Neuroprotective Role of Akt in Hypoxia Adaptation in Andeans

Abstract

Chronic mountain sickness (CMS) is a disease that potentially threatens a large segment of high-altitude populations during extended living at altitudes above 2,500 m. Patients with CMS suffer from severe hypoxemia, excessive erythrocytosis and neurologic deficits. The cellular mechanisms underlying CMS neuropathology remain unknown. We previously showed that iPSC-derived CMS neurons have altered mitochondrial dynamics and increased susceptibility to hypoxia-induced cell death. Genome analysis from the same population identified many ER stress-related genes that play an important role in hypoxia adaptation or lack thereof. In the current study, we showed that iPSC-derived CMS neurons have increased expression of ER stress markers Grp78 and XBP1s under normoxia and hyperphosphorylation of PERK under hypoxia, alleviating ER stress does not rescue the hypoxia-induced CMS neuronal cell death. Akt is a cytosolic regulator of ER stress with PERK as a direct target of Akt. CMS neurons exhibited lack of Akt activation and lack of increased Parkin expression as compared to non-CMS neurons under hypoxia. By enhancing Akt activation and Parkin overexpression, hypoxia-induced CMS neuronal cell death was reduced. Taken together, we propose that increased Akt activation protects non-CMS from hypoxia-induced cell death. In contrast, impaired adaptive mechanisms including failure to activate Akt and increase Parkin expression render CMS neurons more susceptible to hypoxia-induced cell death.

Keywords: Akt; Parkin; cell death; chronic mountain sickness; hypoxia; iPSCs; neurons.

FIGURE 1

ER stress in CMS neurons. (A) A representative blot of Grp78 in iPSC-derived non-CMS neurons (n = 3) and CMS neurons (n = 3) following hypoxia (1% O₂) treatment for 0, 6, and 24 h. Tunicamycin (TM)-treated CMS neurons as a positive control for ER stress. (B) Densitometry analysis of Grp78. (C) A representative blot of XBP1s in iPSC-derived non-CMS neurons (n = 3) and CMS neurons (n = 3) following hypoxia (1% O₂) treatment for 0, 6, and 24 h. (D) Densitometry analysis of XBP1s. (E) A representative blot of PERK in iPSC-derived non-CMS neurons (n = 3) and CMS neurons (n = 3) following hypoxia (1% O₂) treatment for 0, 6, and 24 h. (F) Densitometry analysis of PERK. (G). A representative blot of PERK and cPARP in iPSC-derived CMS neurons following hypoxia (1% O₂) treatment for 0 and 48 h with or without 1 mM 4-PBA. (H) Densitometry analysis of cPARP. * indicates p < 0.05 and ** indicates p < 0.01 as compared to non-CMS neurons at a given time point. ^## indicates p < 0.01 and ^###p < 0.001 as compared to themselves under normoxia.

FIGURE 2

Akt activation in CMS. (A) A representative blot of pAkt and Akt in iPSC-derived non-CMS neurons (n = 3) and CMS neurons (n = 3) following hypoxia (1% O₂) treatment for 0, 6, and 24 h. (B) Densitometry analysis of Akt. (C) A representative blot of Parkin in iPSC-derived non-CMS neurons (n = 3) and CMS neurons (n = 3) following hypoxia (1% O₂) treatment for 0, 6, and 24 h. (D) Densitometry analysis of Parkin. (E) A representative blot of Mfn2 in iPSC-derived non-CMS neurons (n = 3) and CMS neurons (n = 3) following hypoxia (1% O₂) treatment for 0, 6, and 24 h. Red line indicates the ubiquitylated Mfn2 (uMfn2). (F) Densitometry analysis of steady state of Mfn2 and (G) densitometry analysis of uMfn2. * indicates p < 0.05 and ** indicates p < 0.01 as compared to non-CMS neurons at a given time point. ^## indicates p < 0.01 as compared to themselves under normoxia.

FIGURE 3

Neuroprotective role of Akt and Parkin in CMS. (A) A representative blot of pAkt in iPSC-derived CMS neurons (n = 3) following hypoxia (1% O₂), hypoxia + 100 nM insulin and hypoxia + 100 nM insulin + MK2206 treatment for 48 h. (B) A representative blot of Parkin in iPSC-derived CMS neurons (n = 3) after a lentivirus transduction following hypoxia treatment or hypoxia with insulin or 4-PBA. (C) A representative blot of cPARP in iPSC-derived CMS neurons (n = 3) and Parkin-overexpressed CMS neuron (n = 3) following 1 mM 4-PBA, 100 nM insulin and hypoxia (1% O₂) treatment for 48 h. (D) densitometry analysis of cPARP (4-PBA data was summarized in Figure 1H). * indicates p < 0.05 as compared to CMS neurons under hypoxia, ^###p < 0.001 as compared to themselves under normoxia. (E) Schematic overview of current findings. Hypoxic treatment results in an increased ER stress response and lack of Akt activation and Parkin expression in CMS neurons. Failure to activate Akt and expression of Parkin render CMS neurons more susceptible to hypoxia-induced cell death, which can be alleviated by insulin-induced Akt activation and Parkin overexpression. Solid line indicates findings obtained from the current study, and dashed lines indicate findings from literature.