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Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment

ABSTRACT: Since the trabecular meshwork (TM) is central to intraocular pressure (IOP) regulation and glaucoma, a deeper understanding of its genomic landscape is needed. We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for 1) extracellular matrix structure and metabolism (TM1 subtype), 2) secreted ligand signaling to support Schlemm’s canal cells (TM2), and 3) contractile and mitochondrial/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in LMX1B cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from Lmx1bV265D/+ mutant mice (2,491 TM cells). In V265D/+ mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. Mitochondria in TM cells of V265D/+ mice are swollen with a reduced cristae area, further supporting a role for mitochondrial dysfunction in the initiation of IOP elevation in these mice. Importantly, treatment with vitamin B3 (nicotinamide), to enhance mitochondrial function and metabolic resilience, significantly protected Lmx1b mutant mice from IOP elevation.

ORGANISM(S): Mus musculus

PROVIDER: GSE309500 | GEO | 2025/11/10

REPOSITORIES: GEO

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Project description:Purpose: Primary open-angle glaucoma (POAG) is the most common glaucoma with elevated intraocular pressure (IOP). Dysfunction of trabecular meshwork (TM) cells affects the outflow facility of aqueous humor, resulting in high IOP. Phagocytosis is an important function for TM cells, this study aims to identify phagocytosis related genes/proteins and the effect of lovastatin in glaucomatous TM cells. Methods: The phagocytic ability of immortal normal human TM cells (iHTM) and glaucomatous TM cells (GTM3) was detected by fluorescent beads and flow cytometry. RNA sequencing and proteomic analysis were used to identify different genes and proteins that potentially involved in regulating the phagocytic function of TM cells. The expression of low density lipoprotein receptor (LDLR) in iHTM and GTM3 was validated by immunofluorescence staining and Western blot. Lovastatin was used to test the effect of LDLR on the phagocytosis of TM cells. The expression of LDLR and the phagocytic ability was measured by western blot and fluorescent beads respectively. Results: The Phagocytosis of GTM3 was significantly weaker than that of iHTM. The integrated analysis upon the transcriptome and proteome identified 3 differentially expressed genes/proteins closely related to phagocytosis of cells, including DAB2: NM_001244871.1, DNAJC6: NM_001256864.1 and LDLR: NM_000527.4. Their expression in GTM3 was obviously lower than that in iHTM. In addition, we have further illustrated the decreased expression of LDLR in GTM3 by immunofluorescence staining and Western blot. However, Lovastatin was found to increase its expression and the phagocytosis ability of GTM3. Conclusions: Three phagocytosis-related genes/proteins were obviously decreased in GTM3 compared to iHTM, they might be responsible for the decreased phagocytic function of TM cells in glaucoma. The up-regulation of LDLR by Lovastatin can enhanced the phagocytosis of GTM3, suggesting Lovastatin might be a new potential medicine to promote the phagocytosis of TM cells in glaucoma.

Project description:Purpose: The optic nerve head (ONH) is the likely site of initial damage in the glaucomatous eye. Despite the recognition of elevated intraocular pressure (IOP) as a leading risk factor for the development of glaucoma, ocular hypertension (OHT) eyes displaying consistently elevated IOP do not experience ONH damage. This study aims to identify global gene expression variations in glaucomatous ONHs and their relationship to those identified in OHT derived ONHs in order to improve our understanding of IOP-induced ONH damage. Methods: (N=6) ONHs were collected from clinically confirmed glaucoma, OHT and age-matched control donor eyes. Total RNA extracted from ONHs was reverse transcribed and assayed using the Affymetrix Human Exon 1.0 ST array. Differentially expressed genes in glaucoma versus control and OHT derived ONHs were identified using an ANOVA analysis with a 1.25 fold change limit and P-value < 0.05. Quantitative RT-PCR was performed to validate selected differentially expressed genes. Results: Microarray analysis revealed 149 under under-expressed genes in POAG versus control ONHs, many of which are involved in ion transport, axonogenesis and macromolecule catabolic processes. 297 genes were over expressed in OHT versus glaucoma derived ONHs. Mediators of oxidation-reduction and chemical homeostasis were among the most prominent gene groups identified. The over expression of prostaglandin-endoperoxide synthase 2, integrin, beta-like 1 and fibulin 5 in glaucomatous ONHs was confirmed by qRT-PCR. Conclusions: Our data demonstrates marked alteration in global gene expression patterns in the glaucomatous ONH, likely due to extensive tissue injury. The observed overlapping of several differentially expressed genes in glaucoma and OHT derived ONHs suggests the induction of common mechanisms in response to elevated IOP. Preferential over-expression of certain gene groups in OHT but not glaucoma derived ONHs may confer possible protection against IOP-induced ONH damage, which remains to be investigated in future studies. (N=6) Glaucoma, ocular hypertension and age-matched control ONHs were assayed to investigate and compare global gene expression patterns in each sample group.

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Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment

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