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Title: A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging
The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion—critical processes underlying neuronal health and function, and in turn, memory and cognition.  more » « less
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  1. Abstract

    Brain pericytes regulate diverse aspects of neurovascular development and function, including blood‐brain barrier (BBB) induction and maintenance. Primary brain pericytes have been widely employed in coculture‐based in vitro models of the BBB, and a method to generate brain pericytes from human pluripotent stem cells (hPSCs) could provide a renewable, genetically tractable source of cells for BBB modeling and studying pericyte roles in development and disease. Here, we describe a protocol to differentiate hPSCs to NG2+PDGFRβ+αSMAlowbrain pericyte‐like cells in 22‐25 days through a p75‐NGFR+HNK‐1+neural crest intermediate, which mimics the developmental origin of forebrain pericytes. The resulting brain pericyte‐like cells have molecular and functional attributes of brain pericytes. We also provide protocols for maintenance, cryopreservation, and recovery of the neural crest intermediate, and for molecular and functional characterization of the resulting cells. © 2021 Wiley Periodicals LLC.

    This article was corrected on 18 July 2022. See the end of the full text for details.

    Basic Protocol 1: Differentiation of hPSCs to neural crest

    Basic Protocol 2: Differentiation of neural crest to brain pericyte‐like cells

    Support Protocol 1: Flow cytometry analysis of neural crest cells

    Support Protocol 2: Maintenance, cryopreservation, and recovery of neural crest cells

    Support Protocol 3: Molecular characterization of brain pericyte‐like cells

    Support Protocol 4: Cord formation assay with endothelial cells and brain pericyte‐like cells

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