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Differential gene expression and pathway analysis by RNA sequencing in human OPTN(E50K) retinal ganglion cells

ABSTRACT: Retinal ganglion cells (RGCs) serve as the essential connection between the eye and the brain, with this connection disrupted in blinding disorders such as glaucoma. Numerous cellular mechanisms have been associated with glaucomatous neurodegeneration, and useful models for the study of glaucoma allow for the precise analysis of these degenerative phenotypes. Human pluripotent stem cells (hPSCs) serve as powerful tools for the in vitro analysis of human neurodegenerative diseases, particularly those cellular mechanisms underlying degeneration. Thus, efforts of the current study were initially focused upon the use of hPSCs with an E50K mutation in the Optineurin (OPTN) gene underlying glaucomatous neurodegeneration. CRISPR/Cas9 gene editing approaches were used to introduce the OPTN(E50K) mutation into existing lines of hPSCs, as well as the generation of isogenic control lines from existing OPTN(E50K) patient-derived hPSC lines. When grown from glaucomatous sources, hPSC-derived RGCs developed similarly to isogenic controls. Conversely, at later stages of maturation, OPTN(E50K) RGCs exhibited numerous deficits associated with a neurodegenerative phenotype, including neurite retraction, autophagy pathway disruption, apoptosis, and increased excitability. The results of this study provide an extensive analysis of the OPTN(E50K) mutation in hPSC-derived RGCs, with a number of biochemical and molecular alterations associated with functional changes to these RGCs. The opportunity now exists to further explore the precise cellular pathways leading to RGC death in glaucoma, as well as develop novel translational approaches for glaucoma including pharmacological screening and cell replacement therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE145069 | GEO | 2020/02/11

REPOSITORIES: GEO

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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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Differential gene expression and pathway analysis by RNA sequencing in human OPTN(E50K) retinal ganglion cells

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