Gap junction protein connexin 36 proteostatic mechanisms in photoreceptor cells subjected to excitotoxic stress
Monika Katan, Klaudia Mróz, Joanna Lewin-Kowalik, Adrian Smędowski
Connexin 36 (Cx36), a neuronal gap junction protein, is essential for retinal signal transmission but may also contribute to neurodegeneration by facilitating the spread of excitotoxic stress signals. This study investigated the mechanisms involved in Cx36 proteostasis in photoreceptor cells exposed to conditions relevant to retinal disease. Immortalized 661W murine photoreceptor cells were transfected with a plasmid encoding GFP-tagged Cx36. Lysosomal degradation was inhibited using bafilomycin A1, while excitotoxic stress was induced using N-methyl-D-aspartate (NMDA). Immunofluorescence was used to examine markers associated with autophagy, endosomal trafficking, proteasomal degradation, and ubiquitination. Bafilomycin A1 treatment increased the immunofluorescence signals of SQSTM1/p62 and PSMA1 and was associated with increased RAB11A expression, suggesting impaired autophagic clearance and possible compensatory changes in proteasomal and endosomal recycling pathways. NMDA exposure increased ubiquitin staining, particularly in Cx36-overexpressing cells, without producing a marked change in the Cx36 signal distribution. These findings indicate that excitotoxic stress and lysosomal inhibition alter proteostasis-related markers in photoreceptor-like cells. The results suggest that Cx36 handling may be influenced by cellular stress and protein quality-control pathways. However, direct functional evidence of Cx36 turnover, proteasome activation, and autophagic flux remains necessary. Further studies using complementary biochemical methods and in vivo models are required to establish the relevance of these mechanisms to retinal neurodegenerative diseases.