Age-Associated Decline in Autophagy Pathways in the Retinal Pigment Epithelium and Protective Effects of Topical Trehalose in Light-Induced Outer Retinal Degeneration in Mice

Age is a primary risk factor for chronic conditions, including age-related macular degeneration (AMD). Impairments in autophagy processes are implicated in AMD progression, but the extent of autophagy’s contribution and its therapeutic potential remain ambiguous. This study investigated age-associated transcriptomic changes in autophagy pathways in the retinal pigment epithelium (RPE) and evaluated the protective effects of topical trehalose, an autophagy-enhancing small molecule, against light-induced outer retinal degeneration in mice. Transcriptomic analysis of human RPE/choroid and mouse RPE revealed consistent downregulation of autophagy pathways with age, alongside variable changes as AMD severity progressed. Given the age- and AMD-associated perturbation of autophagy pathways, we examined trehalose treatment in vitro, which enhanced autophagic flux and restored mitochondrial respiratory function in primary murine RPE cells exposed to oxidative stress. In vivo, pre-treatment with topical trehalose improved autophagy-lysosome activity in mouse RPE, demonstrated by elevated LC3B turnover and SQSTM1/p62 degradation. Furthermore, trehalose eyedrops protected mice from light-induced damage to the RPE and photoreceptors, preserving outer nuclear layer thickness, RPE morphology, and junctional F-actin organization. Taken together, the data support that age-related decline and severe dysregulation in autophagy contributed to AMD progression. By restoring autophagic flux, topical trehalose demonstrates therapeutic potential to address early autophagy-related pathological changes in AMD. Murine RPE cells used for RNA-seq were isolated following an established protocol. Briefly, eyes were enucleated and cleaned using angled scissors to remove any remaining connective tissue. After removing the cornea and lens, the eyecups were incubated at 37°C in hyaluronidase (Sigma-Aldrich) for 45 min, and in Hank’s balanced salt solution with 10 mM HEPES for a further 30 min before retinas were removed via incision. Subsequently, eyecups were incubated at 37°C in trypsin/EDTA for 45 min before being transferred into HBSS with 20% heat-inactivated FCS. Gently shaking facilitated the detachment of RPE sheets, which were further incubated in trypsin/EDTA for 1 min to generate single-cell suspensions. RNA of RPE cells was extracted using the RNeasy Mini kit (Qiagen). The RNA samples were sent to the Genomics Facility at University of Bristol for ScreenTape assessment to ensure RNA integrity (RIN) >7 prior to downstream processes. Library preparation was conducted using Illumina Stranded Total RNA Prep with Ribo Zero Plus (Illumina), followed by sequencing using the Illumina NextSeq 550 platform with 2×75 bp by paired-end reads at a depth of 16 million reads. Galaxy software was used to assess the quality of reads via FastQC, followed by read trimming via Trimmomatic. Reads were then aligned using HISAT2 and quantified using FeatureCounts. Batch correction of the resulting counts from two independent experiments was performed using R Studio v2023.06.1 with ComBat-seq. Following batch correction, the counts were normalized by DESeq2 package and lowly expressed genes (basemean < 20) were excluded. Normalized data of the remaining 14220 genes were processed using iDEP.96 for logarithmic transformation, quality control, and DEG identification.