The effect of semax and its C-end peptide PGP on expression of the neurotrophins and their receptors in the rat brain during incomplete global ischemia

Neurotrophins regulate key function of nervous tissue cells. Analysis of neurotrophins mRNA expression is an appropriate tool to assess therapeutic efficiency of the anti-stroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide upon mRNAs expression of neurotrophins Ngf, Bdrf, Nt-3 and their receptors TrkA, TrkB, TrkC, p75 in rat frontal lobes, hippocampus and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated 30 min, 1, 2, 4, 8, 12, 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our showed that ischemia causes a significant decrease in gene expression in the hippocampus. Semax and PGP affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides has been observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the semax mechanism of and present certain features of mRNA's expression of neurotrophins and their receptors in experimental conditions.     

Effect of peptide Semax and its C-terminal fragment PGP on Vegfa gene expression during incomplete global cerebral ischemia in rats

Vascular endothelial growth factor (VEGF-A) is hypoxia-inducible signal glycoprotein. VEGF-A induces vascular endothelial cell proliferation, which leads to the reconstitution of the vascular network in brain regions damaged by ischemia. However, this protein is also involved in the processes of inflammation and edema in early stages of ischemia. The synthetic peptide Semax has neuroprotective and anti-inflammatory properties and is actively used in the treatment of cerebral ischemia. We have previously shown that Semax reduces vascular injury and activates the mRNA synthesis of neurotrophins and their receptors during global cerebral ischemia in rats. In this work, we studied the effect of Semax and its C-terminal Pro-Gly-Pro tripeptide on Vegfa mRNA expression in different regions of the rat brain after 0.5, 1, 2, 4, 8, 12 and 24 h, which is the irreversible occlusion of the common carotid arteries. It was shown that ischemia increases the levels of Vegfa mRNA in rat brains (4 h after occlusion in cerebellum, cerebral cortex, and hippocampus; 8 h after occlusion in the cortex and hippocampus; and 24 h after occlusion in the cortex). Treatment with Semax reduces the levels of Vegfa mRNA in the frontal cortex (4, 8 and 12 h after occlusion) and the hippocampus (2 and 4 h after occlusion). The effect of PGP on the Vegfa gene expression was almost negligible. It was shown that Semax prevents the activating effect of hypoxia on the expression of the Vegfa gene at early stages of global cerebral ischemia. In turn, an increase in the level of Vegfa mRNA in the hippocampus 24 h after occlusion and Semax administration apparently reflects the neuroprotective properties of the drug.     

Effect of semax on the temporary dynamics of brain-derived neurotrophic factor and nerve growth factor gene expression in the rat hippocampus and frontal cortex

Semax is a synthetic peptide, which consists of the N-terminal adrenocorticotropic hormone fragment (4-7) (ACTH4-7) and C-terminal Pro-Gly-Pro peptide. Semax promotes neuron survival in hypoxia, increases selective attention and memory storage. It was shown that this synthetic peptide exerted a number of gene expressions, especially brain derived neurotrophic factor gene (Bdnf) and nerve growth factor gene (Ngf). Temporary dynamics of Bdnf and Ngf ex- pression in rat hippocampus and frontal cortex under Semax action (50 mg/kg, single intranasal administration) was studied in this work. It was shown that the studied gene expression levels changed significantly both in the hippocampus and the frontal cortex tissues 20 minutes after the peptide preparation application. The expression levels decreased in the hippocampus and increased in the frontal cortex. Forty minutes after Semax administration both gene expression levels returned to the level typical of control tissues. After that they increased significantly by 90 minutes after experiment start. Bdnf and Ngf expression levels decreased up to the control levels by 8 hours after medicine applying maximum gene expression levels were attained. Thus, Semax administration results in rapid, long-term, and specific activation of Bdnf and Ngf expression changes in different rat brain departments.     

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.     

p75 Neurotrophin receptor mediates neurotrophin activation of NF‐kappa B and induction of iNOS expression in P19 neurons

P19 embryonic carcinoma cells can be differentiated into neurons that form synaptic connections and that produce a variety of neurotransmitters. Results of RT‐PCR indicate that P19 neurons express several neurotrophin receptors (p75^NTR, trkB, and trkC, but not trkA) but they do not express any of the four neurotrophins. Consistent with the presence of trkB but not trkA, BDNF causes rapid phosphorylation of MAP kinases ERK1 and ERK2, but NGF does not. Neurotrophins induce translocation of NF‐κB into the nucleus. All four neurotrophins induce activation of NF‐κB in a biphasic manner. This effect is apparently mediated by p75^NTR, because an inhibitor of trk receptors, K252a, does not inhibit activation of NF‐κB. Instead, K252a itself promotes activation of NF‐κB and this effect is additive with the effect of neurotrophins. Inhibition of reactive oxygen intermediates with PDTC completely abolishes basal activity of NF‐κB and strongly inhibits activation of NF‐κB by neurotrophins, indicating an important role of reactive oxygen intermediates in the pathway by which neurotrophins activate NF‐κB. NF‐κB is known to promote expression of the iNOS gene. We found that all four neurotrophins increased iNOS mRNA levels, resulting in increased accumulation of iNOS protein. In contrast, none of the neurotrophins stimulated nNOS mRNA or protein synthesis. PDTC abolishes constitutive and neurotrophin‐induced expression of iNOS mRNA and protein and abolishes constitutive expression of nNOS mRNA, suggesting that reactive oxygen intermediates promote expression of nNOS. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 191–203, 2003     

Glutamate (mGluR-5) gene expression in brain regions of streptozotocin induced diabetic rats as a function of age: role in regulation of calcium release from the pancreatic islets in vitro

Metabotrophic glutamate receptors (mGluRs) modulate cellular activities involved in the processes of differentiation and degeneration. In this study, we have analysed the expression pattern of group-I metabotropic glutamate receptor (mGlu-5) in cerebral cortex, corpus striatum, brainstem and hippocampus of streptozotocin induced and insulin treated diabetic rats (D+I) as a function of age. Also, the functional role of glutamate receptors in intra cellular calcium release from the pancreatic islets was studied in vitro. The gene expression studies showed that mGlu-5 mRNA in the cerebral cortex increased siginficantly in 7 weeks old diabetic rats whereas decreased expression was observed in brainstem, corpus striatum and hippocampus when compared to control. 90 weeks old diabetic rats showed decreased expression in cerebral cortex, corpus striatum and hippocampus whereas in brainstem the expression increased significantly compared to their respective controls. In 7 weeks old D+I group, mGlu-5 mRNA expression was significantly decreased in cerebral cortex and corpus striatum whereas the expression increased significantly in brainstem and hippocampus. 90 weeks old D+I group showed an increased expression in cerebral cortex, while it was decreased significantly in corpus striatum, brainstem and hippocampus compared to their respective controls. In vitro studies showed that glutamate at lower concentration (10^-7 M) stimulated calcium release from the pancreatic islets. Our results suggest that mGlu-5 receptors have differential expression in brain regions of diabetes and D+I groups as a function of age. This will have clinical significance in management of degeneration in brain function and memory enhancement through glutamate receptors. Also, the regulatory role of glutamate receptors in calcium release has immense therapeutic application in insulin secretion and function.     

Structure–function relationships in the neurotrophin family

The study of structure–function relationships in the neurotrophin family has in recent years increased our understanding of several important aspects of neurotrophin function. Site-directed mutagenesis studies have localized amino acid residues important for binding to the low-affinity (p75^LNGFR), as well as to the members of the Trk family of tyrosine kinase receptors. A cluster of positively charged residues has been shown to form a surface for binding to p75^LNGFR in all four neurotrophins. Differences in the spatial distribution of these charges among the different neurotrophins may explain some of their distinct binding properties. Elimination of these positive charges drastically reduces binding to P75^LNGFR but not to the Trk family members, and it does not impair the biological properties of the neurotrophins in vitro, arguing that binding to and activation of Trk receptors is sufficient to mediate the biological responses of neurotrophins. In contrast. the binding sites to Trk receptors appear to be formed by discontinuous stretches of amino acid residues distributed throughout the primary sequence of the molecule. These include the N-terminus, some of the variable loop regions and a β-strand. Despite their apparent distribution, when viewed in the three-dimensional structure of NGF, these residues appear grouped on one side of the neurotrophin dimer, delineating a continuous surface extending approximately parallel to the twofold symmetry axis of the molecule. Two symmetrical surfaces are formed along the axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. In the neurotrophin family, co-evolution of cognate ligands and Trk receptors has developed specific contacts through different residues in the same variable regions of the neurotrophins. Thus, binding specificity is determined by the cooperation of distinct active and inhibitory binding determinants that restrict ligand-receptors interactions. Binding determinants to the Trk receptors can be manipulated independently in a rational fashion to create neurotrophin analogues with novel ligand-binding properties. In this way, second-generation chimeric neurotrophins with multiple specificities (pan-neurotrophins) have been engineered which may have valuable applications in the treatment of neurodegeneration and nerve damage. 1994 John Wiley & Sons, Inc.     

Expression of Nerve Growth Factor,p75, and trk in the Somatosensory and Motor Cortices of Mature Rats: Evidence for Local Trophic Support Circuits

Neurotrophins such as nerve growth factor (NGF) are critical for the maintenance of CNS neurons. We determined the expression of NGF and the neurotrophin receptors p75 and trk in the somatosensory and motor cortices of mature rats with immuno-histochemical techniques. Sections of mature rat cortex were processed immunohisto-chemically with primary antibodies directed against NGF, p75, or trk. The distribution of immunoreactive elements was examined, and stereological techniques were used to determine the density and size of immunoreactive cell bodies. Some sections processed for trk immunoreactivity were examined with an electron microscope.

From the size and morphology of the labeled cells, it appeared that only neurons in the gray matter were NGF-positive. NGF was detected in one-third of the neurons in layers II-III, V, and VI of both somatosensory cortex and motor cortex; however, fewer than 1 in 12 of the layer IV neurons was NGF-positive. With the notable exception of layer V, few cell bodies (2–10% of the total population) were p75– or trk-immunoreactive. Layer Vb was replete with receptor-positive cell bodies; more than one-third of the layer Vb neurons were p75– or trk-positive. All labeled cells appeared to be pyramidal neurons. The distribution of p75 labeling with the two anti-p75 antibodies was indistinguishable. In addition, the neuropil in the supragranular laminae was p75– or trk-positive. Electron microscopy showed that trk immunoreactivity was also expressed by dendrites. Only rarely were immunoreactive axons detected.

In summary, NGF is expressed by cortical neurons throughout cortex, and neurotrophin receptors are widely produced by postsynaptic targets. Thus, NGF appears to participate in an intracortical autoregulatory system. The strong expression of neurotrophin receptors by pyramidal neurons in layer Vb (the origin of brainstem and spinal cord projections) suggests that the neurotrophins are especially critical for the regulation of corticofugal projection systems.     

Expression of BDNF and TrkB Phosphorylation in the Rat Frontal Cortex During Morphine Withdrawal are NO Dependent

Nitric oxide (NO) mediates pharmacological effects of opiates including dependence and abstinence. Modulation of NO synthesis during the induction phase of morphine dependence affects manifestations of morphine withdrawal syndrome, though little is known about mechanisms underlying this phenomenon. Neurotrophic and growth factors are involved in neuronal adaptation during opiate dependence. NO-dependent modulation of morphine dependence may be mediated by changes in expression and activity of neurotrophic and/or growth factors in the brain. Here, we studied the effects of NO synthesis inhibition during the induction phase of morphine dependence on the expression of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF1) as well as their receptors in rat brain regions after spontaneous morphine withdrawal in dependent animals. Morphine dependence in rats was induced within 6 days by 12 injections of morphine in increasing doses (10–100 mg/kg), and NO synthase inhibitor L-N^G-nitroarginine methyl ester (L-NAME) (10 mg/kg) was given 1 h before each morphine injection. The expression of the BDNF, GDNF, NGF, IGF1, and their receptors in the frontal cortex, striatum, hippocampus, and midbrain was assessed 40 h after morphine withdrawal. L-NAME treatment during morphine intoxication resulted in an aggravation of the spontaneous morphine withdrawal severity. Morphine withdrawal was accompanied by upregulation of BDNF, IGF1, and their receptors TrkB and IGF1R, respectively, on the mRNA level in the frontal cortex, and only BDNF in hippocampus and midbrain. L-NAME administration during morphine intoxication decreased abstinence-induced upregulation of these mRNAs in the frontal cortex, hippocampus and midbrain. L-NAME prevented from abstinence-induced elevation of mature but not pro-form of BDNF polypeptide in the frontal cortex. While morphine abstinence did not affect TrkB protein levels as well as its phosphorylation status, inhibition of NO synthesis decreased levels of phosphorylated TrkB after withdrawal. Thus, NO signaling during induction of dependence may be involved in the mechanisms of BDNF expression and processing at abstinence, thereby affecting signaling through TrkB in the frontal cortex.     

Effect of long-term cold adaptation on the mRNA expression of serotonin 5-HT1A and 5-HT2A receptors

The mRNA expression of serotonin receptors 5-HT1A and 5-HT2A was investigated by the quantitative method RT-PCR in rats adapted to cold (5 weeks at +4 -(+6) degrees C) and in control (5 weeks at +20-22 degrees C). Four brain regions were examined: frontal cortex, hypothalamus, hippocampus, and midbrain. The influence of cold adaptation on the mRNA expression of 5-HT15 receptor was not found to be absent. The mRNA expression of 5-HT2A receptor changed under long-term cold exposure. These changes in different brain regions were various: in hypothalamus, there was an increase of the 5-HT2A receptor mRNA expression; in the cortex, a decrease; in the hippocampus and midbrain, significant changes of the mRNA expression were absent. The findings appear bo te adaptive and, according to their localization in the central nervous system, regulatory. They also suggest involvement of brain serotoninergic system in mechanism of adaptive reorganization of temperature regulation.     

At Least Three Neurotransmitter Systems Mediate a Stress-Induced Increase in c-fos mRNA in Different Rat Brain Areas

1. Protooncogene c-fos mRNA levels were determined in the rat cerebral cortex, hippocampus, and cerebellum after exposure to a combined forced swimming and confinement stress. The stress resulted in an increase in c-fos mRNA levels in all three brain areas.2. In an effort to elucidate the neurotransmitter systems involved in this stress-induced increase, animals were injected, prior to exposure to the stress, with either diazepam, MK-801, or propranolol.3. In both the cerebral cortex and the hippocampus the stress-induced increase in c-fos mRNA was inhibited by MK-801, suggesting that it is mediated via NMDA receptors. In the hippocampus, propranolol had a similar effect, indicating that -adrenergic receptors are also involved in the stress-induced increase in c-fos mRNA.4. On the other hand, the increase in c-fos mRNA produced by the stress of the injection was inhibited in the cerebral cortex by diazepam or propranolol and in the hippocampus only by diazepam. Furthermore, administration of MK-801 resulted in an increase in c-fos mRNA in the hippocampus of the nonstressed animals. In the cerebellum no one of the three drugs employed affected c-fos mRNA levels in either stressed or nonstressed animals.5. Our results thus show that various forms of stress activate, in different brain areas, neurons with either NMDA, -adrenergic, and/or GABA-A receptors.     

Effects of venlafaxine on the expression level and methylation status of genes involved in oxidative stress in rats exposed to a chronic mild stress

Recent human and animal studies indicate that oxidative and nitrosative stress may play a role in the aetiology and pathogenesis of depression. This study investigates the effect of chronic administration of the serotonin-norepinephrine reuptake inhibitor, venlafaxine, on the expression and methylation status of SOD1, SOD2, GPx1, GPx4, CAT, NOS1 and NOS2 in the brain and blood of rats exposed to a chronic mild stress (CMS) model of depression. Separate groups of animals were exposed to CMS for 2 or 7 weeks; the second group received saline or venlafaxine (10 mg/kg/d, IP) for 5 weeks. After completion of both stress conditions and drug administration, the mRNA and protein expression of selected genes and the methylation status of their promoters were measured in peripheral mononuclear blood cells (PBMCs) and in brain structures (hippocampus, amygdala, hypothalamus, midbrain, cortex, basal ganglia) with the use of TaqMan Gene Expression Assay, Western blot and methylation-sensitive high-resolution melting techniques. CMS caused a decrease in sucrose consumption, and this effect was normalized by fluoxetine. In PBMCs, SOD1, SOD2 and NOS2 mRNA expression changed only after venlafaxine administration. In brain, CAT, Gpx1, Gpx4 and NOS1 gene expression changed following CMS or venlafaxine exposure, most prominently in the hippocampus, midbrain and basal ganglia. CMS increased the methylation of the Gpx1 promoter in PBMCs, the second Gpx4 promoter in midbrain and basal ganglia, and SOD1 and SOD2 in hippocampus. The CMS animals treated with venlafaxine displayed a significantly higher CAT level in midbrain and cerebral cortex. CMS caused an elevation of Gpx4 in the hippocampus, which was lowered in cerebral cortex by venlafaxine. The results indicate that CMS and venlafaxine administration affect the methylation of promoters of genes involved in oxidative and nitrosative stress. They also indicate that peripheral and central tissue differ in their response to stress or antidepressant treatments. It is possible that that apart from DNA methylation, a crucial role of expression level of genes may be played by other forms of epigenetic regulation, such as histone modification or microRNA interference. These findings provide strong evidence for thesis that analysis of the level of mRNA and protein expression as well as the status of promoter methylation can help in understanding the pathomechanisms of mental diseases, including depression, and the mechanisms of action of drugs effective in their therapy.