Rapamycin treatment increases hippocampal cell viability in an mTOR-independent manner during exposure to hypoxia mimetic, cobalt chloride

Abstract

Background: Cobalt chloride (CoCl 2) induces chemical hypoxia through activation of hypoxia-inducible factor-1 alpha (HIF-1α). Mammalian target of rapamycin (mTOR) is a multifaceted protein capable of regulating cell growth, angiogenesis, metabolism, proliferation, and survival. In this study, we tested the efficacy of a well-known mTOR inhibitor, rapamycin, in reducing oxidative damage and increasing cell viability in the mouse hippocampal cell line, HT22, during a CoCl 2-simulated hypoxic insult. Results: CoCl 2 caused cell death in a dose-dependent manner and increased protein levels of cleaved caspase-9 and caspase-3. Rapamycin increased viability of HT22 cells exposed to CoCl 2 and reduced activation of caspases-9 and-3. Cells exposed to CoCl 2 displayed increased reactive oxygen species (ROS) production and hyperpolarization of the mitochondrial membrane, both of which rapamycin successfully blocked. mTOR protein itself, along with its downstream signaling target, phospho-S6 ribosomal protein (pS6), were significantly inhibited with CoCl 2 and rapamycin addition did not significantly lower expression further. Rapamycin promoted protein expression of Beclin-1 and increased conversion of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, suggesting an increase in autophagy. Pro-apoptotic protein, Bcl-2 associated × (Bax), exhibited a slight, but significant decrease with rapamycin treatment, while its anti-apoptotic counterpart, B cell lymphoma-2 (Bcl-2), was to a similar degree upregulated. Finally, the protein expression ratio of phosphorylated mitogen-activated protein kinase (pMAPK) to its unphosphorylated form (MAPK) was dramatically increased in rapamycin and CoCl 2 co-treated cells. Conclusions: Our results indicate that rapamycin confers protection against CoCl 2-simulated hypoxic insults to neuronal cells. This occurs, as suggested by our results, independent of mTOR modification, and rather through stabilization of the mitochondrial membrane with concomitant decreases in ROS production. Additionally, inhibition of caspase-9 and-3 activation and stimulation of protective autophagy reduces cell death, while a decrease in the Bax/ Bcl-2 ratio and an increase in pMAPK promotes cell survival during CoCl 2 exposure. Together these results demonstrate the therapeutic potential of rapamycin against hypoxic injury and highlight potential pathways mediating the protective effects of rapamycin treatment.

Diminished apoptosis in hypoxic porcine retina explant cultures through hypothermia

“…Divalent metal ions, such as cobalt, induce a disturbance of the mitochondrial respiration chain and stimulate the rupture of the outer cell membrane, resulting in ROS production and oxidative stress 6 , 8 , 22 . Our results suggest that CoCl 2 not only mimics hypoxia through the stabilization of HIF-1α, but also leads to a strongly elevated level of ROS, indicating that it triggers oxidative stress.…”

Impairment of Autophagy Causes Superoxide Formation and Caspase Activation in 661 W Cells, a Cell Line for Cone Photoreceptors, under Hyperglycemic Conditions

“…However, it decreased the caspase 3/7 activities to the levels of the Z-VAD-FMK control. These differences may reflect its neuroprotective effects independent of the mTOR pathway [ 32 ]. These issues need to be investigated further.…”

Quantitative Proteomics Reveals the Beneficial Effects of Low Glucose on Neuronal Cell Survival in an in vitro Ischemic Penumbral Model

“…For nuclear protein extraction, Qproteome Cell Compartment Kit (Qiagen) was used. Protein separation and band signal detection were conducted as previously described (Zimmerman et al, 2018 ). Briefly, the protein lysates (about 20 μg per well) were separated using 4–12% Bis-Tris NuPAGE gels (Invitrogen) and then transferred to PVDF membranes using a Bio-Rad Mini Trans-Blot system.…”