Lingling Zhu

Hypoxia‐driven proliferation of embryonic neural stem/progenitor cells – role of hypoxia‐inducible transcription factor‐1α

We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus inu2003vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs inu2003vitro and that hypoxia‐inducible factor (HIF)‐1α, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20%u2003O2, 10%u2003O2, and 3%u2003O2) for 3u2003days. The results showed that hypoxia, especially 10%u2003O2, promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF‐1α mRNA and protein expression detected by RT‐PCR and western blot significantly increased in NPCs subjected to 10%u2003O2. To further elucidate the potential role of HIF‐1α in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF‐1α, and the pSilenceru20031.0‐U6 plasmid as RNA interference vector targeting HIF‐1α mRNA was used to knock down HIF‐1α. We found that overexpression of HIF‐1α caused the same proliferative effect on NPCs under 20%u2003O2 as under 10%u2003O2. In contrast, knockdown of HIF‐1α inhibited NPC proliferation induced by 10%u2003O2. These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF‐1α was critical in this process.

Oxygen, a Key Factor Regulating Cell Behavior during Neurogenesis and Cerebral Diseases.

Oxygen is vital to maintain the normal functions of almost all the organs, especially for brain which is one of the heaviest oxygen consumers in the body. The important roles of oxygen on the brain are not only reflected in the development, but also showed in the pathological processes of many cerebral diseases. In the current review, we summarized the oxygen levels in brain tissues tested by real-time measurements during the embryonic and adult neurogenesis, the cerebral diseases, or in the hyperbaric/hypobaric oxygen environment. Oxygen concentration is low in fetal brain (0.076–7.6u2009mmHg) and in adult brain (11.4–53.2u2009mmHg), decreased during stroke, and increased in hyperbaric oxygen environment. In addition, we reviewed the effects of oxygen tensions on the behaviors of neural stem cells (NSCs) in vitro cultures at different oxygen concentration (15.2–152u2009mmHg) and in vivo niche during different pathological states and in hyperbaric/hypobaric oxygen environment. Moderate hypoxia (22.8–76u2009mmHg) can promote the proliferation of NSCs and enhance the differentiation of NSCs into the TH-positive neurons. Next, we briefly presented the oxygen-sensitive molecular mechanisms regulating NSCs proliferation and differentiation recently found including the Notch, Bone morphogenetic protein and Wnt pathways. Finally, the future perspectives about the roles of oxygen on brain and NSCs were given.

miR-210 suppresses BNIP3 to protect against the apoptosis of neural progenitor cells.

MiR-210 is a hypoxia-inducible factor (HIF)-1 target gene and is the most consistently and predominantly upregulated miRNA in response to hypoxia in various cancer cell lines. Our recent study shows that hypoxia increased miR-210 expression in neural progenitor cells (NPCs) in a time-dependent manner. However, the role of miR-210 in NPCs remains unknown. Following the identification of the miR-210 putative target genes, we demonstrated that the Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3), which is regulated by HIF-1 and activates cell death, is regulated by miR-210 in NPCs under hypoxia. Moreover, the over-expression of miR-210 decreased apoptosis in NPCs, and the inhibition of miR-210 expression remarkably increased the number of TUNEL-positive NPCs by 30% in response to hypoxia. Importantly, miR-210 mimics reduced both BNIP3 protein expression and the translocation of AIF into the nucleus, which reduced cell death, whereas miR-210 inhibitors reversed this process, leading to cell death during hypoxia. Taken together, we report a novel feedback loop of BNIP3 regulation in NPCs under hypoxia. HIF-1 is activated under hypoxia and then induces the expression of both BNIP3 and miR-210. The upregulation of miR-210 then directly suppresses BNIP3 expression to maintain the survival of NPCs under hypoxia. This negative feedback regulation might partially contribute to protection against hypoxia-induced cell death via the inhibition of AIF nuclear translocation.

Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain

It has been shown that induction of HSP70 by administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury. The present study was performed to determine the effect of GGA on the survival of mice and on brain damage under acute hypobaric hypoxia. The data showed that the mice injected with GGA survived significantly longer than control animals (survival time of 9.55u2009±u20093.12xa0min, nu2009=u200916 vs. controls at 4.28u2009±u20094.29xa0min, nu2009=u200915, Pu2009<u20090.005). Accordingly, the cellular necrosis or degeneration of the hippocampus and the cortex induced by sublethal hypoxia for 6xa0h could be attenuated by preinjection with GGA, especially in the CA2 and CA3 regions of the hippocampus. In addition, the activity of nitric oxide synthase (NOS) of the hippocampus and the cortex was increased after exposure to sublethal hypoxia for 6xa0h but could be inhibited by the preinjection of GGA. Furthermore, the expression of HSP70 was significantly increased at 1xa0h after GGA injection. These results suggest that administration of GGA improved survival rate and prevented acute hypoxic damage to the brain and that the underlying mechanism involved induction of HSP70 and inhibition of NOS activity.

Hypoxia promotes dopaminergic differentiation of mesenchymal stem cells and shows benefits for transplantation in a rat model of Parkinson's disease.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into dopaminergic (DAergic) neurons, which is one of the major cell types damaged in Parkinson’s disease (PD). For this reason, MSCs are considered a potential cell source for PD therapy. It has been proved that hypoxia is involved in the proliferation and differentiation of stem cells. In this study, we investigated the effect of hypoxia on MSC proliferation and DAergic neuronal differentiation. Our results demonstrate that 3% O2 treatment can enhance rat MSC proliferation by upregulation of phosphorylated p38 MAPK and subsequent nuclear translocation of hypoxia inducible factor (HIF)-1α. During neural differentiation, 3% O2 treatment increases the expression of HIF-1α, phosphorylated ERK and p38 MAPK. These changes are followed by promotion of neurosphere formation and further DAergic neuronal differentiation. Furthermore, we explored the physiological function of hypoxia-induced DAergic neurons from human fetal MSCs by transplanting them into parkinsonian rats. Grafts induced with hypoxia display more survival of DAergic neurons and greater amelioration of behavioral impairments. Altogether, these results suggest that hypoxia can promote MSC proliferation and DAergic neuronal differentiation, and benefit for intrastriatal transplantation. Therefore, this study may provide new perspectives in application of MSCs to clinical PD therapy.

DNA demethylation regulates the expression of miR‐210 in neural progenitor cells subjected to hypoxia

Several studies have identified a set of hypoxia‐regulated microRNAs, among which is miR‐210, whose expression is highly induced by hypoxia in various cancer cell lines. Recent studies have highlighted the importance of miR‐210 and its transcriptional regulation by the transcription factor hypoxia‐inducible factor‐1 (HIF‐1). We report here that the expression of miR‐210 was highly induced in neural progenitor cells (NPCs) subjected to hypoxia. Specifically, treating hypoxic NPCs with the DNA demethylating agent 5‐aza‐2′‐deoxycytidine significantly increased the expression of miR‐210, even under normoxia; however, the activity of hypoxia‐inducible factor‐1 was unaffected. Further analysis of the miR‐210 sequence revealed that it is embedded in a CpG island. Bisulfite sequencing of the miR‐210 CpG island from NPCs grown under hypoxic conditions showed 24% CpG methylation in NPCs exposed to 20% O2, 18% in NPCs exposed to 3% O2, and 12% in NPCs exposed to 0.3% O2. In addition, the activity of DNA methyltransferases (DNMTs) in NPCs decreased after exposure to hypoxia. Specifically, the expression of DNMT3b decreased significantly after exposure to 0.3% O2. Thus, these results demonstrate that DNA demethylation regulates miR‐210 expression in NPCs under both normoxia and hypoxia.

Methylene Blue Reduces Acute Cerebral Ischemic Injury via the Induction of Mitophagy.

The treatment of stroke is limited by a short therapeutic window and a lack of effective clinical drugs. Methylene blue (MB) has been used in laboratories and clinics since the 1890s. Few studies have reported the neuroprotective role of MB in cerebral ischemia-reperfusion injury. However, whether and how MB protects against acute cerebral ischemia (ACI) injury was unclear. In this study, we investigated the effect of MB on this injury and revealed that MB protected against ACI injury by augmenting mitophagy. Using a rat middle cerebral artery occlusion (MCAO) model, we demonstrated that MB improved neurological function and reduced the infarct volume and necrosis after ACI injury. These improvements depended on the effect of MB on mitochondrial structure and function. ACI caused the disorder and disintegration of mitochondrial structure, while MB ameliorated the destruction of mitochondria. In addition, mitophagy was inhibited at 24 h after stroke and MB augmented mitophagy. In an oxygen-glucose deprivation (OGD) model in vitro, we further revealed that the elevation of mitochondrial membrane potential (MMP) by MB under OGD conditions mediated the augmented mitophagy. In contrast, exacerbating the decline of MMP during OGD abolished the MB-induced activation of mitophagy. Taken together, MB promotes mitophagy by maintaining the MMP at a relatively high level, which contributes to a decrease in necrosis and an improvement in neurological function, thereby protecting against ACI injury.

The protective role of 5-HMF against hypoxic injury.

In an attempt to find new types of anti-hypoxic agents from herbs, we identified 5-hydroxymethyl-2-furfural (5-HMF) as a natural agent that fulfills the criterion. 5-HMF, the final product of carbohydrate metabolism, has favorable biological effects such as anti-oxidant activity and inhibiting sickling of red blood cells. The role of 5-HMF in hypoxia, however, is not yet. Our pilot results showed that pretreatment with 5-HMF markedly increased both the survival time and the survival rate of mice under hypoxic stress. The present study was aimed to further investigate the protective role of 5-HMF and the underlying mechanisms in hypoxic injury using ECV304 cells as an in vitro model. ECV304 cells pretreated with or without 5-HMF for 1xa0h were exposed to hypoxic condition (0.3% O2) for 24xa0h and then cell apoptosis, necrosis, the changes of mitochondrial membrane potential (MMP) and the expressions of phosphorylation- extracellular signal-regulated kinase (p-ERK) were investigated. Pretreatment with 5-HMF markedly attenuated hypoxia-induced cell necrosis and apoptosis at late stage (pu2009<u20090.01). Furthermore, pretreatment with 5-HMF rescued both the decline of the MMP and the increase of p-ERK protein under hypoxia. In a word, these results indicated that 5-HMF had protective effects against hypoxic injury in ECV304 cells, and its effects on MMP and p-ERK may be involved in the mechanism.

CHL1 negatively regulates the proliferation and neuronal differentiation of neural progenitor cells through activation of the ERK1/2 MAPK pathway.

Neural recognition molecules of the immunoglobulin superfamily play important roles in the development and regeneration of nervous system. Close Homologue of L1 (CHL1) is a member of the L1 family of recognition molecules which are expressed during neuronal development, suggesting a potential role in neural progenitor cells (NPCs). Here, we investigated the role of CHL1 in the proliferation and differentiation of NPCs both in vivo and in vitro, and the possible mechanism involved. The number of BrdU-positive cells in the subventricular zone (SVZ) significantly increased in CHL1-/- mice compared with CHL1+/+ mice. Moreover, there were more Tuj1-positive cells in the cortical plate region in CHL1-/- mice than in CHL1+/+ controls. To further examine the function of CHL1 in the proliferation and differentiation of NPCs, NPCs from CHL1-/- mice versus littermate wild-type mice were isolated and cultured in vitro. NPCs derived from CHL1-/- mice showed increased proliferation and self-renewal ability compared with CHL1+/+ mice. In the course of differentiation, CHL1 deficiency enhanced neuronal differentiation in the absence of growth factors. Furthermore, CHL1 deficiency on the proliferation of NPCs is accompanied by means of enhanced activation of ERK1/2 mitogen-activated protein kinase (MAPK) and the inhibitor of ERK1/2 MAPK eliminates the effect of CHL1 deficiency on the proliferation of NPCs. Our results first describe the negative modulation of the proliferation and neuronal differentiation of NPCs by CHL1/ERK1/2 MAPK signaling.

The protective role of 5-hydroxymethyl-2-furfural (5-HMF) against acute hypobaric hypoxia

Our previous study showed that pretreatment with 5-hydroxymethyl-2-furfural (5-HMF) led to protection against hypoxic injury via a p-ERK-mediated pathway in vitro. Whether the protection of 5-HMF against hypoxia is effective in vivo is unknown. The present study is aimed to verify the role of 5-HMF in acute hypobaric hypoxia using Kunming mice as an in vivo model and further investigate the underlying mechanisms. Mice pretreated with or without 5-HMF for 1xa0h were exposed to acute hypobaric hypoxic condition for 6xa0h and then the survival time, the survival rate, the permeability of blood–brain barrier (BBB), the histological analysis in hippocampus and cortex, and the phosphorylation level of mitogen-activated protein kinases (ERK, JNK, and p38) were investigated. The results showed that 5-HMF significantly increased the survival time and the survival rate of mice. Accordingly, pretreatment with 5-HMF markedly attenuated acute hypobaric hypoxia-induced permeability of BBB (Pu2009<u20090.01). In addition, the cellular damage extent of the hippocampus and the cortex induced by hypoxia for 6xa0h was also attenuated by pretreatment with 5-HMF, especially in the hippocampus CA1 region. Furthermore, the activation of ERK rather than JNK and p38 was involved in the protection of 5-HMF against acute hypobaric hypoxia. In summary, 5-HMF enhanced the survival capability of mice and decreased acute hypoxic damage to the brain, which may be associated with the effects on BBB and p-ERK.