Abstract
Purpose : Protein folding chaperones have critical roles in proteostasis. In the human ageing brain and neurodegenerative disease, there is a significant decrease in the expression of these proteins. This study aimed to investigate their expression in relation to ageing in the retinal pigment epithelium (RPE).
Methods : The publicly available microarray transcriptome dataset GSE29801 of RPE/choroid was re-analysed to assess the ageing effect on gene expression in RPE/choroid. A linear model using limma was applied to find the differential expressed genes with age, and the respective significant genes were further subjected to gene set enrichment analysis. Fifteen RNA samples of human RPE/choroid (47-83 years old) were used to validate the significance of ageing for DNAJB1 (HSP40) gene. DNAJB1 expression was also assessed in ARPE-19 cells cultured on extracellular matrix exposed to glycolaldehyde to induce glycation end products (AGE) or exposed to lipid peroxidation (LPE) by qPCR and Western blot. Two-way ANOVA was performed for statistical analysis.
Results : The transcriptome data of 96 human RPE/choroid (9-93 years old) was analysed by limma. The analysis showed 620 genes differentially expressed with ageing (277 upregulated and 343 downregulated). Among the differentially expressed genes, gene-set enrichment analysis showed co-chaperone activity as a significant gene set (FDR<0.05), including down-regulation of DNAJB1, DNAJB4 and DNAJA1. The negative correlation of DNAJB1 transcription with ageing was validated in an independent set of 15 RPE/choroid samples by qPCR (p<0.05). There was an increase in mRNA expression but lower protein expression of DNAJB1 in ARPE-19 cells cultured in the presence of AGE and LPE (p<0.05).
Conclusions : Differential expression of the chaperone DNAJB1 in RPE/choroid of increased ageing was characterised using a publicly available human transcriptome RPE/choroid dataset, in-house human RNA samples extracted from human RPE/choroid and AGE-/LPE-exposed ARPE-19 cells. The results indicate the downregulation of DNAJB1 in ageing RPE. In the presence of AGE and LPE, DNAJB1 protein level is reduced in ARPE-19 cells, possibly due to increased turnover, but the cells might subsequently compensate by increasing mRNA expression of DNAJB1. Our results suggested that DNAJB1 could be a critical regulator of proteostasis in ageing RPE/AMD similar to the ageing brain and other neurodegenerative diseases.
Methods : The publicly available microarray transcriptome dataset GSE29801 of RPE/choroid was re-analysed to assess the ageing effect on gene expression in RPE/choroid. A linear model using limma was applied to find the differential expressed genes with age, and the respective significant genes were further subjected to gene set enrichment analysis. Fifteen RNA samples of human RPE/choroid (47-83 years old) were used to validate the significance of ageing for DNAJB1 (HSP40) gene. DNAJB1 expression was also assessed in ARPE-19 cells cultured on extracellular matrix exposed to glycolaldehyde to induce glycation end products (AGE) or exposed to lipid peroxidation (LPE) by qPCR and Western blot. Two-way ANOVA was performed for statistical analysis.
Results : The transcriptome data of 96 human RPE/choroid (9-93 years old) was analysed by limma. The analysis showed 620 genes differentially expressed with ageing (277 upregulated and 343 downregulated). Among the differentially expressed genes, gene-set enrichment analysis showed co-chaperone activity as a significant gene set (FDR<0.05), including down-regulation of DNAJB1, DNAJB4 and DNAJA1. The negative correlation of DNAJB1 transcription with ageing was validated in an independent set of 15 RPE/choroid samples by qPCR (p<0.05). There was an increase in mRNA expression but lower protein expression of DNAJB1 in ARPE-19 cells cultured in the presence of AGE and LPE (p<0.05).
Conclusions : Differential expression of the chaperone DNAJB1 in RPE/choroid of increased ageing was characterised using a publicly available human transcriptome RPE/choroid dataset, in-house human RNA samples extracted from human RPE/choroid and AGE-/LPE-exposed ARPE-19 cells. The results indicate the downregulation of DNAJB1 in ageing RPE. In the presence of AGE and LPE, DNAJB1 protein level is reduced in ARPE-19 cells, possibly due to increased turnover, but the cells might subsequently compensate by increasing mRNA expression of DNAJB1. Our results suggested that DNAJB1 could be a critical regulator of proteostasis in ageing RPE/AMD similar to the ageing brain and other neurodegenerative diseases.
| Original language | English |
|---|---|
| Publication status | Published - 1 Jun 2022 |
| Event | 2022 ARVO Annual Meeting - event held in Denver and also virtually, Denver, United States Duration: 1 May 2022 → 4 May 2022 |
Conference
| Conference | 2022 ARVO Annual Meeting |
|---|---|
| Country/Territory | United States |
| City | Denver |
| Period | 1/05/22 → 4/05/22 |
