Abstract
Purpose :
Left unbuffered, reactive oxygen species in the ocular lens can lead to protein aggregation and age-related nuclear cataracts. Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) is a transcription factor that responds to oxidative stress and upregulates the transcription of antioxidant genes to protect cells from the continual production of reactive oxygen species. While proteostasis is maintained by diverse families of heat shock proteins, the interplay between the oxidative and proteostatic stress responses in the lens has not been investigated.
Methods :
After phenotyping, we performed RNA-sequencing on multiple zebrafish lines that have compromised function of Nrf2 and/or the two paralogs of the small heat-shock protein αB-crystallin. Total RNA was extracted from homogenized 1-year-old pooled lens tissue from 3 fish and the resulting cDNA library was sequenced on an Illumina NovaSeq 6000 instrument. Proteomics was also performed and compared to transcriptomic results to identify protein networks involved in these pathways. Briefly, the cortical and nuclear regions of 11 and 7-month-old unpooled zebrafish lenses were separated via centrifugation and homogenized. Samples were analyzed via data-independent acquisition (DIA) LC-MS/MS on an Exploris 480 instrument to sequence all precursors within defined m/z windows and probe deeply into the zebrafish lens proteome.
Results :
Surprisingly, our results showed that the loss of Nrf2 mitigated the phenotypic consequences of the αBa-crystallin knockout. Additionally, transcriptomic analysis revealed that this genotype leads to upregulation of the cholesterol biosynthesis pathway in the lens. Baseline WT proteomics identified over 1,000 protein groups, surpassing published zebrafish lens proteomic datasets. Other potential redox pathways that could be involved in this interaction include the synthesis of glutathione. However, further experiments are needed to delineate the intersection between the cholesterol synthesis pathway and Nrf2 in the lens.
Conclusions :
These results establish a previously undiscovered intersection between the oxidative stress and chaperone responses in the lens. Using this integrated multi-omics approach, multiple molecular pathways were revealed, identifying novel targets for further investigation of age-related nuclear cataracts.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.