Retina’s Electrical Wiring Under Attack: New Study Reveals How Connexin 36 Degrades Under Stress

In a discovery that could reshape how scientists approach blinding diseases like glaucoma and age-related macular degeneration, researchers have uncovered the molecular machinery that governs the fate of connexin 36, a critical gap junction protein in the retina, when photoreceptor cells face excitotoxic stress. The study, published in Acta Biochimica Polonica, reveals that this neuronal gap junction protein—long known for its role in synchronizing electrical signals across retinal neurons—is actively managed by two competing protein degradation systems, and that blocking one triggers a compensatory response from the other.

Connexin 36 (Cx36) forms electrical synapses that allow direct cytoplasmic continuity between adjacent retinal neurons, enabling the rapid exchange of ions and small metabolites. In the outer retina, Cx36-containing gap junctions mediate rod-cone coupling and regulate visual signal integration, while in the inner retina, they contribute to electrical coupling between retinal ganglion cells and amacrine cells. These channels are essential for temporal precision in visual processing, enhancing signal-to-noise ratio and firing synchrony. However, under pathological conditions such as excitotoxicity, the same gap junctions that normally support healthy signal transmission can become conduits for the spread of toxic signals—including calcium overload and pro-apoptotic molecules—from injured neurons to their healthy neighbors.

The research team, led by Adrian Smędowski at the Medical University of Silesia in Katowice, Poland, used immortalized 661W murine photoreceptor cells transfected with GFP-tagged Cx36 to visualize the protein’s intracellular distribution under controlled stress conditions. The 661W cell line, derived from mouse cone photoreceptors, expresses cone-specific markers and retains light responsiveness through cGMP modulation, making it a physiologically relevant model for studying retinal photoreceptor biology. The researchers chose this model because it provides a more relevant retinal phenotype compared to other neuron-like cell lines, though they acknowledge that findings from an immortalized cell line must ultimately be validated in intact retinal tissue.

To probe the degradation pathways responsible for Cx36 turnover, the team employed two complementary pharmacological approaches. First, they used bafilomycin A1, a macrolide antibiotic that disrupts autophagic flux by blocking V-ATPase-dependent acidification of lysosomes and inhibiting autophagosome-lysosome fusion. Second, they exposed the cells to N-methyl-D-aspartate (NMDA) to induce excitotoxic stress, mimicking the glutamate-mediated overactivation that contributes to neuronal damage in retinal neurodegenerative diseases. By applying these interventions separately and in combination with Cx36 overexpression, the researchers could dissect the relative contributions of the autophagy-lysosome pathway and the ubiquitin-proteasome system (UPS) to connexin proteostasis.

The results revealed a striking pattern of proteostatic reorganization. Following bafilomycin A1 treatment, immunofluorescence analysis showed intracellular accumulation of the autophagy adaptor protein SQSTM1/p62, with signal intensities increasing approximately 1.5- to 2-fold compared to controls (n = 3, p < 0.05). Under normal conditions, p62 selectively binds polyubiquitinated protein aggregates and delivers them to autophagosomes via interaction with LC3, facilitating their degradation upon fusion with lysosomes. When this pathway is blocked, p62 itself accumulates because it can no longer be cleared. The researchers also observed a marked upregulation of PSMA1, a structural subunit of the 20S proteasome core particle, suggesting a potential compensatory shift toward proteasomal degradation when lysosomal clearance is compromised.

Perhaps most intriguingly, the team detected elevated levels of RAB11A, a small GTPase associated with the recycling endosome pathway. RAB11A controls the return of internalized receptors from recycling endosomes to the plasma membrane and contributes to vesicular trafficking processes underlying endosomal sorting. Its increased expression may reflect an adaptive cellular response to impaired protein degradation, potentially contributing to altered intracellular trafficking and redistribution of proteins—including Cx36—under stress conditions. Notably, the researchers did not observe changes in other endosomal markers such as EEA1, RAB5A, or RAB9A, suggesting that the recycling pathway specifically, rather than the broader endocytic machinery, is recruited in response to lysosomal blockade.

When the researchers turned to the excitotoxicity model, they found that NMDA treatment was associated with increased ubiquitin accumulation, particularly in Cx36-overexpressing cells. Ubiquitin serves as a molecular tag that marks damaged or misfolded proteins for degradation by the 26S proteasome through a cascade involving E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. The enhanced ubiquitin signal in Cx36-expressing cells exposed to NMDA suggests that excitotoxic conditions compromise Cx36 stability and promote its ubiquitination, indicating enhanced proteostatic stress specifically in cells bearing higher loads of the gap junction protein. Interestingly, Cx36 immunoreactivity remained predominantly cytoplasmic with a punctate distribution pattern, and no major redistribution was observed following NMDA treatment.

The implications of these findings extend beyond basic cell biology. Previous studies have demonstrated that blocking gap junctions under excitotoxic or ischemic conditions can improve retinal ganglion cell survival by up to 70 percent, underscoring the critical role of intercellular communication in propagating neuronal injury. Genetic knockout studies have further shown that eliminating Cx36 significantly reduces retinal ganglion cell death under excitotoxic stress. The current work adds a new dimension to this understanding by suggesting that modulating Cx36 turnover or stabilization—rather than simply upregulating or downregulating its expression—may represent a more effective neuroprotective strategy. By targeting the proteostatic machinery that controls connexin degradation, it may be possible to fine-tune gap junction abundance and coupling strength in ways that preserve normal visual signal transmission while limiting the pathological spread of toxic signals.

The study’s authors are careful to note several important limitations. The immunofluorescence-based approach, while providing unique spatial resolution for assessing intracellular distribution, does not allow precise quantification of total protein expression. Definitive confirmation of impaired autophagic flux would require LC3-II turnover analysis, which was not performed in this study. Similarly, the observed PSMA1 upregulation suggests but does not prove functional proteasome involvement. The use of Cx36 overexpression itself may influence intracellular proteostasis, and the cytoplasmic distribution of the tagged protein likely reflects overexpression-driven accumulation within the endoplasmic reticulum or vesicular compartments rather than functional gap junction localization at the plasma membrane. Future work incorporating Western blotting, LC3-II flux assays, proteasome activity measurements, live-cell imaging, and validation in retinal explants or animal models will be essential to confirm and extend these preliminary observations.

Nevertheless, the study opens a compelling new avenue for investigating retinal neurodegeneration. Both glaucoma and age-related macular degeneration are characterized by progressive dysfunction of proteostatic mechanisms—whether through oxidative stress in the retinal pigment epithelium, mitochondrial dysfunction in retinal ganglion cells, or chronic neuroinflammation. If connexin proteostasis proves to be a modifiable node in these degenerative cascades, targeted pharmacological interventions aimed at specific degradation pathways could offer a precision approach to preserving vision. The researchers suggest that better understanding of these subcellular mechanisms will potentially allow the design of precisely targeted treatments in the future, transforming our approach to diseases that currently rob millions of people worldwide of their sight.

Subject of Research: Proteostatic regulation of the neuronal gap junction protein connexin 36 in photoreceptor cells under excitotoxic stress

Article Title: Gap junction protein connexin 36 proteostatic mechanisms in photoreceptor cells subjected to excitotoxic stress

Article References: Katan, M., Mróz, K., Pacwa, A., Lewin-Kowalik, J., & Smędowski, A. (2026). Gap junction protein connexin 36 proteostatic mechanisms in photoreceptor cells subjected to excitotoxic stress. Acta Biochimica Polonica, 73, Article 16091. https://doi.org/10.3389/abp.2026.16091

Image Credits: AI Generated

DOI: 10.3389/abp.2026.16091

Keywords: connexin 36, gap junctions, photoreceptors, excitotoxicity, autophagy, ubiquitin-proteasome system, retinal degeneration, glaucoma, age-related macular degeneration, p62/SQSTM1, RAB11A, proteostasis