhibit uniform or cell type-specific patterns.

Background: ALG13-CDG is an X-linked N-linked glycosylation disorder caused by pathogenic variants in the glycosyltransferase ALG13, leading to severe neurological manifestations. Despite the clear CNS involvement, the impact of ALG13 dysfunction on human brain glycosylation and neurodevelopment remains unknown. We hypothesize that ALG13-CDG causes brain-specific hypoglycosylation that disrupts neurodevelopmental pathways and contributes directly to cortical network dysfunction. Methods: We generated iPSC-derived human cortical organoids (hCOs) from individuals with ALG13-CDG to define the impact of hypoglycosylation on cortical development and function. Electrophysiological activity was assessed using MEA recordings and integrated with multiomic profiling, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was evaluated in both iPSCs and hCOs. Results: ALG13-CDG hCOs showed reduced glycosylation of proteins involved in ECM organization, neuronal migration, lipid metabolism, calcium homeostasis, and neuronal excitability. These pathway disruptions were supported by proteomic and scRNA-seq data and included altered intercellular communication. Trajectory analyses revealed mistimed neuronal maturation with early inhibitory and delayed excitatory development, indicating an E/I imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst structure, and shortened axonal projections, while transcriptomic and proteomic signatures suggested emerging hyperexcitability. Altered lipid and GlcNAc metabolism, along with skewed X-inactivation, were also observed. Conclusions: Our study reveals that ALG13-CDG is a disorder of brain-specific hypoglycosylation that disrupts key neurodevelopmental pathways and destabilizes cortical network function. Through integrated multiomic and functional analyses, we identify early network hypoactivity, mistimed neuronal maturation, and evolving E/I imbalance that progresses to compensatory hyperexcitability, providing a mechanistic basis for seizure vulnerability. These findings redefine ALG13-CDG as disorders of cortical network instability, offering a new framework for targeted therapeutic intervention.

Inhibition of voltage-gated potassium channel Kv1.3 is a therapeutic strategy to curb microglia-mediated neuroinflammation in neurodegeneration, although the cellular and signaling mechanisms of disease-modification by Kv1.3 blockers are unclear. In this study, we delineate protective mechanisms of Kv1.3 blockade in a mouse model of Alzheimers disease (AD) pathology using comprehensive transcriptomics and proteomics profiling of brain, corresponding with neuropathological effects of two translationally relevant Kv1.3 blockers, namely small molecule PAP-1 and peptide ShK-223. Following 3 months of treatment, both molecules reduced Ab plaque burden. Single nuclear RNA seq (snRNA seq) of brain nuclei showed that PAP-1 disproportionately impacted oligodendrocytes and microglia and increased crosstalk between neurons and astrocytes with endothelial cells. In contrast, ShK-223 had pronounced effects on glutamatergic neurons and astrocytes. Both blockers increased expression of myelination genes in oligodendrocytes and synaptic genes in neurons. Neuroprotective effects of PAP-1 were further confirmed by bulk brain transcriptomics and proteomics whereby PAP-1 increased levels of synaptic, cognitive resilience and mitochondrial proteins, while decreasing glial and immune pathways including STAT1/3 phosphorylation. Using proximity labeling and co-immunoprecipitation, we found that Kv1.3 interacts with STAT1/3 in microglia. Using microglial cell lines and primary microglia, we discovered a preferential functional coupling between Kv1.3 and type 2 but not type 1 IFN signaling. Brain-level disease modification by Kv1.3 blockade was reflected in the cerebrospinal fluid (CSF) via reduced levels of neurofilament-light (NEFL) and resilience protein RPH3A, both of which are increased in human AD CSF. Together, this study demonstrates functional links between Kv1.3 channels and type 2 IFN signaling and reveals distinct cellular effects of Kv1.3 blockers in AD pathology that correspond with reduced neuropathology and neuroinflammation, augmentation of resilience and neuro-vascular pathways, along with biomarkers of therapeutic effect.

Glioblastoma (GBM) is a highly heterogenous and malignant brain tumor, in part because it disrupts normal brain circuits to fuel its own growth and invasion. Thus, there is a need to identify the molecular features of invasive GBM cells and their regulators in the neural microenvironment. To address this in a fully human model, we engrafted patient-derived GBM cells (total n= 15 independent samples) from three sources— fresh neurosurgical resections, cell lines, and whole GBM organoids—into human induced pluripotent stem cell-derived organoids patterned to forebrain, midbrain, and spinal cord identities. GBM cells from all sources infiltrated brain organoids within 2 days post-engraftment, reaching maximal invasion by day 14. Across organoids of distinct spatial and maturational milieu, GBM cells showed a consistent reduction in astrocyte-like states and an enrichment in neuron/glia progenitor-like (NPC-like) states. These NPC-like GBM cells expressed neuronal and synaptic machinery, and tumors enriched in this transcriptomic state prior to engraftment achieved greater organoid coverage, suggesting enhanced infiltration and synaptic integration of this GBM cell type. Although GBM cell states converged across organoid types after engraftment, infiltration was greater in the forebrain than spinal cord. This is likely reflective of synaptic input from deep-layer TBR1⁺ excitatory neurons in the forebrain, as demonstrated by a combination of rabies-based monosynaptic tracing and single-cell transcriptomics. In contrast, inhibitory neurons were the predominant synaptic partners of GBM in the spinal cord. Together, this fully human model of the neural-GBM connectome reveals how neuron-like GBM states and regionally distinct synaptic inputs cooperatively shape tumor invasion.

The heavy metal lead (Pb) is a developmental neurotoxicant associated with cognitive and behavioral deficits, but the cellular mechanisms underlying these impairments remain unclear. Here we show that prenatal Pb exposure biases human radial glia fate, prolonging neurogenesis and suppressing astrogenesis. We used hiPSC-derived cortical organoids, primary human fetal tissue, and in vivo xenografts to demonstrate that Pb exposure alters radial glial differentiation. Pb-exposed organoids contain a higher proportion of neurons and fewer astrocytes. We validated this differentiation bias in primary radial glia from human cortices (GW16-20), observing Pb-associated reductions in astrocyte commitment via genetic lineage tracing. This correlated with increased H3K27me3, a repressive histone modification deposited by the histone methyltransferase complex PRC2, suggesting epigenetic reprogramming as a mechanistic link between Pb and neural cell fate commitment. Our findings indicate that prenatal Pb exposure impacts lineage commitment in the developing brain, which may contribute to cognitive and behavioral impairment.

Measuring splicing and chromatin accessibility simultaneously in frozen tissues remains challenging. Here we combined single-cell isoform RNA sequencing and assay for transposase accessible chromatin (ScISOr–ATAC) to interrogate the correlation between these modalities in single cells in human and rhesus macaque frozen cortical tissue samples. Applying a previous definition of four ‘cell states’ in which the transcriptome and chromatin accessibility are coupled or decoupled for each gene, we demonstrate that splicing patterns in one cell state can differ from those of another state within the same cell type. We also use ScISOr–ATAC to measure the correlation of chromatin and splicing across brain cell types, cortical regions and species (macaque and human) and in Alzheimer’s disease. In macaques, some excitatory neuron subtypes show brain-region-specific splicing and chromatin accessibility. In human and macaque prefrontal cortex, strong evolutionary divergence in one molecular modality does not necessarily imply strong divergence in another modality. Finally, in Alzheimer’s disease, oligodendrocytes show high dysregulation in both chromatin and splicing.

Chronic stress is a major risk factor for neuropsychiatric disorders, acting via increased neuroinflammation and disrupted synaptic plasticity. While non-invasive visual or audiovisual neurostimulation (AV flicker) at 40Hz has been shown to modulate brain immune signaling and improve cognitive performance in mouse models of Alzheimer’s disease, its effects in the context of stress remain unknown. Here we show that AV flicker protects against stress-induced behavioral, microglial, astrocytic, and synaptic changes in a sex- and frequency-specific manner. Male and female mice underwent 28 days of chronic unpredictable stress with concomitant daily AV flicker exposure at 10Hz, 20Hz, or 40Hz. Stress-induced behaviors were most effectively mitigated by 10Hz AV flicker in males and 40Hz AV flicker in females. In the medial prefrontal cortex, AV flicker normalized the balance of mature and immature dendritic spines and counteracted stress-induced molecular changes in neurons, microglia, and astrocytes, including in key neuropsychiatric risk genes. These findings show that frequency optimized AV flicker induces resilience to chronic stress.