BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons.

It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.

Vascular endothelial growth factor promotes transdifferentiation of astrocytes into neurons via activation of the MAPK/Erk-Pax6 signal pathway.

Reactive astrocytes can be transformed into new neurons. Vascular endothelial growth factor (VEGF) promotes the transformation of reactive astrocytes into neurons in ischemic brain. Therefore, in this study, the molecular mechanism of VEGF's effect on ischemia/hypoxia-induced astrocyte to neuron transformation was investigated in the models of rat middle cerebral artery occlusion (MCAO) and in astrocyte culture with oxygen and glucose deprivation (OGD). We found that VEGF enhanced ischemia-induced Pax6, a neurogenic fate determinant, expression and Erk phosphorylation in reactive astrocytes and reduced infarct volume of rat brain at 3 days after MCAO, which effects could be blocked by administration of U0126, a MAPK/Erk inhibitor. In cultured astrocytes, VEGF also enhanced OGD-induced Erk phosphorylation and Pax6 expression, which was blocked by U0126, but not wortmannin, a PI3K/Akt inhibitor, or SB203580, a MAPK/p38 inhibitor, suggesting VEGF enhanced Pax6 expression via activation of MAPK/Erk pathway. OGD induced the increase of miR365 and VEGF inhibited the increase of OGD-induced miR365 expression. However, miR365 agonists blocked VEGF-enhanced Pax6 expression in hypoxic astrocytes, but did not block VEGF-enhanced Erk phosphorylation. We further found that VEGF promoted OGD-induced astrocyte-converted to neuron. Interestingly, both U0126 and Pax6 RNAi significantly reduced enhancement of VEGF on astrocytes-to-neurons transformation, as indicated Dcx and MAP2 immunopositive signals in reactive astrocytes. Moreover, those transformed neurons become mature and functional. We concluded that VEGF enhanced astrocytic neurogenesis via the MAPK/Erk-miR-365-Pax6 signal axis. The results also indicated that astrocytes play important roles in the reconstruction of neurovascular units in brain after stroke.

Studying the Effects of Vitamin A on the Severity of Allergic Rhinitis and Asthma.

Allergic asthma is a complex lung disease characterized by breathlessness, airway inflammation, and obstruction. Allergy and allergic rhinitis (AR) are the main triggers of asthma. Vitamin A is an important supplementary factor for the physiological activation of the immune system. In the present study, we investigated the effects of vitamin A on the exacerbation of allergic asthma symptoms. BALB/c mice were allocated to four groups. Asthma was created in two groups, and in the other two groups, rhinitis was induced. One of the asthma groups and one of the rhinitis groups orally received vitamin A (20 IU/g for 15 days). The levels of Immunoglobulin (Ig) E, histamine, leukotriene B4 (LTB4), Cysteinyl leukotriene receptor (Cys-LT), interleukin (IL)-4, IL-5, IL-13, and IL-35 as well as eosinophil peroxidase activity, were measured. Also, the histopathology of mice lungs was evaluated. The levels of total IgE, LTB4, Cys-LT, IL-4, IL-5, IL-17, and IL-33, eosinophil peroxidase activity, perivascular and peribronchial inflammation significantly decreased in vitamin A-treated asthma and rhinitis groups compared to non-treated groups. Also, IL-13 and histamine levels, hyperplasia of the goblet cell, and hyper-secretion of the mucus insignificantly decreased in vitamin A-treated asthma and rhinitis groups. Asthma and AR are common diseases that are generally developed due to the dysregulation of the immune system. Vitamin A plays an important role in controlling the immunopathologic mechanisms of allergic diseases. Vitamin A could be a useful supplement in managing AR and asthma by decreasing the severity of inflammatory responses. Therefore, control of vitamin A deficiency is recommended in Allergy.

Vascular endothelial growth factor increases the function of calcium-impermeable AMPA receptor GluA2 subunit in astrocytes via activation of protein kinase C signaling pathway.

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.

Endothelial cells promote excitatory synaptogenesis and improve ischemia-induced motor deficits in neonatal mice.

Brain microvascular endothelial cells (BMEC) are highly complex regulatory cells that communicate with other cells in the neurovascular unit. Cerebral ischemic injury is known to produce detectable synaptic dysfunction. This study aims to investigate whether endothelial cells in the brain regulate postnatal synaptic development and to elucidate their role in functional recovery after ischemia. Here, we found that in vivo engraftment of endothelial cells increased synaptic puncta and excitatory postsynaptic currents in layers 2/3 of the motor cortex. This pro-synaptogenic effect was blocked by the depletion of VEGF in the grafted BMEC. The in vitro results showed that BMEC conditioned medium enhanced spine and synapse formation but conditioned medium without VEGF had no such effects. Moreover, under pathological conditions, transplanted endothelial cells were capable of enhancing angiogenesis and synaptogenesis and improved motor function in the ischemic injury model. Collectively, our findings suggest that endothelial cells promote excitatory synaptogenesis via the paracrine factor VEGF during postnatal development and exert repair functions in hypoxia-ischemic neonatal mice. This study highlights the importance of the endothelium-neuron interaction not only in regulating neuronal development but also in maintaining healthy brain function.

[VEGF enhances reconstruction of neurovascular units in the brain after injury].

Vascular endothelial growth factor (VEGF) was originally recognized as a substance predominantly with vascular permeability and angiogenesis. Recently, more and more evidence indicated that VEGF is expressed in the neurons of the developing and adult brains. Functional investigation demonstrated that VEGF shows several important effects on the neuronal development and physiological function. For example, VEGF accelerates the development of neurons and neural dendritic and axon growth. Besides, VEGF directly and acutely regulates the functions of multiple ion channels of the neuron membrane and changes neural excitability. In traumatic or ischemic injured brains, VEGF produces neuroprotection, enhances capacity of adult neurogenesis and transformation of astroglial cells into new neurons, which are fundamental basis for re-establishment of neural network. Based on the knowledge obtained from the literatures, we propose that VEGF may play very important roles in neural plasticity in the normal brain, and the reconstruction of neurovascular units and neural repair in the traumatic injured brain. This review mainly focuses on neural activity and repair roles of VEGF in adult mammalian brains. Further study on the mechanism of VEGF's neurobiological effects in the brain will be helpful for understanding the regulation of brain functions and developing new therapeutic strategy for prevention of neurodegeneration of the brain.

Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia.

The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in promoting the recovery process; however, the underlying mechanisms are still largely unknown. The objective of this study is to reveal the function and mechanism of neuronal-specific cpg15 expressed in astrocytes after ischemia induction, in promoting the recovery of injured neurons. Our findings provided new mechanistic insight into the neurological recovery, which might help develop novel therapeutic options for cerebral ischemia via astrocytic-targeting interference of gene expression.

Magnetofection Based on Superparamagnetic Iron Oxide Nanoparticles Weakens Glioma Stem Cell Proliferation and Invasion by Mediating High Expression of MicroRNA-374a.

Glioma stem cells belong to a special subpopulation of glioma cells that are characterized by strong proliferation, invasion and drug resistance capabilities. Magnetic nanoparticles are nanoscale biological materials with magnetic properties. In this study, CD133(+) primary glioma stem cells were isolated from patients and cultured. Then, magnetic nanoparticles were used to mediate the transfection and expression of a microRNA-374a overexpression plasmid in the glioma stem cells. Transmission electron microscopy detected the presence of significant magnetic nanoparticle substances within the CD133(+) glioma stem cells after transfection. The qRT-PCR and Northern blot results showed that the magnetic nanoparticles could be used to achieve the transfection of the microRNA-374a overexpression plasmid into glioma stem cells and the efficient expression of mature microRNA-374a. The MTT and flow cytometry results showed that the proliferation inhibition rate was significantly higher in cells from the microRNA-374a transfection group than in cells from the microRNA-mut transfection group; additionally, the former cells presented significant cell cycle arrest. The Transwell experiments confirmed that the overexpression of microRNA-374a could significantly reduce the invasiveness of CD133(+) glioma stem cells. Moreover, the high expression of microRNA-374a mediated by the magnetic nanoparticles effectively reduced the tumourigenicity of CD133(+) glioma stem cells in nude mice. The luciferase assays revealed that mature microRNA-374a fragments could bind to the 3'UTR of Neuritin (NRN1), thereby interfering with Neuritin mRNA expression. The qRT-PCR and Western blotting results showed that the overexpression of microRNA-374a significantly reduced the expression of genes such as NRN1, CCND1, CDK4 and Ki67 in glioma stem cells. Thus, magnetic nanoparticles can efficiently mediate the transfection and expression of microRNA expression plasmids in mammalian cells. The overexpression of microRNA-374a can effectively silence NRN1 expression, thereby inhibiting the proliferation, invasion and in vivo tumourigenicity of human glioma stem cells.

Glutathione protects against hepatic injury in a murine model of primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease which may cause complications such as hepatic dysfunction and injury. As an important antioxidant, reduced glutathione (GSH) has been reported protecting against hepatic injury induced by some diseases, but the role of GSH in pSS is poorly understood. This study aims at investigating the role of GSH in hepatic injury during pSS. A murine model of pSS, non-obese diabetic (NOD) mice, was used for GSH administration via tail intravenous injection. Enzyme-linked immunosorbent assay (ELISA) was performed to detect serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as the levels of GSH, tumor necrosis factor, interleukin (IL) 10, integrin alpha M, IL1B, malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase 4, and superoxide dismutases in hepatocyte homogenates. Hematoxylin-eosin staining was performed to observe hepatic histology. The results showed that serum AST and ALT levels were up-regulated in the NOD mice (p = 0.0021 and 0.0048), but were significantly recovered after the GSH administration (p = 0.0081 and 0.0263). The NOD mice exhibited disturbed hepatic tissue structure, which was attenuated by GSH. The GSH administration could also promote the production of GSH in the hepatocytes (p = 0.0264), and control the levels of inflammatory factors and oxidative stress-related factors. These results indicate that GSH has significant effects on protecting against the hepatic injury during pSS, which may be associated with its regulation of the inflammatory factors and oxidative stress-related factors. This study suggests that GSH is a promising therapeutic strategy for controlling hepatic injury during pSS and offers valuable information for further research.

Protective effect of taraxasterol against rheumatoid arthritis by the modulation of inflammatory responses in mice.

Taraxasterol is an effective component of dandelion that has anti-inflammatory effects in vivo and in vitro. The present study was performed to explore whether taraxasterol exhibits a protective effect against rheumatoid arthritis through the modulation of inflammatory responses in mice. Eight-week-old CCR9-deficient mice were injected with a collagen II monoclonal antibody cocktail to create a rheumatoid arthritis model. In the experimental group, arthritic model mice were treated with 10 mg/kg taraxasterol once per day for 5 days. Treatment with taraxasterol significantly increased the pain thresholds and reduced the clinical arthritic scores of the mice in the experimental group compared with those of the model group. Furthermore, treatment with taraxasterol significantly suppressed tumor necrosis factor-α, interleukin (IL)-1ß, IL-6 and nuclear factor-κB protein expression levels compared with those in the rheumatoid arthritis model mice. Taraxasterol treatment also significantly reduced nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels compared with those in the rheumatoid arthritis model group. These observations indicate that the protective effect of taraxasterol against rheumatoid arthritis is mediated via the modulation of inflammatory responses in mice.

Neurogenic effect of VEGF is related to increase of astrocytes transdifferentiation into new mature neurons in rat brains after stroke.

To study the cellular mechanism of vascular endothelial growth factor (VEGF)-enhanced neurogenesis in ischemic brain injury, we used middle cerebral artery occlusion (MCAO) model to induce transient focal ischemic brain injury. The results showed that ischemic injury significantly increased glial fibrillary acidic protein immunopositive (GFAP(+)) and nestin(+) cells in ipsilateral striatum 3 days following MCAO. Most GFAP(+) cells colocalized with nestin (GFAP(+)-nestin(+)), Pax6 (GFAP(+)-Pax6(+)), or Olig2 (GFAP(+)-Olig2(+)). VEGF further increased GFAP(+)-nestin(+) and GFAP(+)-Pax6(+) cells, and decreased GFAP(+)-Olig2(+) cells. We used striatal injection of GFAP targeted enhanced green fluorescence protein (pGfa2-EGFP) vectors combined with multiple immunofluorescent staining to trace the neural fates of EGFP-expressing (GFP(+)) reactive astrocytes. The results showed that MCAO-induced striatal reactive astrocytes differentiated into neural stem cells (GFP(+)-nestin(+) cells) at 3 days after MCAO, immature (GFP(+)-Tuj-1(+) cells) at 1 week and mature neurons (GFP(+)-MAP-2(+) or GFP(+)-NeuN(+) cells) at 2 weeks. VEGF increased GFP(+)-NeuN(+) and BrdU(+)-MAP-2(+) newborn neurons after MCAO. Fluorocitrate, an astrocytic inhibitor, significantly decreased GFAP and nestin expression in ischemic brains, and also reduced VEGF-enhanced neurogenic effects. This study is the first time to report that VEGF-mediated increase of newly generated neurons is dependent on the presence of reactive astrocytes. The results also illustrate cellular mechanism of VEGF-enhanced neural repair and functional plasticity in the brains after ischemic injury. We concluded that neurogenic effect of VEGF is related to increase of striatal astrocytes transdifferentiation into new mature neurons, which should be very important for the reconstruction of neurovascular units/networks in non-neurogenic regions of the mammalian brain.

Regulation of RAGE splicing by hnRNP A1 and Tra2ß-1 and its potential role in AD pathogenesis.

The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full-length membrane-bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with ß-amyloid peptide (Aß) or inhibition of the mRAGE-activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2ß-1 (Tra2ß-1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up-regulation of hnRNP A1 and down-regulation of Tra2ß-1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2ß-1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis. The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, membrane-bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis. Mechanism for imbalanced expression of these two isoforms in AD brain remains elusive. We proposed here a hypothetic model to illustrate that impaired glucose metabolism in AD brain may increase the expression of splicing protein hnRNP A1 and reduce Tra2ß-1, which cause the imbalanced expression of mRAGE and esRAGE.

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia.

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen-glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM-L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte-specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM-L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte-specific NSSR1 cKD mice compared with in wild-type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM-L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1-mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM-L1 and CREB are probably mediators in NSSR1-mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM-L1 and CREB in astrocytes.

VEGF overexpression enhances the accumulation of phospho-S292 MeCP2 in reactive astrocytes in the adult rat striatum following cerebral ischemia.

PURPOSE: Astrocytes can be reactivated after cerebral ischemia by expressing nestin and other characteristic markers of neural stem cells (NSCs). However, the epigenetic features of reactive astrocytes are not well known. Methyl-CpG-binding protein 2 (MeCP2) is a vital transcriptional modulator in brain development. Although the expression and function of some phosphorylated MeCP2 isoforms have been clarified, phospho-serine 292 (pS292) MeCP2 has not yet drawn much attention. In this study, we used western blot analysis and immunohistochemical and immunofluorescent staining to reveal the expressive features of pS292 MeCP2 and MeCP2 in the adult rat striatum following transient middle cerebral artery occlusion (MCAO). RESULTS: We first discovered that the ischemia-induced expression of cytoplasmic pS292 MeCP2 is primarily accumulated in nestin-positive reactive astrocytes in the stroke-injured striatum. Moreover, the enhancement of astrocytic pS292 MeCP2 was correlated with the augmentation of VEGF in astrocytes, as determined by the substantial co-localization of pS292 MeCP2 and VEGF after stroke. Finally, the exogenous overproduction of VEGF further promoted the expression of pS292 MeCP2 in reactive astrocytes, and this effect was accompanied by a marked increase in reactive astrocytes. On the contrary, MeCP2 was predominantly expressed in the neuronal nucleus, and the level of this protein was not significantly altered after ischemic injury and VEGF overproduction. CONCLUSION: Our data provide the first demonstration that overexpression of VEGF enhances the accumulation of pS292 MeCP2 in reactive astrocytes in the ischemic-injured rat striatum, implicating a pS292 MeCP2-related epigenetic role of exogenous VEGF in reactive astrocytes following cerebral ischemia.

Diabetes-induced central neuritic dystrophy and cognitive deficits are associated with the formation of oligomeric reticulon-3 via oxidative stress.

Diabetes is a high risk factor to dementia. To investigate the molecular mechanism of diabetic dementia, we induced type 2 diabetes in rats and examined potential changes in their cognitive functions and the neural morphology of the brains. We found that the diabetic rats with an impairment of spatial learning and memory showed the occurrence of RTN3-immunoreactive dystrophic neurites in the cortex. Biochemical examinations revealed the increase of a high molecular weight form of RTN3 (HW-RTN3) in diabetic brains. The corresponding decrease of monomeric RTN3 was correlated with the reduction of its inhibitory effects on the activity of ß-secretase (BACE1), a key enzyme for generation of ß-amyloid peptides. The results from immunoprecipitation combined with protein carbonyl detection showed that carbonylated RTN3 was significantly higher in cortical tissues of diabetic rats compared with control rats, indicating that diabetes-induced oxidative stress led to RTN3 oxidative damage. In neuroblastoma SH-SY5Y cells, high glucose and/or H2O2 treatment significantly increased the amounts of carbonylated proteins and HW-RTN3, whereas monomeric RTN3 was reduced. Hence, we conclude that diabetes-induced cognitive deficits and central neuritic dystrophy are correlated with the formation of aggregated RTN3 via oxidative stress. We provided the first evidence that oxidative damage caused the formation of toxic RTN3 aggregates, which participated in the pathogenesis of central neuritic dystrophy in diabetic brain. Present findings may offer a new therapeutic strategy to prevent or reduce diabetic dementia.

Bcl-2 enhances the formation of newborn striatal long-projection neurons in adult rat brain after a transient ischemic stroke.

OBJECTIVE: It has been reported that B-cell lymphoma 2 (Bcl-2) enhances neurogenesis as well as supporting axonal growth after injury. In the present study, we investigated whether Bcl-2 overexpression plays a role in the formation of newborn striatonigral projection neurons in the adult rat brain after transient middle cerebral artery occlusion (MCAO). METHODS: We infused human Bcl-2-expressing plasmid (pBcl-2) into the lateral ventricle immediately after 30 min of MCAO, injected 5'-bromodeoxyuridine (BrdU) intraperitoneally to label proliferative cells, and microinjected fluorogold (FG) into the substantia nigra at 11 weeks of reperfusion followed by multiple immunostaining of striatonigral projection neurons at 12 weeks. RESULTS: We found that pBcl-2 treatment significantly increased the number of newborn neurons (BrdU(+)-NeuN(+)) in the striatum ipsilateral to the MCAO. We further detected newborn striatonigral projection neurons (BrdU(+)-FG(+)-NeuN(+)) in the ipsilateral striatum at 12 weeks. More interestingly, the number of newborn striatonigral projection neurons (BrdU(+)-FG(+)) was significantly increased by pBcl-2 treatment compared to that by pEGFP, a control plasmid. CONCLUSION: Taken together, we found that Bcl-2 overexpression in the brain enhanced the generation of newborn striatonigral projection neurons. This provides a potential strategy for promoting the reestablishment of neural networks and brain repair after ischemic injury.

Bcl-2 increases stroke-induced striatal neurogenesis in adult brains by inhibiting BMP-4 function via activation of ß-catenin signaling.

Our previous experiments suggest that treatment with Bcl-2 increases proliferation and differentiation of neuronal progenitors induced by ischemic injury and ameliorates neurological functional deficits after stroke. However, in addition to its traditional anti-apoptotic effect, little is known about the concrete molecular modulation mechanism. In this study, Bcl-2-expressing plasmids were injected into the lateral ventricle of rat brains immediately following a 30-min occlusion of the middle cerebral artery to determine the role of Bcl-2 in adult neurogenesis. Bcl-2 overexpression reduced ischemic infarct and astrogenesis, and enhanced ischemia-induced striatal neurogenesis. We further found that Bcl-2 increased ß-catenin, a key mediator of canonical Wnt/ß-catenin signaling pathway, and reduced bone morphogenetic proteins-4 (BMP-4) expression in the ipsilateral striatum following ischemia. Treatment of stroke with ß-catenin siRNA (i.c.v.) showed that ß-catenin siRNA antagonized Bcl-2 neuroprotection against ischemic brain injury. More interestingly, ß-catenin siRNA simultaneously abolished Bcl-2-mediated reduction of BMP-4 expression and enhancement of neurogenesis in the ipsilateral striatum. This effect is independent of Noggin, the known BMP antagonist. These findings highlight a new regulatory mechanism that Bcl-2 elevates ischemia-induced striatal neurogenesis by down-regulating expression of BMP-4 via activation of the Wnt/ß-catenin signaling pathway in adult rat brains.

1-Methyl-4-phenyl-pyridinium time-dependently alters expressions of oxoguanine glycosylase 1 and xeroderma pigmentosum group F protein in PC12 cells.

OBJECTIVE: To determine if DNA excision repair enzymes oxoguanine glycosylase 1 (OGG1) and xeroderma pigmentosum group F protein (XPF) are involved in the pathogenesis of Parkinson's disease (PD) in a cell model. METHODS: PC12 cells were treated with 1-Methyl-4-phenylpyridine ion (MPP(+)) for various periods of time to induce oxidative DNA damage. MTT assay was used to determine cell viability. Immunocytochemistry with antibody against 8-hydroxy-2'-deoxyguanosine (8-oxodG) was used to evaluate oxidative DNA damage. Immunoblotting was used to detect the protein levels of OGG1 and XPF. RESULTS: MPP(+) treatment (1 mmol/L) for 18 h and 24 h reduced cell viability to 78.6% and 70.3% of the control, respectively, in a time-dependent way. MPP(+) increased the immunoreactivity of 8-oxodG in the cytoplasm at 3 h and in the nucleus at 24 h of treatment. With the treatment of MPP(+), the expression of OGG1 was significantly increased at 1 h, reaching a peak at 3 h, and then it was decreased at 24 h, as compared to that with vehicle treatment. The same effect was exerted on XPF level, except that the XPF level reached a peak at 18 h of MPP(+) treatment. Moreover, the maximally-increased protein level of OGG1 by MPP(+) was approximately 2-fold higher than that of XPF. CONCLUSION: MPP(+) treatment could time-dependently induce increases in OGG1 and XPF expressions in PC12 cells. Also, this study indicates that the base and nucleotide excision repair pathways may be compensatory activated in the early stage of pathogenesis in the cells after MPP(+) treatment.

VEGF enhance cortical newborn neurons and their neurite development in adult rat brain after cerebral ischemia.

To study the effect of VEGF overexpression on development of cortical newborn neurons in the brains after stroke, we injected human VEGF(165)-expressive plasmids (phVEGF) into the lateral ventricle of rat brains with a transient middle cerebral artery occlusion (MCAO). An injection of phVEGF significantly promoted angiogenesis (BrdU(+)-von Willebrand's factor(+)) and reduced infarct volume in the rat brain after MCAO. Single labeling of 5'-bromodeoxyuridine (BrdU) and double staining of BrdU with lineage-specific neuronal markers were used to indicate the proliferated cells and maturation of newborn neurons in the brain section of rats at 2, 4, and 8 weeks after MCAO. The results showed that BrdU positive (BrdU(+)) cells existed in ipsilateral frontal cortex within 8 weeks after MCAO and reached the maximum at 2 weeks of reperfusion. The phVEGF treatment significantly increased BrdU(+) cells compared with the control plasmid (pEGFP) injection. Cortical neurogenesis was indicated by the presence of newborn immature (BrdU(+)-Tuj1(+)), newborn mature (BrdU(+)-MAP-2(+)), and newborn GABAergic (BrdU(+)-GAD67(+)) neurons. All these neurons declined within 8 weeks after MCAO in the controls. Injection of phVEGF significantly increased BrdU(+)-Tuj1(+) neurons at 2 weeks, and BrdU(+)-MAP-2(+) neurons and BrdU(+)-GAD67(+) neurons at 4 and 8 weeks, respectively after MCAO. Moreover, phVEGF treatment significantly increased neurite length and branch numbers of BrdU(+)-MAP-2(+) newborn neurons compared with pEGFP treatment. These results demonstrate that VEGF enhances maturation of stroke-induced cortical neurogenesis and dendritic formation of newborn neurons in adult mammalian brains.

Vascular endothelial growth factor acutely reduces calcium influx via inhibition of the Ca2+ channels in rat hippocampal neurons.

Vascular endothelial growth factor (VEGF) protects neurons against ischemic injury. An overload of intracellular calcium ions (Ca(2+)) caused by the excessive release of glutamate is widely considered to be one of the molecular mechanisms of ischemic neuronal death. In the present study, we investigated whether VEGF could modulate the activity of Ca(2+) channels on the neuronal membrane. We used the Fluo-3 image method assisted by confocal laser scan microscopy to detect any Ca(2+) influx in primary cultured hippocampal neurons. Whole-cell patch-clamp techniques were used to record the activity of the high-voltage-activated (HVA) Ca(2+) currents in the CA1 pyramidal neurons of hippocampal slices that were freshly prepared from neonatal brains of rats. The results obtained from the Fluo-3 image experiments showed that VEGF pretreatment of cultured neurons at a final concentration of 50, 100, or 200 ng/ml acutely and dose dependently attenuated the Ca(2+) influx induced by application of KCl (60 mM) or glutamate (50 microM). This effect was blocked by SU1498, an antagonist of Flk-1 VEGF receptor. The influx of Ca(2+) returned to basal levels after removal of VEGF. Furthermore, electrophysiological recording data showed that VEGF could acutely reduce the amplitudes of the HVA Ca(2+) currents in a dose- and voltage-dependent manner. The HVA Ca(2+) currents also returned to the levels of the control after removal of VEGF from the system. Taken together, the results obtained from the present study demonstrated that VEGF specifically reduced the influx of Ca(2+) via the inhibitory activity of the HVA Ca(2+) channels in hippocampal neurons.