Effects Of Diazoxide On NOX2 In Rat Cholinergic Neurons Exposed To Aβ1-42

Alzheimer’s disease (AD), a neurodegenerative disease, distinguished by progressive memory/cognitive impairment and personality changes represents the most common type of dementia. Currently, no satisfactory treatment exists for AD. As a result of increasing longevity, and therefore the numbers, of our aging population, the incidence of AD has been growing rapidly. This has become a serious social problem, due to the psychological and economic burdens AD has exerted, not only upon the patients themselves but also their families. Therefore, investigations directed at understanding the mechanisms and pathogenesis of this disorder are imperative for new therapeutic strategies for the treatment and prevention of AD.Selective neuronal loss, senile plaques and neurofibrillary tangles represent the most salient pathological changes of AD. Senile plaques are related with β-amyloid (AP) deposits. The neurotoxicity resulting from Aβ, is associated a number of processes including oxidative modification of lipid membrane systems and proteins, increases in reactive oxygen species (ROS), mitochondrial dysfunction, reductions in ATP levels, cell metabolism dysfunction and oxidative stress. Oxidative stress, which exists in a variety of neurodegenerative diseases, embodies one of the most important pathological mechanisms of AD. Mitochondria have long been considered as the major source of ROS production, but emerging data have indicated that, NADPH oxidase (NOX) also plays an important role in the generation of ROS within the central nervous system.NOX, distributed in almost all organs, tissues and cells, is a group of proteins whose main biological function appears to be ROS generation. NOX2 is an enzyme complex consisting of membrane subunits gp91phox and p22phox, cytosolic subunit p47phox, p67phox, p40phox and the small molecular GTPase-binding protein, Rac. The catalytic subunit gp91phox homolog NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2 are collectively referred to as comprising the NOX family. While the ROS generated by NOX can maintain normal physiological activity of cells involved in the regulation of cell growth, differentiation, apoptosis and cytoskeletal remodeling, under abnormal conditions such as ischemia and hypoxia, mechanical damage, exposure to toxic substances or inflammatory factor stimulation, NOX can be unduly activated, with the result that large amounts of ROS-inducing oxidative stress are produced. The activation of NOX requires a variety of NOX subunits assembling into an active complex to exert their biological function. The transfer of cytoplasmic NOX subunits to the membrane is the basis of NOX activation. The p47phox C-terminal PRR region can be combined with the p67phox C-terminal SH3 region, which then play an important role in assembling the cytoplasmic subunits p67phox, p40phox and Rac. Considerable evidence exists demonstrating that NOX is involved in the pathogenesis of AD, in particular, NOX2. For example, blocking of NOX2 has been shown to produce neuroprotective effects, as revealed in several AD animal model studies. Moreover, while perfusion of the neocortex with Aβ1-40 leads to ROS generation and cerebral vascular adjustment disorders in the wild-type mice, identical Aβ1-40 treatments within NOX2-/-mice did not appear to elevate ROS. Findings from other studies have shown that abolishing NOX2 function has a protective effect on preventing the toxic effects of Aβ plaques and significantly reducing neuropathic oxidative stress from the APP overexpressing Swedish mutation mice (Tg2576, Aβ fragment can cause aggregation) and Cybb-/-mice (NOX2 dysfunctional) hybridizations. In cultured mouse hippocampal neurons, silencing of the p47phox gene or NOX2 as achieved by blocking DPI inhibited superoxide production and neurotoxicity induced by activation of NMDARs. The expression of NOX2 subunits is not yet fully clear in cholinergic neurons in AD.The energy metabolism and electron transfer chain associated with mitochondria can also produce ROS. Under abnormal cellular stress, the resulting metabolic disorders can generate excessive amounts of ROS leading to oxidative stress. The brain contains disproportionate concentrations of mitochondria, being sevenfold greater than that found in the liver or heart. There exists considerable evidence indicating that mitochondria are involved in the pathogenesis of AD, and play an important role in oxidative stress injury. As based upon studies with vascular endothelial cells, the two main sources of ROS, mitochondrial and NOX have been shown to interact with each other, resulting in an exacerbation of oxidative stress and cell apoptosis.Findings from a number of recent studies have shown that the MitoKATP channel could be a trigger and/or effector of various brain preconditioning processes. Pretreatment with MitoKATP channel is associated with neuroprotection, as shown in vivo and in vitro; an effect which may be reversed by 5-HD, a MitoKATP channel antagonist. MitoKATP channel can be activated by diazoxide, which has a protective effect involving mitochondrial membrane depolarization, inhibition of ROS release and maintenance of a stable structure of the mitochondria. Diazoxide can significantly reduce rotenone-induced PC 12 cell death, and also inhibits hydrogen peroxide-induced neuronal apoptosis by stabilizing the mitochondrial membrane potential as shown in primary cultured cerebellar granule neurons. MitoKATP channel can stabilize mitochondrial structure and inhibit mitochondrial ROS release. Whether MitoKATP channel activation can influence the NOX2 expression in cholinergic neurons in AD pathology is not yet clear.Object:In this study, primary cultured rat cholinergic neurons treated with Aβ1-42 were used as a model for AD pathology. To examine some of the potential mechanisms of this pathology in this model, diazoxide was used as a MitoKATP channel activator and DPI as a NOX2 blocker, to address the following issues:1. To investigate the mechanism of oxidative stress induced by Aβ1-42, the effect of Aβ1-42 on cellular ROS generation, cell activity and the major subunits of NOX2 expression were examined.2. To investigate whether NOX2 is involved in the pathological process of AD, the effect of DPI on cellular ROS generation and cell activity induced by Aβ1-42 were examined.3. To investigate the mechanism of diazoxide in preventing oxidative stress in the pathological process of AD, the pre-activation of MitoKATP channel on cellular ROS generation, cell activity and the major subunits of NOX2 expression induced by Aβ1-42 were examined.Methods:1. Primary culture and identification of cholinergic neurons. Embryos at 17-19 days of gestation were removed from pregnant Wistar rats. The basal forebrain was dissected and digested with 0.125% trypsogen. Proliferation growth medium composed of a 1:9 mixture of fetal bovine serum and DMEM, and a 2% B27 supplement was added to these cells, which were then treated with cytarabine (5μM) when cultured for 3-5 days in order to inhibit the proliferation of gliocyte. Choactase was used as marker protein for identifying cholinergic neurons which were identified by immunocytochemistry.2. Generation of experimental groups. The cells were randomly divided into six groups:controls, Aβ1-42, diazoxide (DZ), diazoxide+Aβ1-42(DZ+Aβ), DPI (a NOX2 inhibitor) and DPI+Aβ1-42. Cells in the Aβ1-42 group were cultured for seven days and treated with Aβ1-42 for 24 or 72h. Cells in the DZ+Aβ and DPI+Aβ groups were pretreated with diazoxide or DPI (1μM) for one hour prior to treatment with Aβ1-42 for 24 or 72h. The DZ and DPI groups were pretreated with diazoxide or DPI (1μM) for one hour, and then treated with equal volumes of PBS instead of Aβ1-42 for 24 or 72h.3. Cell viability. Cultured cholinergic neurons were subjected to the tratments described above for 24 or 72 hours. Cell viability was then assessed using a MTT assay.4. Detection of ROS and MDA. The levels of intracellular reactive oxygen species (ROS) were determined by the change in fluorescence resulting from the oxidation of the fluorescent probe DCFH-DA. Cells were collected to examine MDA content as determined following reaction with thiobarbituric acid using the MDA assay kit.5. Expression of gp91phox and p47phox. The expressions of gp91phox and p47phox as assessed after exposure to the different treatment conditions for 24 or 72h were detected using double immuofluorescence and western blot analysis.Results:1. Choactase was used as marker protein for identifying cholinergic neurons. For identification of cholinergic neurons, cells were cultured for 7 d in proliferation growth medium. Then immunocytochemistry was performed using anti-Choline acetyltransferase. Over 90% of the cells were cholinergic neurons.2. Aβ1-42 induced cytotoxicity in cholinergic neurons as assessed with the MTT assay. Aβ1-42 induced cytotoxicity and significantly reduced cell viability (P<0.05 at 24h and P<0.001 a 72h) as compared to controls. Pretreatment of cholinergic neurons with diazoxide or DPI resulted in a reduction of Aβ1-42-induced cytotoxicity and significantly increased cell viability.3. Double immunofluorescence and western blot analyses revealed that gp91phox and p47phox expression were up-regulated in the group treated with Aβ1-42 (2μM) for 24h as compared with controls (P<0.01), while no obvious changes were observed in the DZ+Aβ (24h) group. Exposure to Aβ1-42 for 72h, increased further the expressions of these two subunits as compared with controls (P<0.001). However, this upregulation was significantly diminished by pretreatment with diazoxide. These results suggest that NOX2 subunit expression is regulated by diazoxide treatment.4. To examine whether diazoxide pretreatment can inhibit oxidative stress induced by Aβ1-42, the effects of diazoxide on ROS and MDA levels in cholinergic neurons treated with Aβ1-42 were assessed. Exposure of cholinergic neurons to Aβ1-42 for 24 or 72h resulted in significant increases of ROS and MDA levels. Cells pretreated with diazoxide or DPI showed significant reductions in ROS and MDA levels. These results indicate that diazoxide or DPI can inhibit Aβ1-42-mediated ROS and MDA production in cholinergic neurons.Conclusion:1. Aβ1-42 upregulates gp91phox and p47phox expressions in cholinergic neurons, resulting in increased intracellular ROS generation and oxidative stress.2. DPI can inhibit oxidative stress of Aβ1-42 through inhibiting NOX2 activity in cholinergic neurons.3. Diazoxide preconditioning can counteract Aβ1-42 induced oxidative stress and associated cell death by reducing levels of ROS and MDA, in part, by alleviating NOX2 expression.