Biology subjects

Bubnys, A.

Publications and source records attributed to Bubnys, A..

5 recordsLinked to original sources

bioRxiv2025

Genome-wide CRISPR screen reveals Wnt signaling defects regulate lipid accumulation in APOE4 oligodendrocytes

APOE4 is the largest genetic risk factor for late-onset Alzheimers disease, but the cellular mechanisms by which APOE variants influence risk of disease remain incompletely understood. We have previously found that APOE4 expression led to the intracellular accumulation of lipid droplets in oligodendrocytes, causing decreased myelination. However, the mechanisms by which APOE4 alters lipid metabolism are not fully understood. Here, we leveraged a genome-wide CRISPR screen and ATAC-sequencing in human induced pluripotent stem cell (iPSC)-derived oligodendrocytes to dissect APOE4s lipid-associated mechanisms of action. Using these approaches, we identified decreased Wnt signaling, and overactive GSK3b activity, as regulators of lipid droplet accumulation in oligodendrocytes. Genetic and pharmacological inhibition of GSK3b reduced lipid droplets in APOE4 oligodendrocytes, and increased myelination in three-dimensional iPSC-derived brain organoids. Finally, we show that pharmacological inhibition of GSK3b reduces lipid droplets and improves myelination in APOE4;PS19 Tau transgenic mice. Together, our results provide a framework for understanding the mediation of APOE4-related changes to oligodendrocyte lipid metabolism and myelination.

neuroscience↗

bioRxiv2025

Vascular-Perfusable Human 3D Brain-on-Chip

Development and delivery of treatments for neurological diseases are limited by the tight and selective human blood-brain barrier (BBB). Although animal models have been important research and preclinical tools, the rodent BBB exhibits species differences and fails to capture the complexity of human genetics. Microphysiological systems incorporating human-derived cells hold great potential for modeling disease and therapeutic development, with advantages in screening throughput, real-time monitoring, and tunable genetic backgrounds when combined with induced pluripotent stem cell (iPSC) technology. Existing 3D BBB-on-chip systems have incorporated iPSC-derived endothelial cells but not the other major brain cell types from iPSCs, each of which contributes to brain physiology and disease. Here we developed a 3D Brain-Chip system incorporating endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia from iPSCs. To enable this multicellular 3D co-culture in-chip, we designed a GelChip microfluidic platform using a 3D printing-based approach and dextran-based engineered hydrogel. Leveraging this platform, we co-cultured and characterized iPSC-derived brain-on-chips and modeled the brain microvasculature of APOE4, the strongest known genetic risk factor for sporadic Alzheimers disease. These 3D brain-on-chips provide a versatile system to assess BBB vascular morphology and function, investigate downstream neurological effects in disease, and screen therapeutics to optimize delivery to the brain. Significance StatementThe blood-brain barrier (BBB) is both a contributing factor to neurological disease and a major obstacle to its treatment, yet human-relevant models remain limited. Most existing brain-on-chip systems incorporate only subsets of BBB cell types and cannot capture the full cellular complexity of the human neurovascular unit. Here, we establish a vascular-perfusable 3D Brain-Chip using human induced pluripotent stem cell-derived brain cells including endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia. This system enables systematic analysis of human genetic risk factors, such as APOE4 in Alzheimers disease, and provides a powerful platform to investigate BBB function and dysfunction and accelerate the development of more effective neurological therapies.

neuroscience↗

bioRxiv2023

Engineered 3D Immuno-Glial-Neurovascular Human Brain Model

Patient-specific, human-based cellular models integrating a biomimetic blood-brain barrier (BBB), immune, and myelinated neuron components are critically needed to enable accelerated, translationally relevant discovery of neurological disease mechanisms and interventions. By engineering a novel brain-mimicking 3D hydrogel and co-culturing all six major brain cell types derived from patient iPSCs, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model with in vivo-like hallmarks inclusive of neuronal activity, functional connectivity, barrier function, myelin-producing oligodendrocyte engagement with neurons, multicellular interactions, and transcriptomic profiles. We implemented the model to study Alzheimers Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Unlike the co-emergent fate specification of glia and neurons in organoids, miBrains integrate independently differentiated cell types, a feature we harnessed to identify that APOE4 in astrocytes promotes neuronal tau pathogenesis and dysregulation through crosstalk with microglia.

neuroscience↗

bioRxiv2022

Single-cell multi-region dissection of brain vasculature in Alzheimer's Disease

Cerebrovascular breakdown occurs early in Alzheimers Disease (AD), but its cell-type-specific molecular basis remains uncharacterized. Here, we characterize single-cell transcriptomic differences in human cerebrovasculature across 220 AD and 208 control individuals and across 6 brain regions. We annotate 22,514 cerebrovascular cells in 11 subtypes of endothelial, pericyte, smooth muscle, perivascular fibroblast, and ependymal cells, and how they differ in abundance and gene expression between brain regions. We identify 2,676 AD-differential genes, including lower expression of PDGFRB in pericytes, and ABCB1 and ATP10A in endothelial cells. These AD-differential genes reveal common upstream regulators, including MECOM, EP300, and KLF4, whose targeting may help restore vasculature function. We find coordinated vasculature-glial-neuronal co-expressed gene modules supported by ligand-receptor pairs, involved in axon growth/degeneration and neurogenesis, suggesting mechanistic mediators of neurovascular unit dysregulation in AD. Integration with AD genetics reveals 125 AD-differential genes directly linked to AD-associated genetic variants (through vasculature-specific eQTLs, Hi-C, and correlation-based evidence), 559 targeted by AD-associated regulators, and 661 targeted by AD-associated ligand-receptor signaling. Lastly, we show that APOE4-genotype associated differences are significantly enriched among AD-associated genes in capillary and venule endothelial cells, and subsets of pericytes and fibroblasts, which underlie the vascular dysregulation in APOE4-associated cognitive decline. Overall, our multi-region molecular atlas of differential human cerebrovasculature genes and pathways in AD can help guide early-stage AD therapeutics.

neuroscience↗

bioRxiv2019

Hindbrain V2a neurons impose rhythmic activity on motor neurons in an in vitro reticulospinal circuit

The reticulospinal system is an evolutionarily conserved pathway among vertebrates that relays locomotor control signals from the hindbrain to the spinal cord. Recent studies have identified specific hindbrain cell types that participate in this circuit, including Chx10+ neurons of the medullary reticular formation, which project to the spinal cord and are active during periods of locomotion. To create a system in which reticulospinal neurons communicate with spinal motor effectors, we have constructed an in vitro model using two purified excitatory neuronal subtypes: HB9+ spinal motor neurons and Chx10+ hindbrain neurons. Cultured separately, these neurons exhibit cell type-specific patterns of activity; the Chx10+ cultures developed regular, synchronized bursts of activity that recruited neurons across the entire culture, whereas motor neuron activity consisted of an irregular pattern. A combination of the two subtypes produced cultures in which Chx10+ neurons recruited the motor neurons into synchronized network bursts, which were dependent on AMPA receptors. In addition to demonstrating that the activity of in vitro networks can depend on the developmental identity of their constituent neurons, we provide a new model with genetically specified nerve cell types to study the activity of a reticulospinal circuit.\n\nSignificance statementModels of the brain that use cultured neurons are usually comprised of a complex mixture of different kinds of cells, making it hard to determine how each cell type contributes to the overall pattern of activity. We made a simplified culture containing two cell types known to form a reticulospinal circuit in vivo. While in isolated cultures, each cell type had a distinct pattern of activity, in coculture the activity of one cell type came to dominate, indicating that the patterns observed in complex neuronal cultures arise in part from the distinctive properties of the constituent neurons.

neuroscience↗