Fengyan Zhao

mTOR activates hypoxia-inducible factor-1α and inhibits neuronal apoptosis in the developing rat brain during the early phase after hypoxia–ischemia

The mammalian target of rapamycin (mTOR) exerts neuroprotective effects under hypoxic or ischemic conditions. To explore whether mTOR participates in neuroprotective signaling through regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and neuronal apoptosis in developing rat brain with hypoxia-ischemia (HI), we operated on postnatal day 10 rats by ligating the common carotid artery followed by exposure to systemic hypoxia. Brains were collected at various intervals to detect the expression of mTOR, phosphorylated mTOR (p-mTOR), HIF-1α, VEGF and cleaved caspase 3 (CC3), using immunohistochemistry and Western blot analysis. We also used terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) to detect neuronal apoptosis. The p-mTOR protein expression increased at 2h after HI, peaked at 8h, lasted 24h, and then dropped to the basal level. Also, the expression of HIF-1α and VEGF was significantly enhanced and peaked at 8h after HI. Up-regulated expression of CC3 was observed at 2h, peaked at 24h, and lasted 72h after HI. Increased neuronal apoptosis is associated with reduced HIF-1α and VEGF expression. Furthermore, pretreatment with rapamycin, a mTOR specific inhibitor, significantly inhibited HIF-1α and VEGF protein after HI. The expression of CC3 and the number of TUNEL-positive cells were up-regulated at 8h and down-regulated at 24h after HI in the rapamycin-treated group. Our findings suggest that mTOR may participate in the regulation of HIF-1α, VEGF and neuronal apoptosis, serving neuroprotective functions after HI in developing rat brain.

MiR-139-5p inhibits HGTD-P and regulates neuronal apoptosis induced by hypoxia–ischemia in neonatal rats

Human growth transformation dependent protein (HGTD-P) is a newly identified protein that promotes neuronal apoptosis in hypoxia-ischemia brain damage (HIBD) in neonatal rats. However, the mechanisms regulating HGTD-P expression are not clear. Here we describe microRNAs targeted to HGTD-P and examine their effects on regulating neuronal apoptosis in HIBD. We use samples from cultured neurons after oxygen-glucose deprivation (OGD) and postnatal day 10 rat brains after hypoxia-ischemia (HI). RT-PCR, Western blotting, and immunostaining are used to detect the expression of HGTD-P and cleaved caspase 3, as well as real-time PCR detects microRNA expression. MicroRNA agomir is used to inhibit the expression of HGTD-P, and DAPI, TUNEL, and TTC staining are employed to detect cell apoptosis and brain damage. Moreover, in vitro processing assay is used to examine the mechanism by which HI down-regulates miR-139-5p expression. We found that miR-139-5p is down-regulated in neurons and rat brains after HI treatment. The expression pattern of miR-139-5p correlates inversely with that of HGTD-P. Furthermore, miR-139-5p agomir inhibits neuronal apoptosis and attenuates HIBD, which is concurrent with down-regulation of HGTD-P. Moreover, pre-miR-139 processing activity decreases in extracts from OGD neurons, and OGD neuronal extracts attenuates the processing of pre-miR-139 by Dicer. In conclusion, HI induces inhibitors which block the processing step of pre-miR-139, resulting in the down-regulation of mature miR-139-5p. The down-regulation of miR-139-5p plays a critical role in the up-regulation of HGTD-P expression. MiR-139-5p agomir attenuates brain damage when used 12h after HI, providing a longer therapeutic window than anti-apoptosis compounds currently available.

Telomerase Reverse Transcriptase Upregulation Attenuates Astrocyte Proliferation and Promotes Neuronal Survival in the Hypoxic–Ischemic Rat Brain

Background and Purpose— Telomerase reverse transcriptase (TERT) is tightly related to the resistance of cells to stress and injury. However, little is known about the roles of TERT in the nervous system. We try to investigate the effects of TERT on the function of astrocytes in developing rat brains subjected to hypoxia–ischemia. Methods— TERT expression was detected in rat brains with hypoxia–ischemia. In in vitro study, the function of astrocytes with TERT overexpression was measured, and the effects of astrocyte on neuronal apoptosis were examined. Moreover, overexpression or inhibition of TERT was conducted in vivo by gene transduction. Astrocyte proliferation was examined through Ki67 staining. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining and brain infarct volume calculation were used to detect neuronal injury. Results— Both TERT mRNA and protein were upregulated in neurons within 2 days but shifted to astrocytes at Day 3 after hypoxia–ischemia. Astrocyte proliferation was inhibited with TERT overexpression due to the upregulation of cell-cycle regulatory protein p15. Meanwhile, the apoptosis of neurons increased, whereas neurons were cocultured with conditioned media from astrocytes with TERT inhibition compared with TERT overexpression due to the decrease of neurotrophin-3 expression in astrocytes. Furthermore, Ki67-positive astrocytes and neuronal injury were found to be inhibited in TERT-overexpressing rat brains with hypoxia–ischemia. Conclusions— TERT attenuates astrocyte proliferation and promotes neuronal survival in the developing rat brain after hypoxia–ischemia, partly through its enhancement of p15 and neurotrophin-3 expression in astrocytes.

Involvement of the Akt/GSK-3β/CRMP-2 pathway in axonal injury after hypoxic–ischemic brain damage in neonatal rat

Akt has been demonstrated as a survival kinase in brain after hypoxia-ischemia (HI). Previous studies have shown that glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein 2 (CRMP-2) signaling pathway could be regulated by Akt for axonal-dendritic polarity. CRMP-2 is associated also with microtubule-mediated trafficking. However, whether Akt could regulate GSK-3β/CRMP-2 pathway and the possible effects of this regulation is unclear in developing brain after HI. In this study, we detected the expression of total and phosphorylated Akt, GSK-3β, and CRMP-2, as well as the axonal injury marker amyloid precursor protein (APP) by utilizing an HI model in postnatal 10-day rats. Axonal loss was determined by Bielschowsky silver impregnation, and histological injury was evaluated by hematoxylin and eosin (H&E) staining. We found that the phosphorylation of Akt was accompanied by phosphorylation of GSK-3β and dephosphorylation of CRMP-2 after HI. Furthermore, Akt inhibition significantly decreased the phosphorylation of GSK-3β and dephosphorylation of CRMP-2. Moreover, the down-regulation of dephosphorylated CRMP-2 was associated with increased axonal injury (increased APP expression and axonal loss). Our findings suggest that the Akt/GSK-3β/CRMP-2 pathway mediates axonal injury in neonatal rat brain after HI.

Microarray Profiling and Co-Expression Network Analysis of LncRNAs and mRNAs in Neonatal Rats Following Hypoxic-ischemic Brain Damage.

Long noncoding RNAs (lncRNAs) play critical roles in cellular homeostasis. However, little is known about their effect in developing rat brains with hypoxic-ischemic brain damage (HIBD). To explore the expression and function of lncRNA in HIBD, we analyzed the expression profiles of lncRNAs in hypoxic-ischemic (HI) brains and sham control using microarray analysis. The results showed a remarkable difference in lncRNA between HI and sham brains. A total of 322 lncRNAs were found to be differentially expressed in HI brains, compared to sham control. Among these, BC088414 was one of the most significantly urpregulated lncRNAs. In addition, 375 coding genes were differentially expressed between HI brains and sham control. Pathway and gene ontology analysis indicated that the upregulated coding genes mostly involved in wounding, inflammation and defense, whereas the downregulated transcripts were largely associated with neurogenesis and repair. Moreover, coding non-coding co-expression network analysis showed that the BC088414 lncRNA expression was correlated with apoptosis-related genes, including Casp6 and Adrb2. Silencing of lncRNA BC088414 in PC12 cells caused reduced mRNA level of Casp6 and Adrb2, decreased cell apoptosis and increased cell proliferation. These results suggested lncRNA might participate in the pathogenesis of HIBD via regulating coding genes.

miR-199a-3p Inhibits Aurora Kinase A and Attenuates Prostate Cancer Growth: New Avenue for Prostate Cancer Treatment

Prostate cancer (PCa) is the most common solid tumor malignancy in men that severely influences quality of life. Surgery is most often the recommended treatment for PCa, but radical prostatectomy can cause significant urinary adverse effects. Therefore, finding effective biochemical treatments for PCa remains a necessity. Aurora kinase A has been shown to be involved in PCa progression, thus making it a good target for PCa therapy. miRNAs are important regulators of gene expression, with some miRNAs specifically involved in carcinogenesis. Therefore, herein, we identified miRNAs targeted to aurora kinase A and examined their effects on the growth of PCa. We used primary samples from PCa patients and PCa cell lines as research subjects. We demonstrate that miR-199a-3p is down-regulated in PCa tissues compared with normal prostate tissues, with the expression pattern inversely correlated with the expression pattern of aurora kinase A. We find that miR-199a-3p agomir inhibits aurora kinase A and attenuates xenograft tumor growth of PCa. Moreover, we demonstrate that down-regulation of miR-199a-3p results from enhancement of the methylation of miR-199a gene in PCa. Furthermore, we find that the expression level of miR-199a-3p is inversely correlated to tumor stage and Gleason score of PCa. Revealing novel mechanisms for oncogene inhibition by miRNA-mediated pathways offers new avenues for PCa treatment.

Nitric oxide synthase in hypoxic or ischemic brain injury.

Abstract Hypoxic or ischemic stress causes many serious brain injuries, including stroke and neonatal hypoxia ischemia encephalopathy. During brain hypoxia ischemia processes, nitric oxide (NO) may play either a neurotoxic or a neuroprotective role, depending upon factors such as the NO synthase (NOS) isoform, the cell type by which NO is produced, and the temporal stage after the onset of the hypoxic ischemic brain injury. Excessive NO production can be neurotoxic, leading to cascade reactions of excitotoxicity, inflammation, apoptosis, and deteriorating primary brain injury. In contrast, NO produced by endothelial NOS plays a neuroprotective role by maintaining cerebral blood flow and preventing neuronal injury, as well as inhibiting platelet and leukocyte adhesion. Sometimes, NO-derived inducible NOS and neuronal NOS in special areas may also play neuroprotective roles. Therefore, this review summarizes the different roles and the regulation of the three NOS isoforms in hypoxic or ischemic brain injury as revealed in research in recent years, focusing on the neurotoxic role of the three NOS isoforms involved in mechanisms of hypoxic or ischemic brain injury.

The neuroprotective role and mechanisms of TERT in neurons with oxygen-glucose deprivation.

Telomerase reverse transcriptase (TERT) is reported to protect neurons from apoptosis induced by various stresses including hypoxia-ischemia (HI). However, the mechanisms by which TERT exerts its anti-apoptotic role in neurons with HI injury remain unclear. In this study, we examined the protective role and explored the possible mechanisms of TERT in neurons with HI injury in vitro. Primary cultured neurons were exposed to oxygen and glucose deprivation (OGD) for 3h followed by reperfusion to mimic HI injury in vivo. Plasmids containing TERT antisense, sense nucleotides, or mock were transduced into neurons at 48h before OGD. Expression and distribution of TERT were measured by immunofluorescence labeling and western blot. The expression of cleaved caspase 3 (CC3), Bcl-2 and Bax were detected by western blot. Neuronal apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The mitochondrial reactive oxygen species (ROS) were measured by MitoSOX Red staining. Fluorescent probe JC-1 was used to measure the mitochondrial membrane potential (ΔΨm). We found that TERT expression increased at 8h and peaked at 24h in neurons after OGD. CC3 expression and neuronal apoptosis were induced and peaked at 24h after OGD. TERT inhibition significantly increased CC3 expression and neuronal apoptosis after OGD treatment. Additionally, TERT inhibition decreased the expression ratio of Bcl-2/Bax, and enhanced ROS production and ΔΨm dissipation after OGD. These data suggest that TERT plays a neuroprotective role via anti-apoptosis in neurons after OGD. The underlying mechanisms may be associated with regulating Bcl-2/Bax expression ratio, attenuating ROS generation, and increasing mitochondrial membrane potential.

Umbilical cord blood mesenchymal stem cells co-modified by TERT and BDNF: A novel neuroprotective therapy for neonatal hypoxic-ischemic brain damage

Hypoxic‐ischemic brain damage (HIBD), a leading cause of perinatal disability and death, has limited therapeutic options. Stem cell therapy has been demonstrated as a potential novel therapy for neurological disorders. Compared with other types of stem cells, umbilical cord blood mesenchymal stem cells (UCB‐MSCs) have several unique characteristics, such as a higher rate of cell proliferation and clonality. However, the limited life span of UCB‐MSCs hinders their clinical application. Therefore, efforts are urgently needed to circumvent this disadvantage. Telomerase reverse transcriptase (TERT), which promotes cell proliferation and survival, plays a protective role in hypoxic‐ischemic (HI) brain injury. Thus, it is reasonable to propose that UCB‐MSCs modified by exogenous TERT expression might have a longer lifespan and increased viability. Moreover, brain‐derived neurotrophic factor (BDNF), a neurotrophin that regulates development, regeneration, survival and maintenance of neurons, facilitates post‐injury recovery when administered by infusion or virus‐mediated delivery. Therefore, TERT‐ and BDNF‐modified UCB‐MSCs may have a longer lifespan and also maintain neural differentiation, thus promoting the recovery of neurological function following hypoxic‐ischemic brain damage (HIBD) and thereby representing a new effective strategy for HIBD in neonates.

Proapoptotic Role of Human Growth and Transformation-Dependent Protein in the Developing Rat Brain After Hypoxia-Ischemia

Background and Purpose— Human growth and transformation-dependent protein (HGTD-P) is a new proapoptotic protein and an effector of cell death induced by hypoxia-ischemia (HI). The function of HGTD-P has been investigated in human prostate cancer cells and mouse neurons cultured in vitro. However, whether HGTD-P is involved in regulating the apoptosis of rat neurons is not clear, and the relevance of HGTD-P in HI animal models is still unknown. Therefore, in the present study, we tried to elucidate the role that HGTD-P plays in apoptosis of rat neurons subjected to HI, both in culture and in the developing rat brain in vivo. Methods— Samples from primary cultured neurons and postnatal day 10 rat brains with HI were collected. RT-PCR, Western blotting, and immunocytochemistry were used to detect the expression and distribution of rat HGTD-P, cleaved caspase 3, and apoptosis- inducing factor (AIF). MTT assay, DAPI, TUNEL, and flowcytometry were used to detect cell viability and apoptosis. Results— We found that HI upregulated the mRNA and protein levels of HGTD-P in rat neurons in vitro and in vivo. Antisense oligonucleotides (AS) targeted to HGTD-P inhibited the expression of HGTD-P, thus rescuing neuronal viability and attenuating neuronal apoptosis. In addition, we found that HGTD-P played its proapoptotic role by activating caspase 3 and inducing the translocation of AIF to nuclear. Conclusions— Our findings show that HGTD-P plays a proapoptotic role in the developing rat brain after HI and that it may be a potential target in treating HI-induced brain damage.