Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin

Alma Kokott-Vuong,Aaron Voigt,Pardes Habib,Jörg B Schulz,Arno Reich,Jennifer Jung,Michael Huber,Rozina Noristani,Nele Kirschfink,Aaron T Fehr

doi:10.3390/ijms222111586

Abstract

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

Highlights

Hypoxia is a key underlying condition of various devastating diseases including pulmonary hypertension, ischemic heart failure as well as global and focal cerebral ischemia [1,2,3]
To assess the impact of TNF receptor-associated protein 1 (TRAP1) on the mortality rates of D. melanogaster after severe hypoxia, we subjected wild-type flies (Canton-S) as well as Trap1-deficient flies (Trap1 heterozygous (Trap1+/−) and Trap1 homozygous (Trap1−/−), respectively) to severe hypoxia in a self-designed hypoxia chamber under controlled conditions followed by a reperfusion period of 120 h (Figure 1a–d)
While a hypoxia duration of 3 h induced a mortality rate of 47.12% in Trap−/−, only 38.18% Trap1+/− flies died after 120 h of reperfusion and 11.82% of Canton-S died in the same period (Figure 1f)

Summary

Introduction

Hypoxia is a key underlying condition of various devastating diseases including pulmonary hypertension, ischemic heart failure as well as global and focal cerebral ischemia [1,2,3]. Occlusion of a cerebral artery results in a rapid deprivation of oxygen and nutrients leading to neuronal cell death [3,5]. Excitotoxicity, oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial impairment are the most frequently observed mechanisms after hypoxia/ischemia [6]. Post-hypoxic oxidative stress and excitotoxicity, caused by a substantial release of glutamate, lead to an accumulation of Ca2+ in mitochondria [6]. Elevated Ca2+ levels open the mitochondrial permeability transition pore (MPTP) releasing cytochrome C (CytC) into the cytosol and inducing a breakdown of the membrane potential accompanied by an osmotic swelling of mitochondria [6,7]. Cytosolic CytC forms a complex with the initiator caspase 9, which in turn activates the effector caspases 3 or 7 resulting in apoptosis [7,8,9,10,11]

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Conclusion