Activation Of Aldehyde Dehydrogenase2(ALDH2) Protects Against Ischemic Stroke

Stroke is an age-related disease and the second most common cause of death.Ischemic stroke is the largest subtype, with an estimated incidence more than80%of stroke worldwide. The morbidity of stroke may be reduced with an improved control of risk factors. Unfortunately, these identified risk factors explain only approximately60%of the attributable risk, and there may be some important risk factors that have not yet been identified. In comparison, more than90%of ischemic heart disease can be explained by identifiable risk factors. The unknown risk factors for stroke, combined with unsatisfactory control of known risk factors (e.g. hypertension), may explain the continued high prevalence of stroke. Thus, identification of new risk factors may result in the development of new strategies for the treatment of stroke.Recently, we used fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) methods to detect the differentially-expressed protein in the brain of spontaneously hypertensive rats (SHR), and their substrain, stroke-prone spontaneously hypertensive rats (SHR-SP). As the susceptibility to stroke varies greatly between SHR and SHR-SP, the comparison of SHR-SP with SHR would be beneficial to find new risk factors against stroke beyond hypertension. More than2000spots were detected in the CyDye-stained gels using DeCyder software. Among them, aldehyde dehydrogenase2(ALDH2) attracted very much our attention.ALDH2is a mitochondrial protein and is the key enzyme in the ethanol metabolism, converting acetaldehyde (produced by ethanol dehydrogenase) to acetic acid. In addition, ALDH2detoxifies other aromatic and aliphatic aldehydes, including4-hydroxy-2-nonenal (4-HNE), which is produced during oxidative stress as a result of lipid peroxidation. However, little is known about the protective effect of ALDH2against stroke, and the causal role of4-HNE on stroke. Light or moderate ethanol consumption has been shown protective against stroke, whether ALDH2involved in the protection of ethanol is unclear. Here we show that ALDH2has potential as a therapeutic target, and mediate the protective effect of ethanol against stroke and the4-HNE might be an important risk factor for stroke.RESULTSALDH2is down regulated in SHR-SPWe used fluorescent2D-DIGE methods to show the differentially-expressed protein in the brain between SHR and SHR-SP. Among these2000spots,25protein spots were downregulated, and18were upregulated in SHR-SP. All these43protein spots were identified with tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight mass spectrometry (MALDI-TOF/TOF MS). We found that compared with SHR, ALDH2was down regulated by32%in SHR-SP, which was confirmed by western blot. There was no difference in the expression of ALDH2between SHR and its normotensive control, Wistar Kyoto (WKY) rat.Neuroprotection of ALDH2under ischemic stress in neuronsIn cultured primary neurons, activation of ALDH2by its agonist Aldal (10μM) reduced the neuronal apoptosis induced by oxygen-glucose deprivation (OGD) for12h (16.7±0.62%vs.28.9±0.12%in vehicle,P<0.05), which was assessed by flow cytometric analysis of Annexin V and propidium iodide (PI) staining. In contrast, the inhibition of ALDH2by Cyanamide (Cya,1mM) aggravated neuronal apoptosis induced by OGD (45.2±3.26%vs.28.9±0.12%in vechicle,P<0.05). Hoechst and TUNEL staining to assess apoptosis yielded similar results.We also determined the role of ALDH2in neuron survival using ALDH2overexpression and knockdown methods in cultured neurons. Primary neurons were infected with lentiviral vector encoding ALDH2(LV-ALDH2) or lentiviral vectors encoding short hairpin RNA (sh-RNA) targeting ALDH2(sh-ALDH2). The control was infected with corresponding empty lentiviral vector encoding green fluorescent protein (LV-GFP or sh-Scramble, vector control). For all experiments, transfection efficiency was maintained at over90%determined by flow cytometer, and no detectable cellular toxicity was observed. The expression of ALDH2in LV-ALDH2was increased about2-fold and in sh-ALDH2was about0.16-fold. Annexin V and PI staining assay showed that LV-ALDH2 neurons exhibited resistance to cell apoptosis under OGD (12.6±0.2%vs.57.8±2.1%in LV-GFP,P<0.01). In contrast, sh-ALDH2neurons were susceptible to apoptosis under OGD (62.1±2.4%vs.46.1±0.7%in sh-Scramble,P<0.01). Similar effects were observed by using TUNEL and Hoechst staining analysis to identify neuronal apoptosis. Overexpression of ALDH2reduced OGD-induced neuronal injury, and knockdown of ALDH2aggravated OGD-induced neuronal injury.ALDH2protects against ischemic cerebral injury induced by middle cerebral arterial occlusion (MCAO)Sprague-Dawley (SD) rats were subjected to MCAO after being administrated with ALDH2agonist Aldal or its antagonist Cya by intracerebroventricular injection (i.c.v.).In the presence of Aldal (50μg/5μl), the ALDH2activity in brain is elevated by109%and the infarct size decreased by40%; Whereas the Cya (1mg/5μl) reduced ALDH2activity by about60%and enlarged the infarct size by30%.The protective effect of ALDH2against ischemic stroke was further investigated using overexpression and knockdown of ALDH2in the local brain of SD rats. LV-ALDH2, sh-ALDH2or their empty lentiviral vectors (LV-GFP or sh-Scramble, vector control) were stereotaxically injected into the cortex and hippocampus of rats at four sites (2×106TU/site). MCAO was performed three weeks after injection, and animals were sacrificed24hours after MCAO for various examinations.Lentivirus-mediated ALDH2expression in the local brain was first confirmed by immunochemical analysis of GFP. General view of LV-GFP infection in rat brain7days after injection showed that the region of green fluorescent is in coincidence with the four sites of injection. Furthermore, as detected by colocalization with the neuron-specific marker Neu-N, LV-GFP efficiently infected neurons. Injection of LV-ALDH2and sh-ALDH2led to an approximately1.7-fold (overexpression) and0.4-fold (knockdown) of ALDH2protein levels in targeted brain regions, respectively, compared with their control. ALDH2overexpression significantly reduced infarct size from38%in LV-GFP rats to12%, and ameliorated the neurological function; whereas converse effects were observed in sh-ALDH2rats, infarct size increased from37%in sh-Scramble rats to49%, and neurological function was also lightly worsen.The4-HNE is persistently high expressed in SHR-SP and negatively related to the life span of SHR-SPTo investigate the role of4-HNE on stroke, we first detected the level of4-HNE in serum and brain of WKY, SHR and SHR-SP. Compared to age-matched WKY, the serum4-HNE was only significantly higher in SHR-SP, but not in SHR. The expression of4-HNE in the brain of WKY, SHR and SHR-SP yielded similar results. We also determined serum4-HNE level of male SHR-SP (n=27) at age of8-month and recorded their survival time. It was found that serum4-HNE level significantly and inversely correlated with the survival time of SHR-SP (r=-0.602,P<0.001). The results imply a possibility of using serum4-HNE level as a biomarker for the occurrence of stroke.The4-HNE is persistently highly expressed in stroke and stroke prone patientsTwo clinical observations have been conducted to investigate the relationship between serum4-HNE and stroke. In the first observation, we determined4-HNE level in serum of control subjects (n=30) and stroke patients at3stages:(1) very early stage, within3days (n=23);(2) early stage,7-14days after onset of stroke (n=15) and (3) recovery period,3-6months after stroke (n=13). Comparisons of baseline characteristics between those groups showed no important differences among groups. All the stroke patients at3different stages have significantly higher level of serum4-HNE than control subjects (15.5±2.8,18.2±3.3,14.2±1.7vs.9.1±1.9mg/ml,P<0.001). In the second observation, the serum samples selected from a cohort study with1242participants during the period from2004to2011, which has been described elsewhere. During8-year follow-up,21subjects suffered from stroke. From the participants without stroke, we randomly selected45subjects as control, which have similar baseline health care with1242participants. We measured the physical condition and serum4-HNE level of these21stroke subjects and45control participants. There were no significant differences between these two groups in sociodemographic and health care characteristics. Interestingly, the people who developed stroke during follow up exhibited significantly higher4-HNE level than control subjects (12.1±1.6vs.9.6±1.6mg/ml,P=0.0007).The4-HNE induced-cerebral injury was abolished by ALDH2We first investigated whether4-HNE causes cerebral injury.In cultured primary neurons, adding4-HNE increased apoptosis on neurons under OGD (44.6±7.2%vs.29.8±4.8%in vehicle,P<0.01), which was revealed by flow cytometric analysis or Hoechst and TUNEL staining. In SD rats, intracerebroventricular injection of4-HNE (31.25μg/5μl) also significantly augmented the infarction size induced by MCAO (51.2±4.5%vs.32.8±2.7%in vehicle, P<0.01).Because4-HNE could be detoxified by ALDH2, we hypothesized that activating ALDH2would reduce the accumulation of4-HNE, which might alleviate the cerebral injury induced by4-HNE. Indeed, in cultured neurons, the increased expression of4-HNE induced by OGD was completely abolished by ALDH2agonist Aldal, and significantly enhanced by ALDH2inhibitor Cya. Rats treated with4-HNE (31.25μg/5μl, i.c.v.)30minutes before MCAO had45.9%larger infarct size (56.6±5.2%vs.38.8±9.2%) and62%higher level of4-HNE (968±57.0vs.596±21.6ng/ml) in LV-GFP group. The overexpression of ALDH2significantly reduced the infarct size (12.3±6.7%vs.38.8±9.2%in LV-GFP group), completely abolished the enlarged infarct size induced by4-HNE in MCAO rats (13.2±6.9%vs.12.3±6.7%without4-HNE), and prevented the increase of4-HNE in serum. The neurological score also had the similar trends in these different groups. These data indicated that the removal of4-HNE may be the key protective mechanism of ALDH2against ischemic damage.Moderate ethanol consumption protects against stroke and prolong the life span of SHR-SPSD rats were administrated with regular liquid diet with6%ethanol (vol/vol) or isocaloric pair-feeding regimen (control) during6-week. Although36%ethanol consumption aggravated the cerebral injury induced by MCAO, the moderate ethanol intake (6%) significantly decreased the infarct size (P<0.01), and ameliorated the neurological function. Moderate ethanol intake for6weeks did not increased the expression of ALDH2in the brain or liver, it did significantly increase the activity of ALDH2(138±38.9%vs.100±17.2%,P<0.05). Using2DE technologies, we observed some shift in isoelectric point distribution of cerebral ALDH2induced by ethanol, most likely due to the ALDH2phosphorylation. We also found that life-long treatment with6%ethanol delayed the onset of lethal stroke in SHR-SP. The survival time was increased by approximately33%in ethanol group compared with control group (Log rank8.9984, P=0.0027). In normal SD rats,6%ethanol did not affect the production of4-HNE in serum (159±54.0vs.170±45.8ng/ml). In rats with MCAO, ethanol significantly decreased the serum4-HNE level (464±29.3vs.608±53.1ng/ml) as well as tissue4-HNE level in the ischemic brain area revealed by western blot and immunohistochemistry methods.ALDH2mediates the protective effect of ethanol against ischemic strokeAs ethanol increased the activity of ALDH2, we next determined whether ALDH2mediated the protection of ethanol against stroke. Ethanol significantly decreased the infarct size in rats with MCAO (25.1±3.4%vs35.2±2.3%). But this effect was abolished by the ALDH2inhibitor, Cya (1mg/5μl). Then the knockdown of ALDH2in the local brain was performed in SD rats and followed by MCAO3weeks later. The expression of ALDH2in the local brain was diminished and the infarct size induced by MCAO was significantly enlarged. Moreover, the reduction of infarct size by ethanol was also completely abolished by the knockdown of ALDH2. In addition, ethanol-induced4-HNE reduction was also attenuated by the knockdown of ALDH2.Epsilon isozyme of protein kinase C (PKCε) is required for the neuroprotective effect of ethanolWe next designed a series of experiments to investigate whether PKCε mediates the activation of ALDH2and the protective effect of ethanol or ALDH2against stroke. In cultured neurons, ethanol (30mM) significantly enhanced the phosphorylation of PKCs by68%, and promotes the direct binding of ALDH2and PKCε, but had no effect on PKCa. Then we investigated the role of PKCε in neuron survival using PKCε overexpression and knockdown methods in cultured neurons. Primary neurons were infected with lentiviral vector encoding PKCs (LV-PKCs) or lentiviral vectors encoding short hairpin RNA targeting PKCε (sh-PKCε). The expression of PKCε was significantly increased in LV-PKCε infection group, and significantly decreased in sh-ALDH2infection. Annexin V and PI staining assay showed that LV-PKCε neurons exhibited resistance to cell apoptosis under OGD (12.5±0.1%vs.38.2±0.1%). In contrast, sh-PKCε neurons were susceptible to apoptosis under OGD (55.6±2.9%vs.46.3±0.7%).We also used LV-PKCε to increase the expression of PKCε, and sh-PKCε to decrease it in the local brain. All these lentiviral were stereotaxically injected into the cortex and hippocampus of rats at four sites (2×106TU/site). Then MCAO was performed three weeks after injection. The over-expression of PKCε significantly decreased the infarct size of MCAO rats (26.2±4.1%vs.34.8±3.9%in LV-GFP) and improved their neurological function (2.0±0.0vs.2.8±0.3in LV-GFP). This protective effect of PKCε against stroke was completely abolished by the knockdown of ALDH2(49.8±14.7%in LV-PKεs+sh-ALDH2vs.34.8±4.5%in LV-GFP+sh-ALDH2). The knockdown of PKCε decreased the activity of ALDH2by68%in the brain, and completely removed the activation of ALDH2induced by ethanol. It also abolished the ethanol induced protection against cerebral infarction (42.0±12.7%vs.38.8±6.9%without ethanol), and the reduction of4-HNE (1054.1±107.9vs.952.3±71.6ng/ml without ethanol).Here are our conclusions:1.ALDH2protects the brain from ischemic damage, most likely through its detoxification effect on4-HNE.2.4-HNE might be an important risk factor for stroke.3. PKCε protects the brain from ischemic damage, through mediating activation of ALDH2.4.The neuroprotective effects of ethanol may proceed through PKCε mediated activation of ALDH2to diminish the accumulation of4-HNE.