Xingshun Xu

Carvacrol, a Food-Additive, Provides Neuroprotection on Focal Cerebral Ischemia/Reperfusion Injury in Mice

Carvacrol (CAR), a naturally occurring monoterpenic phenol and food additive, has been shown to have antimicrobials, antitumor, and antidepressant-like activities. A previous study demonstrated that CAR has the ability to protect liver against ischemia/reperfusion injury in rats. In this study, we investigated the protective effects of CAR on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that CAR (50 mg/kg) significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. This neuroprotection was in a dose-dependent manner. Post-treatment with CAR still provided protection on infarct volume when it was administered intraperitoneally at 2 h after reperfusion; however, intracerebroventricular post-treatment reduced infarct volume even when the mice were treated with CAR at 6 h after reperfusion. These findings indicated that CAR has an extended therapeutic window, but delivery strategies may affect the protective effects of CAR. Further, we found that CAR significantly decreased the level of cleaved caspase-3, a marker of apoptosis, suggesting the anti-apoptotic activity of CAR. Finally, our data indicated that CAR treatment increased the level of phosphorylated Akt and the neuroprotection of CAR was reversed by a PI3K inhibitor LY-294002, demonstrating the involvement of the PI3K/Akt pathway in the anti-apoptotic mechanisms of CAR. Due to its safety and wide use in the food industry, CAR is a promising agent to be translated into clinical trials.

Protective role of autophagy in AGE-induced early injury of human vascular endothelial cells.

Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetes mellitus and atherosclerosis by promoting vascular endothelial cell proliferation, migration, damage and death. In this study, we examined the role of autophagy in HUVECs exposed to AGE-modified bovine serum albumin (AGE-BSA). HUVECs incubated with AGE-BSA for 6 h showed an increase in the formation of acidic vesicular organelles and autophagosomes. AGE-BSA-induced upregulation of microtubule associated protein 1 light chain 3-II (LC3-II), a marker of autophagy, was abolished by pretreatment with the autophagy inhibitor 3-methyladenine (3-MA), and was increased by rapamycin, an autophagy inducer. The increase of lactate dehydrogenase (LDH) leakage induced by AGE-BSA was increased by 3-MA, but not rapamycin. An oxidative inhibitor, α-tocopherol, decreased not only the AGE-BSA-induced increase of reactive oxygen species, but also the upregulation of LC3-II protein levels. These results suggest that AGE-BSA increases the level of autophagy, which is protective against HUVEC injury, and that ROS play a role in this activation of autophagy.

A natural coumarin derivative esculetin offers neuroprotection on cerebral ischemia/reperfusion injury in mice.

J. Neurochem. (2012) 121, 1007–1013.

Apelin-36, a potent peptide, protects against ischemic brain injury by activating the PI3K/Akt pathway.

Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin-APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.

Homocysteine Triggers Inflammatory Responses in Macrophages through Inhibiting CSE-H2S Signaling via DNA Hypermethylation of CSE Promoter

Hyperhomocysteinemia (HHcy) is an independent risk factor of atherosclerosis and other cardiovascular diseases. Unfortunately, Hcy-lowering strategies were found to have limited effects in reducing cardiovascular events. The underlying mechanisms remain unclear. Increasing evidence reveals a role of inflammation in the pathogenesis of HHcy. Homocysteine (Hcy) is a precursor of hydrogen sulfide (H2S), which is formed via the transsulfuration pathway catalyzed by cystathionine β-synthase and cystathionine γ-lyase (CSE) and serves as a novel modulator of inflammation. In the present study, we showed that methionine supplementation induced mild HHcy in mice, associated with the elevations of TNF-α and IL-1β in the plasma and reductions of plasma H2S level and CSE expression in the peritoneal macrophages. H2S-releasing compound GYY4137 attenuated the increases of TNF-α and IL-1β in the plasma of HHcy mice and Hcy-treated raw264.7 cells while CSE inhibitor PAG exacerbated it. Moreover, the in vitro study showed that Hcy inhibited CSE expression and H2S production in macrophages, accompanied by the increases of DNA methyltransferase (DNMT) expression and DNA hypermethylation in cse promoter region. DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages. In sum, our findings demonstrate that Hcy may trigger inflammation through inhibiting CSE-H2S signaling, associated with increased promoter DNA methylation and transcriptional repression of cse in macrophages.

RIP1 mediates the protection of geldanamycin on neuronal injury induced by oxygen-glucose deprivation combined with zVAD in primary cortical neurons.

J. Neurochem. (2012) 120, 70–77.

THE INHIBITION OF CDK5 ACTIVITY AFTER HYPOXIA/ISCHEMIA INJURY REDUCES INFARCT SIZE AND PROMOTES FUNCTIONAL RECOVERY IN NEONATAL RATS

Recent studies indicate that over-activation of Cdk5 is a crucial pro-death signal and Cdk5 activity inhibition provides neuroprotection in animal stroke models. However, Cdk5 inhibitors are reported to affect physiological functions of Cdk5 and lead to serious side effects. Therefore, targeting Cdk5 or its activators without affecting physiological functions of Cdk5 is a therapeutic strategy for ischemic brain injury. In this study, we examined Cdk5 activity in a rat hypoxia/ischemia (HI) injury model. Cdk5 expression was not changed after HI injury, but Cdk5 activity significantly increased, which was demonstrated by the increased phorsphorylation-phosphorylation of Tau and glucocorticoid receptor (GR), two downstream signals of Cdk5. We further showed that the levels of Cdk5 activators p35 and p39 decreased after HI injury, while p25, which is converted from p35 and has a higher activator activity on Cdk5, increased markedly after HI injury. P5, a 24-residue mimetic peptide of p35, was reported to specifically inhibit the p25/Cdk5 signal pathway in an Alzheimers disease model. P5-TAT, which can cross the blood-brain barrier and cell membrane facilitated by TAT protein, was used in our study. We found that p5-TAT treatment did not change the levels of p35, p39, and p25, but reduced the phorsphorylation of Tau and GR, suggesting the inhibition of the p25/Cdk5 by the peptide p5-TAT. This was supported by the fact that p5 interacted with Cdk5, but not with Cdk5 activators. In addition, p5-TAT reduced cleaved caspase-3 level, a marker of neuronal apoptosis. We further demonstrated that p5-TAT pre-treatment reduced cerebral infarct volume; even when p5-TAT was delayed to be administered at 24h after HI injury, p5-TAT still promoted long-term functional recovery. Therefore, Cdk5 inhibition by the small peptide p5-TAT or its derivatives is a promising therapeutic strategy for the treatment of ischemic brain injury including hypoxic-ischemic encephalopathy and stroke.

Altering 5-hydroxymethylcytosine modification impacts ischemic brain injury.

Epigenetic modifications such as cytosine methylation and histone modification are linked to the pathology of ischemic brain injury. Recent research has implicated 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) via oxidation by ten-eleven translocation (Tet) enzymes, in DNA methylation-related plasticity. Here we show that 5hmC abundance was increased after ischemic injury, and Tet2 was responsible for this increase; furthermore, inhibiting Tet2 expression abolished the increase of 5hmC caused by ischemic injury. The decrease in 5hmC modifications from inhibiting Tet2 activity was accompanied by increased infarct volume after ischemic injury. Genome-wide profiling of 5hmC revealed differentially hydroxymethylated regions (DhMRs) associated with ischemic injury, and DhMRs were enriched among the genes involved in cell junction, neuronal morphogenesis and neurodevelopment. In particular, we found that 5hmC modifications at the promoter region of brain-derived neurotrophic factor (BDNF) increased, which was accompanied by increased BDNF mRNA, whereas the inhibition of Tet2 reduced BDNF mRNA and protein expression. Finally, we show that the abundance of 5hmC in blood samples from patients with acute ischemic stroke was also significantly increased. Together, these data suggest that 5hmC modification could serve as both a potential biomarker and a therapeutic target for the treatment of ischemic stroke.

Loss of Ahi1 Impairs Neurotransmitter Release and Causes Depressive Behaviors in Mice

Major depression is becoming one of the most prevalent forms of psychiatric disorders. However, the mechanisms of major depression are still not well-understood. Most antidepressants are only effective in some patients and produce some serious side effects. Animal models of depression are therefore essential to unravel the mechanisms of depression and to develop novel therapeutic strategies. Our previous studies showed that Abelson helper integration site-1 (Ahi1) deficiency causes depression-like behaviors in mice. In this study, we characterized the biochemical and behavioral changes in Ahi1 knockout (KO) mice. In Ahi1 KO mice, neurotransmitters including serotonin and dopamine were significantly decreased in different brain regions. However, glutamate and GABA levels were not affected by Ahi1 deficiency. The antidepressant imipramine attenuated depressive behaviors and partially restored brain serotonin level in Ahi1 KO mice. Our findings suggest that Ahi1 KO mice can be used for studying the mechanisms of depression and screening therapeutic targets.

A novel cysteine-sparing NOTCH3 mutation in a Chinese family with CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL.