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RYR1 encodes the type 1 ryanodine receptor, an intracellular calcium release channel (RyR1) on the skeletal muscle sarcoplasmic reticulum (SR). Pathogenic RYR1 variations can destabilize RyR1 leading to calcium leak causing oxidative overload and myopathy. However, the effect of RyR1 leak has not been established in individuals with RYR1 -related myopathies ( RYR1 -RM), a broad spectrum of rare neuromuscular disorders. We sought to determine whether RYR1 -RM affected individuals exhibit pathologic, leaky RyR1 and whether variant location in the channel structure can predict pathogenicity. Skeletal muscle biopsies were obtained from 17 individuals with RYR1 -RM. Mutant RyR1 from these individuals exhibited pathologic SR calcium leak and increased activity of calcium-activated proteases. The increased calcium leak and protease activity were normalized by ex-vivo treatment with S107, a RyR stabilizing Rycal molecule. Using the cryo-EM structure of RyR1 and a new dataset of > 2200 suspected RYR1 -RM affected individuals we developed a method for assigning pathogenicity probabilities to RYR1 variants based on 3D co-localization of known pathogenic variants. This study provides the rationale for a clinical trial testing Rycals in RYR1 -RM affected individuals and introduces a predictive tool for investigating the pathogenicity of RYR1 variants of uncertain significance.

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) regulate exchange between the peripheral circulation and the central nervous system (CNS). During development, these barriers have a selective permeability that differs from adult states, creating both vulnerabilities and therapeutic opportunities. This dissertation addresses fundamental questions about blood-CNS barrier during development. (1) What endothelial and mural cell subtypes are present during early CNS vascular development? (2) How do these subtypes map to regions of differing barrier permeability? (3) What molecular signatures distinguish these regions? Chapter 2 presents a single-cell atlas of mouse brain and spinal cord vasculature at embryonic day (E)13.5 and E18.5, identifying endothelial and mural subtypes that establish arteriovenous zonation. Trajectory analysis revealed maturation programs progressing from proliferation through angiogenesis to transporter-rich barrier states, with region-specific signatures distinguishing brain from spinal cord. Chapter 3 developed a \"BBB scorecard\" quantifying CNS-specific signatures. Combined with permeability mapping and RNA in situ hybridization, permeable zones align with specific vascular subtypes. Col15a1 was identified as a potential marker of permeable territories. Chapter 4 investigated immune-vascular interactions, identifying galectin-9 as a potential mediator and CD206+ macrophages in multiple positions relative to vessels, consistent with transmigration. These findings establish underscore vascular subtype composition and regional specialization in blood-CNS barrier function.

Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development. Key points This protocol extension describes the purification of prenatal human brain endothelial and mural cells with FACS and their utilization in downstream applications, including cell culture, organoid transplantation and single-cell transcriptomics. This simple, efficient protocol has relatively few steps compared with other methods and uses inexpensive reagents. Robust yields of healthy vascular and perivascular cells can be obtained in 6 h. A protocol extension describing the purification of prenatal human brain endothelial and mural cells with FACS and their utilization in downstream applications, including cell culture, organoid transplantation and single-cell transcriptomics.

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45 + cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45 + cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain’s innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants. Chen et al. show that subtypes of immune cells in prenatal human brain promote angiogenesis in the germinal matrix. Conversely, in preterm infants, proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage.