Cell atlas of trabecular meshwork in glaucomatous non-human primates and DEGs related to tissue contraction based on single-cell transcriptomics

Dysfunction of TM, which is the main channel of aqueous humor outflow, can lead to elevated intraocular pressure (IOP) and result in primary open-angle glaucoma (POAG), but the molecular mechanism is still unclear. In this research, single-cell RNA sequencing (scRNAseq) analysis of TM were performed in spontaneous POAG and healthy macaques to compare cellular heterogeneity, thus exploring differentially expressed genes (DEGs) and pathways associated with dysfunction of TM contraction. Here, we show a comprehensive cell atlas of TM upon clustering analysis based on single-cell transcriptomics, which 14 distinct cell types were identified and some of them were associated with tissue contraction. The proportions of each cell types between spontaneous POAG and healthy macaques were different. Multiple genes associated with TM contraction were identified in Beam A, Beam B, Beam C and SMC cell types, with TPM1 and its associated ACTC1 and TNNT1 being first found. TPM1, ACTC 1 and TNNT1 were considered to be important protein in the regulation of tissue contraction, and the expressions of them were confirmed to be located in the sieve-like region of TM, which expressions in POAG models were lower than that in normal models. Altered levels of TPM1 expression have significant impact on TNNT1 and ACTC1 expression downstream of it. In addition, the microstructural alterations in TM of POAG non-human primate were observed, which was compact and collapsed. Thus, our study indicated that TPM1 may be a key target for regulating TM structure, contraction function and resistance of aqueous humor outflow. Overall design: To explore the cell types and related genes and pathways involved in the relaxation and contraction of the sieve pore of TM in POAG, we screened spontaneous POAG monkey models. We analyzed the scRNAseq results and the healthy controls in our study. Having carefully compared the two groups in cell types, cell proportion, and differentially expressed genes, we unveiled the principal pathways regulating the aqueous outflow in the TM tissue.

小梁网（TM）是房水流出的主要通路，其功能异常可导致眼压（intraocular pressure, IOP）升高，进而引发原发性开角型青光眼（primary open-angle glaucoma, POAG），但其具体分子机制目前仍未阐明。
本研究对自发性POAG猕猴与健康猕猴的小梁网组织开展单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析，以比较二者的细胞异质性，进而探索与小梁网收缩功能异常相关的差异表达基因（differentially expressed genes, DEGs）及信号通路。
本研究基于单细胞转录组学进行聚类分析，构建了小梁网的完整细胞图谱，共鉴定出14种不同的细胞类型，其中部分细胞类型与组织收缩功能相关。自发性POAG猕猴与健康猕猴的各细胞类型占比存在显著差异。
研究人员在Beam A、Beam B、Beam C及平滑肌细胞（smooth muscle cell, SMC）类型中鉴定出多个与小梁网收缩相关的基因，其中TPM1及其关联基因ACTC1、TNNT1为首次发现。TPM1、ACTC1及TNNT1被认为是调控组织收缩的关键蛋白，经证实它们的表达定位于小梁网的筛状区域，且在POAG模型中的表达水平显著低于正常模型。TPM1表达水平的改变会对其下游的TNNT1和ACTC1的表达产生显著影响。
此外，研究观察到POAG非人灵长类动物小梁网的微观结构发生改变，表现为组织致密且塌陷。综上，本研究表明TPM1可能是调控小梁网结构、收缩功能及房水流出阻力的关键靶点。
研究设计：为探索POAG模型中小梁网筛孔舒张与收缩相关的细胞类型、关联基因及信号通路，本研究筛选了自发性POAG猕猴模型。本研究对scRNAseq数据及健康对照样本进行了分析，通过细致比较两组在细胞类型、细胞占比及DEGs方面的差异，揭示了调控小梁网组织房水流出的核心信号通路。