The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu)

PLoS One. 2020 Nov 10;15(11):e0241902. doi: 10.1371/journal.pone.0241902. eCollection 2020.

Abstract

Background: Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation.

Methods and results: CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control.

Conclusions: The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia's loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Apoptosis
  • Astrocytes / cytology*
  • Astrocytes / metabolism
  • CRISPR-Cas Systems
  • Cell Proliferation
  • Cells, Cultured
  • Cytochrome P-450 CYP1B1 / genetics*
  • Cytokines / metabolism
  • Glaucoma / congenital*
  • Glaucoma / genetics
  • Humans
  • Male
  • Mesenchymal Stem Cells / cytology*
  • Mesenchymal Stem Cells / metabolism
  • Microglia / cytology*
  • Microglia / metabolism
  • Models, Animal
  • NADP / metabolism
  • Point Mutation*
  • Rats

Substances

  • Cytokines
  • NADP
  • Cyp1b1 protein, rat
  • Cytochrome P-450 CYP1B1

Grants and funding

We thank the King Abdulaziz City for Science and Technology (KACST) for grant No. MS-37-336 and its support and King Abdullah International Medical Research Center (KAMRC) grant (RC17/038/R) for BMA. The funding agencies had no influence on this research, and their role was solely to offer financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.