Abstract
Purpose :
Time-dependent deterioration of retinal structure and function is responsible for visual impairment and can precede pathological conditions. However, the molecular mechanisms underlying the gradual vision loss during aging are poorly understood. Compromised transcriptional integrity is a common signature of mammalian aging. As such, transcriptomic studies have examined the impact of age on whole retina or purified rod cells, the dominant neuronal type in most mammalian retinas. Given the over-representation of rod cells in previous studies, we designed an optimized single-cell RNA sequencing (scRNA-seq) protocol to identify age-associated changes in gene expression of rods and the understudied non-rod retinal neurons.
Methods :
Retinas from 3-, 12-, 18- and 24-month-old mice were enzymatically dissociated and cells from rod-depleted, via CD73 cell surface maker, and whole retinas were taken for analysis. scRNA-seq was performed using the 10X Genomics platform. An optimized Cell Ranger/Seurat pipeline including decontX, DoubletFinder, and SCTransform tools was developed. Cell type and age dependent differential expression analyses were performed using the Wilcoxon Rank Sum test.
Results :
We identified altered transcriptomic profiles of the 6 major neuronal types in the aging mouse retina. Single-cell transcriptomic profiles of ~18,500 cells per age resulted in 36 distinct clusters representing the diversity of the retina. Over 97% rod depletion allowed us to explore underrepresented cell types. The gene number detected per cell was constant among distinct cell types and time points. However, the number of differentially expressed genes increased with age. Common transcriptionally dysregulated genes enriched for mitochondria, protein synthesis and stress-response (upregulated), and energy metabolism, axon growth and guidance, and synaptic transmission (downregulated). Cell type-specific aberrant expression was detected for genes with specialized functions such as the phototransduction cascade.
Conclusions :
By depleting rods, we identified cell type-specific aging-related changes of minor neuronal types in the retina. Numerous dysregulated genes are associated with known retinal pathologies, suggesting that these accumulative changes may contribute to late-onset visual decline. This work contributes to elucidation of molecular markers and cell-type specific neuronal pathways involved in the aging process of the retina.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.