14 March 2009. PTEN-induced putative kinase 1, or Pink1, defends cells from reactive oxygen species (ROS) by controlling mitochondrial calcium levels, according to a paper published in today’s Molecular Cell. The finding helps explain why mutations in Pink1 make substantia nigra neurons sensitive to oxidative damage and cause Parkinson disease. According to scientists from University College London, United Kingdom, Pink1 regulates calcium flow out of the mitochondria, but when mutations cause the system to go awry, ROS build up and endanger cell viability. In another paper this week, researchers from Xiamen University, China, show that Pink1 acts downstream of the transcription factor FOXO3a to battle ROS. That study appears in the March 10 PNAS online.
Pink1, which localizes to mitochondria, is known to protect cells from oxidative stress, but the mechanism is unclear. Sonja Gandhi, first author of the Molecular Cell paper, senior author Andrey Abramov, and colleagues analyzed the effects of Pink1 knockdown on mitochondrial function and calcium maintenance using shRNAi. They focused on human neuroblastoma and fetal stem cells as well as neurons cultured from Pink1 knockout mice. The authors used oxygen consumption levels and uptake of glucose to examine respiration in the Pink1-depleted cells, and found they had lower rates of respiration than control cells expressing Pink1. To measure mitochondrial responses to increased cellular calcium levels, the authors used potassium chloride, which depolarizes the plasma membrane and opens potential-sensitive calcium channels, allowing calcium to flood into the cytosol. They linked Pink1 dysfunction to the opening of the mitochondrial permeability transition pore (mPTP). While transient mPTP opening is normal, long-term pore traffic is associated with cell death (for review, see Crompton, 1999). The pore allows ions through, destroying the membrane potential that the mitochondria rely on to produce ATP.
In Pink1 knockdown cells, KCl treatment causes higher levels of calcium uptake than control cells, and a drop in mitochondrial membrane potential. An mPTP inhibitor protected membrane potential, indicating that mPTP do swing open in response to calcium in Pink1-depleted cells. The Pink1-depleted cells also retained excess calcium in the mitochondria longer than control cells. This suggested to the authors that the mitochondria were impaired in their ability to release calcium, becoming overloaded with calcium ions. When the authors treated cells containing Pink1 with an inhibitor of the putative mitochondrial sodium/calcium exchanger, they accumulated calcium just like the Pink1-depleted cells, suggesting that Pink1 deficiency interferes with the Na+/Ca2+ exchanger, although the identity of this exchanger is unknown.
Pink1 knockdown cells had higher levels of ROS in the cytosol than control cells, and upon KCl treatment, their ROS levels rose higher still. The scientists investigated NADPH oxidase (NOX), an enzyme that produces superoxide, as the possible mediator of this ROS increase. NOX inhibitors blocked the rise in ROS, showing that NOX produces excess ROS in Pink1-deficient cells. When they treated the cells with ROS scavengers, the scientists found that glucose intake and respiration increased to the levels found in control cells, implying that ROS interfere with glucose import.
Dysregulated calcium and increased ROS levels both trigger opening of the mPTP. Once those pores open, mitochondria lose their membrane potential and fail in their function as the cell’s energy center, ultimately leading to necrosis. Opening of the mPTP may also lead to programmed cell death, if the mitochondria release apoptotic factors such as cytochrome c.
Since glucose uptake appeared to be impaired in the Pink1-depleted cells, the authors wondered if they could circumvent the respiratory defect by providing respiration intermediates. Pyruvate and methyl succinate, which supplied the citric acid cycle, restored oxygen consumption to normal levels in Pink1 knockdown cells. Abramov and colleagues are now exploring ways that they might apply this new knowledge in a therapy for Parkinson’s.
Researchers have linked faulty calcium control to Alzheimer disease as well (Abramov et al., 2003; Mattson, 2007). In Parkinson disease, dopaminergic neurons may be particularly vulnerable to calcium dysregulation because as the brain ages, these cells increasingly rely on calcium channels to drive their basal activity (see ARF related news story and Chan et al., 2007).
If Pink1 protects the cell from ROS by mediating mitochondrial calcium, what controls Pink1? In the PNAS paper, joint first authors Yang Mei and Yiru Zhang and senior author Han You found a connection between Pink1 and FOXO transcription factors. FOXO factors, which frequently promote apoptosis, have also been shown to protect cells from oxidative stress (Kops et al., 2002; Nemoto and Finkel, 2002). The scientists sought to discover genes regulated by FOXO, and found Pink1 was one of them. Cell culture experiments confirmed that in lymphocytes, FOXO3a binds the Pink1 gene and upregulates transcription in response to missing growth factors. Without Pink1, those cells underwent apoptosis. FOXO3a also controlled Pink1 in a neuroblastoma cell line, where FOXO3a shRNA reduced Pink1 expression, while a control, scrambled shRNA, did not.
The authors suggest that Pink1 is part of the FOXO-mediated ROS protection pathway. For approximately 12 hours of growth factor starvation, they speculate, Pink1 helps cells survive until such factors are once again available. Beyond that time period, pro-apoptotic factors take over. Loss of defenses against oxidative stress may be a common theme in Parkinson disease, said Tak Mak of Princess Margaret Hospital in Toronto, Canada, another author on the PNAS paper.—Amber Dance.
Gandhi S, Wood-Kaczamar A, Yao Z, Plun-Favreau H, Deas Em, Klupsch K, Downward J, Latchman DS, Tabrizi SJ, Wood NW, Duchen MR, Abramov AY. PINK1-associated Parkinson’s disease is caused by neuronal vulnerability to calcium-induced cell death. Mol Cell 2009 March 13, 33:627-638. Abstract
Mei Y, Zhang Y, Yamamoto K, Xie W, Mak TW, You H. FOXO3a-dependent regulation of Pink1 (Park6) mediates survival signaling in response to cytokine depravation. PNAS 2009 March 10. Abstract