Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking...

Area of Science:

• Cell Biology
• Genomics
• Ophthalmology

Background:

• The retinal pigment epithelium (RPE) plays a critical role in photoreceptor health and waste clearance.
• Dysfunctional RPE contributes to blinding diseases like age-related macular degeneration and retinitis pigmentosa.
• Understanding RPE's response to photoreceptor outer segment (POS) turnover is crucial for ocular health.

Purpose of the Study:

• To comprehensively analyze the transcriptome-level response of human RPE cells to photoreceptor outer segment (POS) phagocytosis.
• To elucidate the temporal dynamics of gene expression related to the autophagy-lysosomal pathway and other proteolytic systems during POS processing.
• To identify transcriptional orchestration patterns indicative of RPE's homeostatic and stress responses.

Main Methods:

• RNA-sequencing (RNA-seq) analysis of the ARPE-19 human RPE cell line cultured on transwells.
• Exposure of RPE cell monolayers to synchronized photoreceptor outer segments (POS).
• Sample collection at multiple time points (4, 6, 24, and 48 hours) post-POS feeding for gene expression analysis.

Main Results:

• RNA-seq identified 44,909 genes, with 83.35% alignment to the human reference genome, including RPE-specific and retinopathy-associated transcripts.
• Differential gene expression analysis revealed a temporal pattern of gene orchestration consistent with POS breakdown via the autophagy-lysosomal pathway.
• Early (4h) upregulation of genes involved in endosome maturation and cargo transport, followed by a shift towards cargo degradation in autolysosomes (24-48h).
• POS exposure also induced transcriptional upregulation in ubiquitin proteasome and chaperone-mediated degradation systems within 4-6 hours, indicating cross-talk.

Conclusions:

• RPE cells exhibit a dynamic and coordinated transcriptional response to POS phagocytosis and degradation.
• The findings provide detailed insights into the molecular mechanisms governing RPE's role in photoreceptor renewal and waste management.
• This study highlights the complex interplay between different cellular proteolytic pathways in maintaining RPE function and potentially preventing retinal disease.