Neuroglobin Is Involved in the Hypoxic Stress Response in the Brain.

Neuroglobin is an oxygen-binding heme protein expressed predominantly in the brain. Despite many years of research, the exact distribution and expression of neuroglobin in the neocortical development and under mild hypoxia stress still remain unclear. Therefore, we aim to explore the expression of neuroglobin during neocortex expansion and under mild hypoxia stress in vivo. We used Kunming mice to examine the expression of Ngb protein during neocortex expansion. In addition, we analyzed the density of Ngb-positive neural stem cells using the Image-Pro PLUS (v.6) computer software program (Media Cybernetics, Inc.). Our data indicated that the density of the neuroglobin-positive neurons in mice cerebral cortex displayed a downward trend after birth compared with high expression of neuroglobin in a prenatal period. Similarly, we identified that neurons were capable of ascending neuroglobin levels in response to mild hypoxic stress compared with the no intervention group. These findings suggest that neuroglobin behaves as a compensatory protein regulating oxygen provision in the process of neocortical development or under physiological hypoxia, further contributing to the discovery of novel therapeutic methods for neurological disorders, which is clinically important.

miR-590-5p Overexpression Alleviates ß-Amyloid-Induced Neuron Damage via Targeting Pellino-1.

Alzheimer's disease (AD) is one common degenerative disorder. However, the effects of miR-590-5p on AD and the mechanism on modulation of AD development were unclear. In this study, the miR-590-5p level in AD patients at mild, moderate, and severe stage as well as APP/PS1 transgenic mice was detected by qRT-PCR. The relationship of miR-590-5p and pellino-1 (PELI1) was identified by double luciferase reporter gene assay. Afterwards, both BV-2 and HT22 cells were exposed to ß-amyloid (Aß) peptides to mimic AD cell model. Then, the roles of miR-590-5p upregulation or PELI1 silence in cell proliferation and apoptosis were explored by CCK-8 assay and TUNEL assay, and the expression of apoptosis-related proteins was detected by western blotting. Furthermore, the involvements of the downstream Traf3/MAPK P38 pathway with the roles of miR-590-5p in AD were measured by western blotting. Our results showed that knockdown of miR-590-5p was found in AD patients, mice model, and Aß-induced cell model. Notably, PELI1 was proved as a target gene of miR-590-5p. miR-590-5p mimic or PELI1 silence significantly promoted cell proliferation and inhibited cell apoptosis, as well as suppressed the activation of Traf3/MAPK P38 pathway both in Aß-induced BV-2 and HT22 cells. The effects of PELI1 overexpression on cell proliferation, apoptosis, and Traf3/MAPK P38 pathway were partly abrogated by miR-590-5p mimic both in BV-2 and HT22 cells. In conclusion, miR-590-5p was expressed at lower levels in AD, and miR-590-5p/PELI1 axis might be involved in the progression of AD by the downstream Traf3/MAPK P38 pathway.

The distribution and density of monocarboxylate transporter 2 in cerebral cortex, hippocampus and cerebellum of wild-type mice.

Monocarboxylates cannot cross the blood-brain barrier freely to participate in brain energy metabolism. Specific monocarboxylate transporters (MCTs) are needed to cross cellular membranes. Monocarboxylate transporter 2 (MCT2) is a major monocarboxylate transporter encoded by the SLC16A7 gene. Recent studies reported that neurodegenerative diseases of the CNS, such as Alzheimer's disease (AD) and Parkinson's disease (PD), were related to energy metabolic impairment. MCT2 also plays an important role in energy metabolism in the CNS. To provide experimental evidence for future research on the role of MCT2 in the pathological process of CNS degenerative diseases, the distribution and density of MCT2 in different subregions of wild-type mouse brain was examined using immunohistochemistry, western blot and immunogold post-embedding electron microscopic techniques. The amount of MCT2 was higher in cerebellum than in cortex and hippocampus on western blots, and there was no statistical difference between cortex and hippocampus. Immunohistochemistry assay revealed the highest density of MCT2 in the CA3 of the hippocampus. The granular cell layer of the cerebellum contained more MCT2 than the molecular layer. The MCT2 density on the end feet of astrocytes of molecular layer was lower than in hippocampus, but the postsynaptic densities (PSDs) of asymmetric synapses in the molecular layer exhibited a high density using immunogold post-embedding electron microscopic techniques.

Regular Exercise Enhances Cognitive Function and Intracephalic GLUT Expression in Alzheimer's Disease Model Mice.

Brain energy metabolic impairment is one of the main features of Alzheimer's disease (AD) and is considered an underlying factor involved in cognitive impairment. Therefore, brain energy metabolism may represent a new therapeutic target for AD medical interventions. Among nutrients providing energy, glucose, the primary energy source, cannot cross the blood-brain barrier freely without specific glucose transporters (GLUTs), which are essential for the maintenance of cerebral energy metabolism homeostasis. Several converging lines of evidence suggest that GLUT1 deficiency in mice leads to synapse reduction and dysregulation coupled with mitochondrial morphological changes. In this study, the results revealed that regular exercise (RE) decreased the expression of amyloid-ß and phosphorylated tau by western blot, and enhanced the spatial learning and exploration ability of AD model mice as assessed by Morris water maze test. Mitochondrial cristae and edges were clear and intact, ATP production in the brain raised, the number of synapses increased, and GLUT1 and GLUT3 expression levels improved in the central nervous system (CNS) in AD model mice after RE. Changes in GLUT1 and GLUT3 expression at the protein level after RE are an important part of energy metabolic adaptation in AD model mice. Learning and memory improvement are highly associated with mitochondrial integrity and sufficient synapses in the CNS. This research suggests that increased brain energy metabolism attributed to RE exhibits promising therapeutic potential for AD.

Short-term pre- and post-operative stress prolongs incision-induced pain hypersensitivity without changing basal pain perception.

BACKGROUND: Chronic stress has been reported to increase basal pain sensitivity and/or exacerbate existing persistent pain. However, most surgical patients have normal physiological and psychological health status such as normal pain perception before surgery although they do experience short-term stress during pre- and post-operative periods. Whether or not this short-term stress affects persistent postsurgical pain is unclear. RESULTS: In this study, we showed that pre- or post-surgical exposure to immobilization 6 h daily for three consecutive days did not change basal responses to mechanical, thermal, or cold stimuli or peak levels of incision-induced hypersensitivity to these stimuli; however, immobilization did prolong the duration of incision-induced hypersensitivity in both male and female rats. These phenomena were also observed in post-surgical exposure to forced swimming 25 min daily for 3 consecutive days. Short-term stress induced by immobilization was demonstrated by an elevation in the level of serum corticosterone, an increase in swim immobility, and a decrease in sucrose consumption. Blocking this short-term stress via intrathecal administration of a selective glucocorticoid receptor antagonist, RU38486, or bilateral adrenalectomy significantly attenuated the prolongation of incision-induced hypersensitivity to mechanical, thermal, and cold stimuli. CONCLUSION: Our results indicate that short-term stress during the pre- or post-operative period delays postoperative pain recovery although it does not affect basal pain perception. Prevention of short-term stress may facilitate patients' recovery from postoperative pain.

GSK1904529A, an insulin-like growth factor-1 receptor inhibitor, inhibits glioma tumor growth, induces apoptosis and inhibits migration.

Malignant gliomas are the most common type of primary malignancy of the central nervous system, with a poor prognosis. The therapeutic options for malignant gliomas are limited and far from satisfactory, and novel treatment strategies are urgently required to improve the outcome of the disease. Insulin­like growth factor (IGF)/IGF­1 receptor (IGF­1R) signaling pathway regulates cell proliferation, motility and survival. The dysregulation of this signaling pathway has been implicated in the development of malignant gliomas. In the present study, GSK1904529A, a small molecule inhibitor of IGF­1R, suppressed glioma cell viability, induced glioma cell apoptosis and inhibited glioma cell migration in vitro. In addition, GSK1904529A inhibited glioma tumor growth and induced tumor cell apoptosis in vivo. In conclusion, the results of the present study suggested GSK1904529A as a promising agent for the treatment of malignant glioma.

The effects of ponatinib, a multi-targeted tyrosine kinase inhibitor, against human U87 malignant glioblastoma cells.

Glioblastoma is one of the most common malignant tumors in the nervous system in both adult and pediatric patients. Studies suggest that abnormal activation of receptor tyrosine kinases contributes to pathological development of glioblastoma. However, current therapies targeting tyrosine kinase receptors have poor therapeutic outcomes. Here, we examined anticancer effects of ponatinib, a multi-targeted tyrosine kinase inhibitor, on glioblastoma cells both in the U87MG cell line and in the mouse xenograft model. We showed that ponatinib treatment reduced cell viability and induced cell apoptosis in a dose-dependent manner in U87MG cells. In addition, ponatinib suppressed migration and invasion of U87MG cells effectively. Furthermore, ponatinib-treated tumors showed an obvious reduction of tumor volume and an increase of apoptosis as compared with vehicle-treated tumors in the mouse xenograft model. These findings support a potential application of ponatinib as a chemotherapeutic option against glioblastoma cells.

Ceramide is involved in alcohol-induced neural proliferation.

Prenatal alcohol exposure, especially during early pregnancy, can lead to fetal alcohol syndrome. The pharmacological and toxicological mechanisms of ethanol are related to the effects of ceramide. In this study, we established an alcohol exposure model in wild-type mice and in knockout mice for the key enzyme involved in ceramide metabolism, sphingomyelin synthase 2. This model received daily intragastric administration of 25% ethanol, and pups were used at postnatal days 0, 7, 14, 30 for experiments. Serology and immunofluorescence staining found that ethanol exposure dose-dependently reduced blood sphingomyelin levels in two genotypes of pups, and increased neural cell proliferation and the number of new neurons in the hippocampal dentate gyrus. Western blot analysis showed that the relative expression level of protein kinase C α increased in two notypes of pups after ethanol exposure. Compared with wild-type pups, the expression level of the important activator protein of the ceramide/ceramide-1-phosphate pathway, protein kinase C α, was reduced in the hippocampus of sphingomyelin synthase 2 knockouts. Our findings illustrate that ceramide is involved in alcohol-induced neural proliferation in the hippocampal dentate gyrus of pups after prenatal ethanol exposure, and the mechanism may be associated with increased pression of protein kinase C α activating the ceramide/ceramide-1-phosphate pathway.

[Alternation of proteins in brain of Parkinson's disease model rats after the transplantation of TH-NTN gene modified bone marrow mesenchymal stem cells].

OBJECTIVE: To explore the effects of tyrosine hydroxylase-neurturin (TH-NTN) gene modified bone marrow mesenchymal stem cell (BMSC) transplantation in Parkinson's disease (PD) model rats and the alternations of correlated proteins. METHODS: The PD rat model was established by the 2-point injection of 6-hydroxydopamine (6-OHDA) into unilateral (right) striatum. Successful modeling rats were separated into PD, BMSC and TH-NTN-BMSC groups. BMSC and TH-NTN-BMSC groups were transplanted into BMSCs and TH-NTN gene modified BMSC cells separately into right striatum. After transplantation, ethology detection in all groups was made with an intraperitoneal injection of apomorphine (APO). Dopamine (DA) and Dihydroxyphenylacetic Acid (DOPAC) in striatum were detected by high performance liquid electrochemical analysis. TH and NTN proteins in right striatum were also analyzed by immunohistochemistry and Western blot. Finally the density of dopamine receptors in post synaptic density of dopaminergic synapses of corpus striatum were compared between each group by post-embedding immunogold electron microscopy. RESULTS: After an injection of APO, rotation frequency decreased in TH-NTN-BMSC group, i.e. (5.7 ± 1.3) circles/min versus (10.8 ± 2.2), (9.9 ± 1.2) circles/min in PD and BMSC groups (P < 0.05). For proteins in right striatum, DA, (0.421 ± 0.113) and DOPAC, (0.093 ± 0.012) nmol/L increased significantly versus (0.208 ± 0.043), (0.043 ± 0.017) nmol/L in PD and (0.231 ± 0.082), (0.044 ± 0.023)noml/L in BMSC groups (P < 0.05). Also a lower density of D2 receptors at (623 ± 96)/µm(2) in TH-NTN-BMSC group versus (923 ± 132)/µm(2) in PD and (860 ± 116)/µm(2) in BMSC groups was also found. CONCLUSION: The combined therapy of TH and NTN genes increases the synthesis of DA and also protects the dopaminergic neurons to achieve double therapeutic effects. It may provide potential innovations of PD genetic therapy.

Transient focal cerebral ischemia/reperfusion induces early and chronic axonal changes in rats: its importance for the risk of Alzheimer's disease.

The dementia of Alzheimer's type and brain ischemia are known to increase at comparable rates with age. Recent advances suggest that cerebral ischemia may contribute to the pathogenesis of Alzheimer's disease (AD), however, the neuropathological relationship between these two disorders is largely unclear. It has been demonstrated that axonopathy, mainly manifesting as impairment of axonal transport and swelling of the axon and varicosity, is a prominent feature in AD and may play an important role in the neuropathological mechanisms in AD. In this study, we investigated the early and chronic changes of the axons of neurons in the different brain areas (cortex, hippocampus and striatum) using in vivo tracing technique and grading analysis method in a rat model of transient focal cerebral ischemia/reperfusion (middle cerebral artery occlusion, MCAO). In addition, the relationship between the changes of axons and the expression of ß-amyloid 42 (Aß42) and hyperphosphorylated Tau, which have been considered as the key neuropathological processes of AD, was analyzed by combining tracing technique with immunohistochemistry or western blotting. Subsequently, we found that transient cerebral ischemia/reperfusion produced obvious swelling of the axons and varicosities, from 6 hours after transient cerebral ischemia/reperfusion even up to 4 weeks. We could not observe Aß plaques or overexpression of Aß42 in the ischemic brain areas, however, the site-specific hyperphosphorylated Tau could be detected in the ischemic cortex. These results suggest that transient cerebral ischemia/reperfusion induce early and chronic axonal changes, which may be an important mechanism affecting the clinical outcome and possibly contributing to the development of AD after stroke.

Bone marrow-derived mesenchymal stem cells increase dopamine synthesis in the injured striatum.

Previous studies showed that tyrosine hydroxylase or neurturin gene-modified cells transplanted into rats with Parkinson's disease significantly improved behavior and increased striatal dopamine content. In the present study, we transplanted tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells into the damaged striatum of Parkinson's disease model rats. Several weeks after cell transplantation, in addition to an improvement of motor function, tyrosine hydroxylase and neurturin proteins were up-regulated in the injured striatum, and importantly, levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid increased significantly. Furthermore, the density of the D2 dopamine receptor in the postsynaptic membranes of dopaminergic neurons was decreased. These results indicate that transplantation of tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells increases dopamine synthesis and significantly improves the behavior of rats with Parkinson's disease.

[Alcohol-induced proliferation of neurons in mouse hippocampal dentate gyrus: a possible role of ceramide].

To investigate the role and mechanism of ceramide (Cer) regulation in alcohol-induced neuronal proliferation and the newborn neurons formation, we used sphingomyelin synthase 2 (predominant enzyme of Cer metabolism) knockout (SMS2(-/-)) and wild type (WT) female mice to establish the model of prenatal alcohol exposure. In 24 h after being given birth (postnatal day 0, P0), the offspring of model mice received blood sphingomyelin (SM) measurement with enzymatic method. On P0, P7, P14 and P30, the proliferation of granule cells in the dentate gyrus and newborn neurons were investigated with immunofluorescent labeling. The expression of protein kinase Cα (PKCα) in the hippocampus was tested with Western blot analysis. The results showed that the SM level of blood in SMS2(-/-) pups was significantly lower than that in WT pups. No matter in SMS2(-/-) or WT mice, the prenatal alcohol exposure down-regulated the SM levels in pups with dose-dependency. In both SMS2(-/-) and WT pups, the number of proliferative neurons and newborn neurons in the dentate gyrus gradually decreased with the growing age. Compared with the WT pups, SMS2(-/-) pups showed significantly more proliferative neurons and newborn neurons in the dentate gyrus. Notably, prenatal alcohol exposure dose-dependently increased proliferative neurons and newborn neurons in the dentate gyrus in both WT and SMS2(-/-) pups. The hippocampal expression of PKCα protein in SMS2(-/-) mice was lower than that in WT mice, and prenatal alcohol exposure could up-regulate the PKCα protein expression in both WT and SMS2(-/-) mice with dose dependency. These results suggest that alcohol exposure during pregnancy can induce the compensatory neural cell proliferation and the production of newborn neurons in offspring, and the Cer-ceramide-1-phosphate (C1P) pathway is involved in alcohol-induced neural cell proliferation. The activation of PKCα may be a key step to start the Cer-C1P pathway and up-regulate the alcohol-induced neural cell proliferation and the newborn neurons formation.

Effect of hypoxic preconditioning on neural cell apoptosis and expression of Bcl-2 and Bax in cerebral ischemia-reperfusion in rats.

In order to investigate the protective effect of hypoxic preconditioning on the cerebral ischemia-reperfusion injury, the expression of Bcl-2 and Bax was detected by using immunohistochemical staining after 3 h cerebral ischemia followed by 1, 6, 12, 24 and 48 h reperfusion respectively in rats treated with or without hypoxic preconditioning before cerebral ischemia. In addition, the apoptosis of neural cells and the behavioral scores for neurological functions recovery were evaluated by TUNEL staining and "crawling method", respectively. Compared with control group (cerebral ischemia-reperfusion without hypoxic preconditioning), the expression of Bcl-2 was significantly increased, but that of Bax decreased in the hypoxic preconditioning group (cerebral ischemia-reperfusion with hypoxic preconditioning), both P < 0.05. The pre-treatment with hypoxic preconditioning could reduce the apoptosis of neural cells and promote the neurological function recovery as compared to control group. It was suggested that hypoxic preconditioning may have protective effects on the cerebral ischemia-reperfusion injury by inhibiting the apoptosis of neural cells, increase the expression of Bcl-2 and decrease the expression of Bax.

[Identification of a novel mutation IVS2-2A-->C of SEDL gene in a Chinese family with X-linked spondyloepiphyseal dysplasia tarda].

OBJECTIVE: To identify the mutation of spondyloepiphyseal dysplasia tarda (SEDL) gene in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda and to make a discussion on the pathogenesis of SEDL at the molecular level. METHODS: In two patients, four exons comprising the SEDL open reading frame as well as their exon/intron boundaries were analyzed by bi-directional direct sequencing of PCR products. The sequencing results were compared against the normal sequences in GenBank to find the mutation. Then the mutation was identified in other members of the family. RESULTS: A nucleotide substitution of the splice acceptor in SEDL intron 2, IVS2 -2A-->C,was detected in two affected individuals (IV(15) V(3)) in the Chinese family with SEDL, but no sequence change occurring on exons 3-6 was detected. The transversion was also identified in four heterozygous carriers. The mutation was not found in two unaffected male individuals and fifteen normal controls. Furthermore, four potential carriers were identified in the family. CONCLUSION: The mutation IVS2 -2A-->C of SEDL gene was firstly determined in the world. The change of the splice acceptor in SEDL intron 2 may cause skipping of exon 3 which is responsible for the disease. Molecular diagnosis can be made by detecting the mutation.