Single-cell transcriptomics reveals pericyte-glia interactions during neurovascular dysfunction in glaucoma

Abstract

Purpose : Neurovascular dysfunction is a feature of glaucomatous neurodegeneration. This study aimed to elucidate cellular and molecular mechanisms that contribute to blood flow abnormalities during ocular hypertension (OHT)

Methods : OHT was induced by intracameral injection of magnetic microbeads in C57BL/6 mice. Single-cell RNA sequencing (scRNA-seq, 10X Genomics) was used to study gene expression changes in retinal pericytes and glia from OHT and sham control mice (OHT=30 618 cells, Sham=31 936 cells, N=6-8 mice/group). Dimensionality reduction identified diverse cellular clusters with diverse biological pathway enrichment. Given the critical role of calcium (Ca²⁺) in pericyte constriction, we used gene set variation analysis (GSVA) to examine alterations in Ca²⁺ signaling in pericytes. Cell-cell interactions were explored using CellChat. Validation was carried out by retinal immunohistochemistry, ELISA, and focused-ion beam electron microscopy (FIB-SEM)

Results : scRNA-seq analysis revealed two retinal pericyte populations: one characterized by high levels of Cacna1, which encodes the L-type Ca²⁺ voltage-gated channel subunit alpha1c; and another expressing Kcnj8, which encodes the Kir6.1 subunit of an ATP-sensitive potassium channel. OHT-induced changes in Ca²⁺-related pathways, notably upregulation of Cacna1, Ca²⁺ channel complex, and cation transporting ATPase complex (GSVA, non-parameteric Wilcoxon rank sum test, p<0.001). Cell-cell communication analysis demonstrated that pericytes increase their interaction with macroglia (astrocytes and Müller cells) during OHT, which was validated by FIB-SEM. In addition, OHT upregulated S100B, which encodes the Ca²⁺ binding protein S100B, in astrocytes and Müller glia (N=8-17 sample/group, student t-test, p<0.001). Quantification of S100B protein levels by ELISA confirmed a substantial increase in OHT compared to sham controls. Immunolabeling validated increased levels of S100B in retinal and optic nerve head astrocytes and Müller cells in glaucomatous eyes

Conclusions : Our findings identify alterations in Ca²⁺-associated pathways in pericytes subjected to OHT, notably L-type Ca²⁺ voltage-gated channel activity, and implicates enhanced glia-pericyte interactions through glia-derived S100B. This study emphasizes the significant role of Ca²⁺ homeostasis and cross-talk among components of the neurovascular unit in blood flow alterations during glaucoma

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.