Abstract
Purpose :
Recent studies suggest that diabetes affects retinal neurons before onset of more noticeable vascular anomalies. Using cultured neurons and adult retinal explants, we tested the hypothesis that Cell Adhesion Molecules (CAMs) regulate growth of neuronal processes or neurites. We also investigated glucose-sensitive transcriptional regulation of neuronal CAMs and CAM interactions within retinal layers.
Methods :
Caspr1 KO was created using CRISPR-Cas9 in Neuro2a and 661W cell lines. Cells were labelled with F-actin and microtubule probes (n=4). Live imaging was done up to 12 hours, and neurite length measured from randomly chosen fields. To check transcriptional regulation of Caspr1, cells were treated with Actinomycin D for 4-6 hours (10µg/ml, n=6). Luciferase reporter assay was performed in cells transfected with Caspr1 promoter reporter construct (n=6). Retinal explants from 10-week old male chicken were cultured in 5mM or 25 mM glucose up to 6 days, cryosectioned 10µm and stained using CAM-specific antibodies for IHC (n=4).
Results :
Caspr1 KO cells show significantly increased neurite lengths (23.4±1.5, 22.2±2.6, 25.6±1.3µm) compared to WT (6±1, 11.2±1.1, 12.8±1µm, p<0.01) at 1, 2 and 3 hours respectively. Neurite lengths converged in WT (22.8±4.1µm) and KO (26.2±1.4µm, p=0.47) after 4 hours up to 10 hours (26.2±4.3µm WT Vs. 27±1.2µm KO, p=0.87). Transcriptional regulation of Caspr1 was confirmed by treating cells with Actinomycin D, which effectively blocked Caspr1 mRNA expression (p=0.002). Luciferase assay shows that hyperglycemia significantly reduces Caspr1 promoter activity (p=0.0004). Retinal explants show partial colocalization of Caspr1 and Contactin1 (Pearson’s coefficient>0.7). Hyperglycemia visibly alters intensity and distribution of CAMs across retinal layers.
Conclusions :
Previously we showed that hyperglycemia affects neurite growth and CAM expression in primary adult retinal neurons. We now show that Caspr1 negatively regulates initiation of neurite outgrowth, and hyperglycemia leads to transcriptional downregulation of Caspr1 and altered distribution of CAMs in the retina. Neuronal CAM interactions and their regulation will be explored further using studies in an animal model of diabetic retinopathy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.