mTOR Signaling and Endoplasmic Reticulum Stress in Mixed Glial Cultures

Oligodendrocytes are the myelinating cells of the central nervous system. The extraordinary protein and lipid synthesis that myelin production entails makes oligodendrocytes especially prone to endoplasmic reticulum (ER) stress. In this experiment, we set out to investigate the dynamics of ER stress in mixed glial cultures, and how the consequences of that stress vary based on the activity of the anabolic PI3K/AKT/mTOR pathway. Previous experiments in isolated oligodendrocyte cultures demonstrated a negative correlation between AKT/mTOR signaling and oligodendrocyte cell viability in the presence of ER stressors. Because ER stress leads to a buildup of improperly processed proteins, decreases in mTOR pathway activity are thought to moderate the excess of misfolded polypeptides. We hypothesized that administration of rapamycin, an inhibitor of mTOR, would ameliorate the effects of ER stress by preventing protein buildup caused by mTOR pathway activity. To test this hypothesis, mixed cultures of oligodendrocytes and astrocytes were isolated from neonatal rat brains and exposed to tunicamycin (TU), an inhibitor of n-linked glycosylation, to induce ER stress. TU was administered alone and after pre-treatment with rapamycin, to investigate how the effects of ER stress change when the activity of that pathway was limited. By assaying cell survival and protein expression, we were able to determine how ER stress, with and without mTOR inhibition, affected cell populations in mixed glial cultures. We found that chronic TU exposure in the absence of rapamycin failed to decrease the percentage of oligodendrocyte-lineage cells, which we attribute to the protective effects of astrocytes. Acute exposure, however, did decrease the percentage of oligodendrocyte-lineage cells in culture. These two trends lead us to speculate that the protective effects astrocytes offer to oligodendrocytes consist of relatively slow-acting growth-factor production and secretion by astrocytes. Adding strength to this contention, we found that the mTOR inhibitor rapamycin did not increase the percentage of oligodendrocyte-lineage cells in cultures exposed to TU. Because mTOR is involved in the function of many growth factors, its inhibition may have hampered the protective functions of astrocytes. While other experiments have investigated the effects of ER toxins on activity of the mTOR pathway, this experiment is more faithful to in vivo conditions because it incorporates meaningful astrocyte-oligodendrocyte interactions. As observed in this experiment, these effects are powerful and serve to improve our understanding of the dynamics of ER stress and mTOR inhibitors in organismal glial cells