Profiling human brain vascular cells using single-cell transcriptomics and organoids.

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.

Targeting ryanodine receptor type 2 to mitigate chemotherapy-induced neurocognitive impairments in mice.

Chemotherapy-induced cognitive dysfunction (chemobrain) is an important adverse sequela of chemotherapy. Chemobrain has been identified by the National Cancer Institute as a poorly understood problem for which current management or treatment strategies are limited or ineffective. Here, we show that chemotherapy treatment with doxorubicin (DOX) in a breast cancer mouse model induced protein kinase A (PKA) phosphorylation of the neuronal ryanodine receptor/calcium (Ca2+) channel type 2 (RyR2), RyR2 oxidation, RyR2 nitrosylation, RyR2 calstabin2 depletion, and subsequent RyR2 Ca2+ leakiness. Chemotherapy was furthermore associated with abnormalities in brain glucose metabolism and neurocognitive dysfunction in breast cancer mice. RyR2 leakiness and cognitive dysfunction could be ameliorated by treatment with a small molecule Rycal drug (S107). Chemobrain was also found in noncancer mice treated with DOX or methotrexate and 5-fluorouracil and could be prevented by treatment with S107. Genetic ablation of the RyR2 PKA phosphorylation site (RyR2-S2808A) also prevented the development of chemobrain. Chemotherapy increased brain concentrations of the tumor necrosis factor-α and transforming growth factor-ß signaling, suggesting that increased inflammatory signaling might contribute to oxidation-driven biochemical remodeling of RyR2. Proteomics and Gene Ontology analysis indicated that the signaling downstream of chemotherapy-induced leaky RyR2 was linked to the dysregulation of synaptic structure-associated proteins that are involved in neurotransmission. Together, our study points to neuronal Ca2+ dyshomeostasis via leaky RyR2 channels as a potential mechanism contributing to chemobrain, warranting further translational studies.

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Intracellular calcium leak as a therapeutic target for RYR1-related myopathies.

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.