Glutamate and neurotrophic factors in neuronal plasticity and disease.

In adult rat brains, brain-derived neurotrophic factor (BDNF) rhythmically oscillates according to the light-dark cycle and exhibits unique functions in particular brain regions. However, little is known of this subject in juvenile rats. Here, we examined diurnal variation in BDNF and neurotrophin-3 (NT-3) levels in 14-day-old rats. BDNF levels were high in the dark phase and low in the light phase in a majority of brain regions. In contrast, NT-3 levels demonstrated an inverse phase relationship that was limited to the cerebral neocortex, including the visual cortex, and was most prominent on postnatal day 14. An 8-h phase advance of the light-dark cycle and sleep deprivation induced an increase in BDNF levels and a decrease in NT-3 levels in the neocortex, and the former treatment reduced synaptophysin expression and the numbers of synaptophysin-positive presynaptic terminals in cortical layer IV and caused abnormal BDNF and NT-3 rhythms 1 week after treatment. A similar reduction of synaptophysin expression was observed in the cortices of Bdnf gene-deficient mice and Ca(2+)-dependent activator protein for secretion 2 gene-deficient mice with abnormal free-running rhythm and autistic-like phenotypes. In the latter mice, no diurnal variation in BDNF levels was observed. These results indicate that regular rhythms of BDNF and NT-3 are essential for correct cortical network formation in juvenile rodents.

Increasing the level of neurotrophins within the central nervous system may have therapeutic efficacy in patients with various neurological diseases. Earlier we have demonstrated that myelin basic protein (MBP)-primed T cells induce the expression of various proinflammatory molecules in glial cells via cell-to-cell contact. Here we describe that after Th2 polarization by gemfibrozil or other drugs, MBP-primed T cells induced the expression of neurotrophic molecules such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not proinflammatory molecules in microglia and astroglia via cell-to-cell contact. MBP-primed Th2 cells expressed alpha5 and beta3 integrins and functional blocking antibodies against both alpha5 and beta3 integrins inhibited the ability of MBP-primed Th2 cells to induce glial neurotrophins. On the other hand, glial cells expressed PDGF-Rbeta and neutralization of this glial receptor abrogated the ability of Th2 cells to induce neurotrophins in glia. Activation of glial cAMP response element-binding protein (CREB) by MBP-primed Th2 cell contact and inhibition of contact-mediated expression of neurotrophins by antisense knockdown of glial CREB suggest that MBP-primed Th2 cell-glia contact induces the expression of neurotrophins through glial activation of CREB. Accordingly, blocking of either alpha5beta3 integrins on T cells or PDGF-Rbeta on glial cells impaired the ability of MBP-primed Th2 cells to induce glial activation of CREB. Furthermore, we demonstrate that these MBP-primed Th2 cells entered into the central nervous system and increased the expression of neurotrophins in vivo in the brain. This study illuminates the importance of alpha5beta3 and PDGF-Rbeta in guiding the novel neurotrophic property of neuroantigen-primed T cells via activation of CREB that may be of therapeutic importance in various neurological disorders.

A Hypothesized Role for Dendritic Remodeling in the Etiology of Mood and Anxiety Disorders

Methotrexate, which is used to treat many malignancies and autoimmune diseases, affects brain functions including hippocampal-dependent memory function. However, the precise mechanisms underlying methotrexate-induced hippocampal dysfunction are poorly understood. To evaluate temporal changes in synaptic plasticity-related signals, the expression and activity of N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, extracellular signal-regulated kinase 1/2, cAMP responsive element-binding protein, glutamate receptor 1, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor were examined in the hippocampi of adult C57BL/6 mice after methotrexate (40 mg/kg) intraperitoneal injection. Western blot analysis showed biphasic changes in synaptic plasticity-related signals in adult hippocampi following methotrexate treatment. N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, and glutamate receptor 1 were acutely activated during the early phase (1 day post-injection), while extracellular signal-regulated kinase 1/2 and cAMP responsive element-binding protein activation showed biphasic increases during the early (1 day post-injection) and late phases (7–14 days post-injection). Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression increased significantly during the late phase (7–14 days post-injection). Therefore, methotrexate treatment affects synaptic plasticity-related signals in the adult mouse hippocampus, suggesting that changes in synaptic plasticity-related signals may be associated with neuronal survival and plasticity-related cellular remodeling.

Several lines of evidence show a relationship between alterations in the mechanisms that control the expression of neurotrophic factors and mood disorders (1). In particular, support for the role of brain-derived neurotrophic factor (BDNF) in the pathogenesis of depression and related deficits in neuronal plasticity comes from evidence that a reduction of BDNF expression has been found in postmortem brains and serum of depressed subjects and that the BDNF gene is required for the response to antidepressant drugs. With the aim to contribute to the characterization of the molecular and neuronal systems involved in the pathogenesis of depression and in the mechanism of action of the antidepressant treatments, here we use the outbread Roman High- (RHA) and Roman Low-Avoidance (RLA) rat lines, psychogenetically selected for rapid versus poor acquisition of active avoidance, respectively, and bearing several behavioral characteristics closely resembling the cardinal symptoms of depression (2), to investigate on the immunochemical occurrence of BDNF in selected areas of the RHA and RLA rat brain by means of western blot (WB) and immunohistochemistry. WB analysis indicates that the relative levels of BDNF patently and markedly differed in the hippocampus, where they were significantly lower by 58% in RLA vs RHA rats (p = 0.0014). In the remaining examined areas, namely the prefrontal cortex, the caudate-putamen complex proper, the core and shell regions of the nucleus accumbens and the ventral tegmental area, the relative BDNF levels did not show statistically significant differences. In tissue sections, BDNF-like immunoreactive (LI) material labelled neuronal cell bodies, proximal processes and varicose nerve fibers, with an uneven distribution in telencephalic cerebral cortex, hippocampus, amygdala, nucleus accumbens, caudate-putamen complex proper, thalamus and ventral tegmentum of the midbrain. Densitometric analysis of immunostained brain sections were used to quantify differences among the two rat lines. The results obtained provide a morphological evidence for a differential expression of BDNF in specific areas of RLA vs RHA rat brains and may form the morphological basis to understand the regulation of the trophic machinery in depression.

Metabotropic glutamate receptor subtypes (mGluR1 to -8) act as pre- and postsynaptic regulators of neurotransmission in the central nervous system. Regulation of neurotransmission via metabotropic glutamate receptors has recently been implicated in the pathophysiology of anxiety and stress-related disorders including depression. Among metabotropic glutamate receptor subtypes, the group III metabotropic glutamate receptor subtype 7 (mGluR7) shows the highest evolutionary conservation (Flor et al., 1997; Makoff et al., 1996), which suggests that this receptor could play an important physiological role. Cryan et al. (2003) have demonstrated that mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) show antidepressant and anxiolytic-like effects in a variety of stress-related paradigms, including the forced swim stress and the stress-induced hyperthermia tests. Furthermore, the same group has recently developed mGluR7 knockdown using siRNA, which further supported the critical role of mGluR7 in anxiety- and stress-related behaviors (Thakker et al., 2005). Since the hypothalamic-pituitary-adrenal (HPA) axis regulates stress responses, it was investigated in this thesis whether the levels of selected mRNA transcripts and endocrine hormones were altered in mGluR7 deficient mice in the HPA axis. Over all, mGluR7-/- mice showed only moderately lower serum levels of corticosterone and adrenocorticotropic hormone (ACTH) compared to mGluR7+/+ mice. However, strong evidence has been found for up-regulation of glucocorticoid receptor (GR)-dependent feedback suppression of the HPA axis in mice with mGluR7 deficiency: (i) mRNA transcripts of GR were significantly higher in the hippocampus of mGluR7-/- animals, (ii) similar increases were seen for 5-HT1A receptor transcripts which are thought to be directly controlled by the GR transcription factor and finally (iii) mGluR7-/- mice showed elevated sensitivity to dexamethasone-induced suppression of serum corticosterone when compared with mGluR7+/+ animals. These results indicate that mGluR7 deficiency causes dysregulation of HPA axis parameters which may account, at least in part, for the phenotype of mGluR7-/- mice in animal models for anxiety and stress-related disorders. In addition, the data given here show that protein levels of brain-derived neurotrophic factor (BDNF) are elevated in the hippocampus of mGluR7-/- mice which will be discussed at the latter part of this thesis in the context of the stress-resistant phenotype found in those animals. It can be concluded that genetic ablation of mGluR7 in mice interferes at multiple sites in the neuronal circuitry and molecular pathways implicated in anxiety and stress-related disorders. Metabotropic glutamate receptor subtypes (mGluR1 to -8) act as pre- and postsynaptic regulators of neurotransmission in the central nervous system. Regulation of neurotransmission via metabotropic glutamate receptors has recently been implicated in the pathophysiology of anxiety and stress-related disorders including depression. Among metabotropic glutamate receptor subtypes, the group III metabotropic glutamate receptor subtype 7 (mGluR7) shows the highest evolutionary conservation (Flor et al., 1997; Makoff et al., 1996), which suggests that this receptor could play an important physiological role. Cryan et al. (2003) have demonstrated that mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) show antidepressant and anxiolytic-like effects in a variety of stress-related paradigms, including the forced swim stress and the stress-induced hyperthermia tests. Furthermore, the same group has recently developed mGluR7 knockdown using siRNA, which further supported the critical role of mGluR7 in anxiety- and stress-related behaviors (Thakker et al., 2005). Since the hypothalamic-pituitary-adrenal (HPA) axis regulates stress responses, it was investigated in this thesis whether the levels of selected mRNA transcripts and endocrine hormones were altered in mGluR7 deficient mice in the HPA axis. Over all, mGluR7-/- mice showed only moderately lower serum levels of corticosterone and adrenocorticotropic hormone (ACTH) compared to mGluR7+/+ mice. However, strong evidence has been found for up-regulation of glucocorticoid receptor (GR)-dependent feedback suppression of the HPA axis in mice with mGluR7 deficiency: (i) mRNA transcripts of GR were significantly higher in the hippocampus of mGluR7-/- animals, (ii) similar increases were seen for 5-HT1A receptor transcripts which are thought to be directly controlled by the GR transcription factor and finally (iii) mGluR7-/- mice showed elevated sensitivity to dexamethasone-induced suppression of serum corticosterone when compared with mGluR7+/+ animals. These results indicate that mGluR7 deficiency causes dysregulation of HPA axis parameters which may account, at least in part, for the phenotype of mGluR7-/- mice in animal models for anxiety and stress-related disorders. In addition, the data given here show that protein levels of brain-derived neurotrophic factor (BDNF) are elevated in the hippocampus of mGluR7-/- mice which will be discussed at the latter part of this thesis in the context of the stress-resistant phenotype found in those animals. It can be concluded that genetic ablation of mGluR7 in mice interferes at multiple sites in the neuronal circuitry and molecular pathways implicated in anxiety and stress-related disorders.

The outbred Roman High- (RHA) and Roman Low-Avoidance (RLA) rats were psychogenetically selected for rapid vs. poor acquisition of active avoidance, respectively, and differ in many behavioural traits. Thus, RHA rats are impulsive, novelty seekers, and proactive copers, whereas RLA rats display behavioural traits that resemble some of the cardinal symptoms of depression (1). Beyond the monoamine hypothesis, compelling evidence suggests that mood disorders are characterized by reduced neuronal plasticity. Thus, it has been shown that exposure to stress and antidepressant treatments modulate the expression of neurotrophic factors, and that these changes show an anatomical specificity (2). To characterize the molecular and neuronal systems involved in the pathogenesis of stress-induced depression and in the mechanism of action of antidepressant treatments, we performed western blot (WB) and immunohistochemistry studies to assess the localization of the brain-derived neurotrophic factor (BDNF) in the hippocampus of RHA and RLA rats, both under basal conditions and after exposure to an acute stressor, i.e., the Forced Swim Test (FST). WB analyses showed that, under basal conditions, the relative levels of BDNF were lower in RLA vs. RHA rats, whereas, after FST, the relative levels of BDNF were markedly higher in the hippocampus of RLA vs. RHA rats. In brain tissue sections, BDNF-like immunoreactive material labeled neuronal cell bodies, proximal processes and varicose nerve fibers. Densitometric analysis used to compare immunostained brain sections from the two rat lines showed that, under basal conditions and upon FST, major differences were limited to the hippocampus proper, mostly to the CA3 and CA2 sectors, whereas the dentate gyrus (DG) showed no line-related differences. These results are at variance with previous studies showing that the expression of BDNF in the hippocampus is reduced in depressed patients and in rats exposed to stressors. In conclusion, the present study provides morphological evidence for an unexpected differential regional expression of BDNF in the hippocampus of RLA vs. RHA rats and supports the view that acute stress stimulates neuronal plasticity in genetic animal models of depression. This work was supported by grants from L.R. 07/2007, RAS project 2012.

Obesity and low physical activity changes the redox state and neurotrophin secretion by leukocytes. However, the role of exercise on brain derived neurotrophic factor (BDNF) production and oxidative stress markers of peripheral blood mononuclear cells (PBMC) remains unknown. This study aimed to verify the impact of acute maximal exercise on oxidative stress markers and the BDNF production by stimulated PBMC from sedentary and physically active obese men. PBMC from twelve sedentary obese (SED group) and twelve regular exercisers (EXE group) obese men were collected before, immediately and 1-h after maximal exercise. PBMC were stimulated with lipopolysaccharide (LPS) to evaluate the BDNF and nitrite production, lipid peroxidation, and antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities. PBMC from EXE group presented higher BDNF production (P=0.03) and lower TBARS levels than SED group at baseline. Maximal exercise increased BDNF and nitrite production, and lipid peroxidation immediately and 1-h after the bout in both groups. The EXE group presented higher superoxide dismutase activity immediately after bout and higher catalase activity 1-h after bout in PBMC. On the other hand, PBMC from SED group had lower superoxide dismutase activity immediately after exercise. Furthermore, PBMC from EXE group presented higher BDNF production and SOD activity and lower TBARS concentrations than SED group immediately after maximal exercise. Maximal exercise changes the redox state and enhances BDNF production by LPS-stimulated PBMC in obese individuals.

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

Brain derived neurotrophic factor and neurotrophic factor 3 modulate neurotransmitter receptor expressions on developing spiral ganglion neurons

Locally produced in human granulosa cells of the developing follicle, bone morphogenetic protein 2 (BMP2) plays a crucial role in the regulation of ovarian folliculogenesis and luteal formation. Brain-derived neurotrophic factor (BDNF) is an intraovarian neurotrophic factor that has been shown to promote oocyte maturation and subsequent fertilization competency. At present, little is known regarding the intracellular regulation, assembly and secretion of endogenous BDNF in human granulosa cells. The aim of this study was to explore the effect of BMP2 on the expression and production of BDNF in human granulosa cells and the molecular mechanisms underlying this effect. An immortalized human granulosa cell line (SVOG) and primary human granulosa-lutein (hGL) cells were utilized as in vitro study models. Our results showed that BMP2 significantly increased the mRNA and secreted levels of BDNF. Additionally, BMP2 upregulated the expression of furin at the transcriptional and translational levels. Knockdown of endogenous furin partially attenuated the BMP2-induced increase in BDNF production, indicating that furin is involved in the maturation process of BDNF. Using pharmacological (kinase receptor inhibitors) and siRNA-mediated inhibition approaches, we demonstrated that BMP2-induced upregulation of BDNF and furin expression is most likely mediated by the activin receptor-like kinase (ALK)2/ALK3-SMAD4 signaling pathway. Notably, analysis using clinical samples revealed that there was a positive correlation between follicular fluid concentrations of BMP2 and those of BDNF. These results indicate that BMP2 increases the production of mature BDNF by upregulating the precursor BDNF and promoting the proteolytic processing of mature BDNF. Finally, we also investigated the effects of BMP2 on ovarian steroidogenesis and the results showed that BMP2 treatment significantly increased the accumulated level of estradiol (by upregulating the expression of FSH receptor and cytochrome P450 aromatase), whereas it decreased the accumulated level of progesterone (by downregulating the expression of LH receptors and steroidogenic acute regulatory protein) in primary hGL cells. Our findings provide a novel paracrine mechanism underlying the regulation of an intraovarian growth factor in human granulosa cells.

Brain derived neurotrophic factor (BDNF), signaling via its receptor Tropomyosin receptor kinase B (TrkB) plays a pivotal role in establishing and maintaining the structure and function of neurons within the central nervous system. Deficits in BDNF/TrkB signaling are reported to contribute to the pathogenesis of multiple major disorders, such as Alzheimer’s disease. While manipulating the BDNF/TrkB signaling has been shown to be a viable approach to ameliorate some of the symptoms in a variety of neurological and psychiatric disorders, effective delivery of BDNF into the brain is challenging due to its poor pharmacokinetic profile and clinical translation has fallen short. This triggered the development of BDNF mimetics which specifically activate the TrkB receptor. This work investigated whether a recently identified fully human TrkB agonist antibody (ZEB85) exerts similar biological functions to BDNF. It was shown that treatment with ZEB85 leads to TrkB phosphorylation and increases expression of the activity-dependent immediate early gene c-Fos. Further, ZEB85 significantly increases the neurite complexity of developing hippocampal neurons. Parvalbumin positive interneurons from hippocampal cultures deprived of BDNF have a severely altered dendritic phenotype which can be completely rescued by the TrkB agonist antibody. Moreover, ZEB85 leads to changes in spine number and morphology in mature neurons. Under pathological conditions, treatment with ZEB85 completely prevents amyloid-beta induced dendritic spine loss. Lastly, prolonged application of ZEB85 over several days in hippocampal organotypic slice cultures prepared from heterozygous bdnf knockout mice, known to have deficits in the induction and maintenance of long-term potentiation (LTP) improved these parameters. Taken together, ZEB85 indeed exerts some of the biological functions of BDNF as seen in TrkB activation and structural and functional changes of healthy and diseased neurons. However, when directly compared to BDNF, the effect of ZEB85 is less pronounced. Thus, this work provides an initial characterization of a new promising TrkB agonist suggesting its potential value for therapeutical applications.

The brain-derived neurotrophic factor (BDNF) plays an important role in the development and function of the nervous system.BDNF controls the neuronal survival, differentiation, growth of dendrites and axons, formation of synapse, neuronal plasticity and the basic process of learning and memory through a variety of ways, the dysregulation of which is probably the important molecular mechanism responsible for the onset of autism spectrum disorder.The research advance on preclinical research and clinical research between BDNF and autism spectrum disorder is reviewed in this paper. Key words: Brain-derived neurotrophic factor; Autism spectrum disorder; Pathological mechanism

Objective To evaluate the effects of neural stem cells (NSCs) overexpressing brain-derived neurotrophic factor (BDNF) on the levels of neurotrophic factors and microglia activation in hippocampus after brain irradiation. Methods Hippocampal NSCs were isolated from fetal rat brain and infected with GFP-lentivirus and GFP-BDNF-lentivirus. SD rats were randomized into four groups: control group, irradiated group (R group), GFP-modified NSCs transplantation group with irradiation (R+ NSCs group), and GFP-BDNF modified NSCs transplantation group with irradiation (R+ BDNF-NSCs group). NSCs were transplanted into the bilateral hippocampus of rats one month after whole brain irradiation at a single dose of 20 Gy. The expressions of BDNF, glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in hippocampus were detected at 2 and 8 weeks after transplantation. The activation of microglia was observed by immunofluorescence. Results At 2 and 8 weeks after transplantation, the expressions of BDNF and NGF proteins in hippocampus of R+ BDNF-NSCs group were significantly higher than those of R group (P 0.05). Conclusions The transplantation of NSCs overexpressing BDNF promotes the production of BDNF and NGF, which improves the level of neurotrophic factors in hippocampus after radiation. Key words: Radiation-induced cognitive dysfunction; Neurotrophic factors; Microglia; Neural stem cells

Brain-derived neurotrophic factor (BDNF) is a key molecule essential for neural plasticity and development, and is implicated in the pathophysiology of various central nervous system (CNS) disorders. It is now documented that BDNF is synthesized not only in neurons, but also in astrocytes which actively regulate neuronal activities by forming tripartite synapses. Inwardly rectifying potassium (Kir) channel subunit Kir4.1, which is specifically expressed in astrocytes, constructs Kir4.1 and Kir4.1/5.1 channels, and mediates the spatial potassium (K+) buffering action of astrocytes. Recent evidence illustrates that Kir4.1 channels play important roles in bringing about the actions of antidepressant drugs and modulating BDNF expression in astrocytes. Although the precise mechanisms remain to be clarified, it seems likely that inhibition (down-regulation or blockade) of astrocytic Kir4.1 channels attenuates K+ buffering, increases neuronal excitability by elevating extracellular K+ and glutamate, and facilitates BDNF expression. Conversely, activation (up-regulation or opening) of Kir4.1 channels reduces neuronal excitability by lowering extracellular K+ and glutamate, and attenuates BDNF expression. Particularly, the former pathophysiological alterations seem to be important in epileptogenesis and pain sensitization, and the latter in the pathogenesis of depressive disorders. In this article, we review the functions of Kir4.1 channels, with a focus on their regulation of spatial K+ buffering and BDNF expression in astrocytes, and discuss the role of the astrocytic Kir4.1-BDNF system in modulating CNS disorders.