Glycogen synthase kinase‐3 levels and phosphorylation undergo large fluctuations in mouse brain during development

Beurel E, Mines MA, Song L, Jope RS. Glycogen synthase kinase‐3 levels and phosphorylation undergo large fluctuations in mouse brain during development.
Bipolar Disord 2012: 14: 822–830. © 2012 John Wiley & Sons A/S.Published by Blackwell Publishing Ltd. Objectives: Dysregulated glycogen synthase kinase‐3 (GSK3) may contribute to the pathophysiology of mood disorders and other diseases, and appears to be a target of certain therapeutic drugs. The growing recognition of heightened vulnerability during development to many psychiatric diseases, including mood disorders, led us to test if there are developmental changes in mouse brain GSK3 and its regulation by phosphorylation and by therapeutic drugs. Methods: GSK3 levels and phosphorylation were measured at seven ages of development in the mouse cerebral cortex and hippocampus. Results: Two periods of rapid transitions in GSK3 levels were identified: a large rise between postnatal days 1 and 2 and three weeks of age, where GSK3 levels were as much as fourfold higher than adult mouse brain levels, and a rapid decline between 2–4 and eight weeks of age, when adult levels were reached. Inhibitory serine‐phosphorylation of GSK3, particularly GSK3β, was extremely high in the one‐day postnatal mouse brain, and rapidly declined thereafter. These developmental changes in GSK3 were equivalent in the male and female cerebral cortex, and differed from other signaling kinases, including Akt, extracellular‐regulated kinases 1/2, c‐Jun N‐terminal kinase, and p38 levels and phosphorylation. In contrast to the adult mouse brain, where administration of lithium or fluoxetine rapidly and robustly increased serine‐phosphorylation of GSK3, in young mice these responses were blunted or absent. Conclusions: High brain levels of GSK3 and large fluctuations in its levels and phosphorylation in the juvenile and adolescent mouse brain raise the possibility that they may contribute to destabilized mood regulation induced by environmental and genetic factors.     

Antidepressants are neuroprotective against nutrient deprivation stress in rat hippocampal neurons

There is ample evidence that depression and stress can be ameliorated through the use of physical exercise and/or antidepressant drugs. Both have been shown to promote neuroprotection against atrophy of dendrites and neuronal death through the activation of pro-survival signaling pathways, such as that of phosphatidyl inositol 3' kinase (PI-3K) and mitogen-activated protein kinase (MAPK). Depriving neurons in culture of several vital nutrients provides a viable model of neuronal stress, trauma or insult that occurs in vivo. Therefore, we sought to evaluate if various antidepressants are indeed neuroprotective in this model of nutrient deprivation stress. In addition, we evaluated if three key pro-survival pathways (PI-3K, MAPK, protein kinase A) are necessary for such neuroprotection. We used quantitative Western blotting to evaluate the immunoreactivity levels of brain-derived neurotrophic factor, PI-3K, phospho-protein kinase B (P-Akt), phospho-MAPK and phospho-cyclic AMP response element-binding protein, and live/dead cytotoxicity assay to evaluate cell survival. We demonstrate that in the ideal conditions of nutrient supplement, norepinephrine, serotonin and three antidepressants increased all six outcome measures; however, in the absence of such nutrients, only P-Akt levels showed signs of decreasing. In the presence of pro-survival pathway inhibitor, however, five out of the six outcome measures decreased (not P-Akt), relative to those of the ideal conditions of nutrient supplement. Thus, pro-survival pathway integrity, which more directly affects gene expression, is more important than the presence of externally placed nutrients for cell survival. We discuss our results in the context of receptor and pathway cross-talk, indicating that pharmacological rescue of neuronal atrophy/death in the face of mood disorders requires that pro-survival pathways remain intact.     

TrkB‐mediated activation of the phosphatidylinositol‐3‐kinase/Akt cascade reduces the damage inflicted by oxygen–glucose deprivation in area CA3 of the rat hippocampus

The selective vulnerability of hippocampal area CA1 to ischemia‐induced injury is a well‐known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen–glucose deprivation–reperfusion (OGD‐RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain‐derived neurotrophic factor (BDNF) or 7,8‐dihydroxyflavone (7,8‐DHF) exhibited reduced depression of the electrical activity triggered by OGD‐RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD‐RP. The protective effect of TrkB activation was limited to area CA3, as OGD‐RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3‐kinase/Akt (PI‐3‐K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD‐RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase‐3 activity in slices pretreated with BDNF and exposed to OGD‐RP. We propose that PI‐3‐K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.     

Effects of exercise and muscle type on BDNF,NT‐4/5, and TrKB expression in skeletal muscle

Muscle‐derived neurotrophins are thought to contribute to the adaptation of skeletal muscle to exercise, but the effects of brief exercise interventions on BDNF, NT‐4/5, and trkB are not understood. RNA was extracted for RT‐PCR from soleus and medial gastrocnemius of Sprague‐Dawley rats exercised on a treadmill at speeds up to 20 m/min at 5% incline for 5 or 10 days. BDNF expression was elevated in soleus following 5 days (184%, P < 0.001) but not 10 days of exercise. NT‐4/5 and trkB were not affected at either time‐point. BDNF mRNA was significantly higher in soleus at rest when compared with medial gastrocnemius (193%, P < 0.05). No significant effects of muscle type were detected for NT‐4/5 and trkB. Our results indicate differential control of BDNF expression between soleus and medial gastrocnemius following 5 days of exercise. BDNF may be a protein with an uncharacterized contribution to the acute adaptation of skeletal muscle to exercise, whereas NT‐4/5 shows no response. Muscle Nerve, 2009     

Transforming growth factor-beta1 enhances expression of brain-derived neurotrophic factor and its receptor, TrkB, in neurons cultured from rat cerebral cortex.

The effects of transforming growth factor (TGF)-beta1 on expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, in neurons cultured from the cerebral cortex of 18-day-old embryonic rats were examined. BDNF mRNA was significantly increased from 24-48 hr after the TGF-beta1 treatment over 20 ng/ml. Accumulation of BDNF protein in the culture medium was also potentiated by TGF-beta1, although the intracellular content of BDNF was nearly unchanged. The enhancement of BDNF mRNA expression was suppressed by the co-presence of decorin, a small TGF-beta-binding proteoglycan that inhibits the biological activities of TGF-betas. mRNA expression of full-length TrkB, the bioactive high-affinity receptor for BDNF, was also upregulated after treatment with TGF-beta1. These observations suggest that: 1) TGF-beta1 potentiates BDNF/TrkB autocrine or local paracrine system; and 2) the neurotrophic activity of TGF-beta1 is partly responsible for the BDNF induced by TGF-beta1 itself. To test this latter possibility, we examined the neuronal survival activity of TGF-beta1 with or without K252a, a selective inhibitor of Trk family tyrosine kinases. TGF-beta1 significantly enhanced neuronal survival, but the co-presence of K252a completely suppressed the activity, demonstrating the involvement of Trk receptor signaling in TGF-beta1-mediated neuronal survival in cultured rat cortical neurons. These results seem to be in line with recent findings by other investigators that some neurotrophic factors including BDNF require TGF-betas as a cofactor to exert their neurotrophic activities.     

Differential expression of molecular markers of synaptic plasticity in the hippocampus, prefrontal cortex, and amygdala in response to spatial learning, predator exposure, and stress-induced amnesia

We have studied the effects of spatial learning and predator stress-induced amnesia on the expression of calcium/calmodulin-dependent protein kinase II (CaMKII), brain-derived neurotrophic factor (BDNF) and calcineurin in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC). Adult male rats were given a single training session in the radial-arm water maze (RAWM) composed of 12 trials followed by a 30-min delay period, during which rats were either returned to their home cages or given inescapable exposure to a cat. Immediately following the 30-min delay period, the rats were given a single test trial in the RAWM to assess their memory for the hidden platform location. Under control (no stress) conditions, rats exhibited intact spatial memory and an increase in phosphorylated CaMKII (p-CaMKII), total CaMKII, and BDNF in dorsal CA1. Under stress conditions, rats exhibited impaired spatial memory and a suppression of all measured markers of molecular plasticity in dorsal CA1. The molecular profiles observed in the BLA, mPFC, and ventral CA1 were markedly different from those observed in dorsal CA1. Stress exposure increased p-CaMKII in the BLA, decreased p-CaMKII in the mPFC, and had no effect on any of the markers of molecular plasticity in ventral CA1. These findings provide novel observations regarding rapidly induced changes in the expression of molecular plasticity in response to spatial learning, predator exposure, and stress-induced amnesia in brainregions involved in different aspects of memory processing.     

iPlasticity: Induced juvenile‐like plasticity in the adult brain as a mechanism of antidepressants

The network hypothesis of depression proposes that mood disorders reflect problems in information processing within particular neural networks. Antidepressants (AD), including selective serotonin reuptake inhibitors (SSRI), function by gradually improving information processing within these networks. AD have been shown to induce a state of juvenile‐like plasticity comparable to that observed during developmental critical periods: Such critical‐period‐like plasticity allows brain networks to better adapt to extrinsic and intrinsic signals. We have coined this drug‐induced state of juvenile‐like plasticity ‘iPlasticity.’ A combination of iPlasticity induced by chronic SSRI treatment together with training, rehabilitation, or psychotherapy improves symptoms of neuropsychiatric disorders and issues underlying the developmentally or genetically malfunctioning networks. We have proposed that iPlasticity might be a critical component of AD action. We have demonstrated that iPlasticity occurs in the visual cortex, fear erasure network, extinction of aggression caused by social isolation, and spatial reversal memory in rodent models. Chronic SSRI treatment is known to promote neurogenesis and to cause dematuration of granule cells in the dentate gyrus and of interneurons, especially parvalbumin interneurons enwrapped by perineuronal nets in the prefrontal cortex, visual cortex, and amygdala. Brain‐derived neurotrophic factor (BDNF), via its receptor tropomyosin kinase receptor B, is involved in the processes of synaptic plasticity, including neurogenesis, neuronal differentiation, weight of synapses, and gene regulation of synaptic formation. BDNF can be activated by both chronic SSRI treatment and neuronal activity. Accordingly, the BDNF/tropomyosin kinase receptor B pathway is critical for iPlasticity, but further analyses will be needed to provide mechanical insight into the processes of iPlasticity.     

Hippocampal neurotrophin levels after injury: Relationship to the age of the hippocampus at the time of injury

Aging impairs the competence of the hippocampus for synaptic reorganization after injury. This potentially is due to the inability of the aging hippocampus to up-regulate the critical neurotrophic factors for prolonged periods after injury to levels at which they can stimulate neurite outgrowth and facilitate synaptic reorganization. We hypothesize that the concentrations of neurotrophins in the hippocampus after injury depend on the age at the time of injury. We quantified the concentrations of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) in the hippocampus of young, middle-aged, and aged Fischer 344 rats at 4 days after kainic acid (KA)-induced injury. In comparison with the age-matched intact hippocampus, the KA-lesioned hippocampus exhibited increased levels of BDNF and NGF in all three age groups. In contrast, the NT-3 concentration was unaltered after KA lesion. Notwithstanding similar percentage increases in BDNF after injury, the lesioned middle-aged and aged hippocampus contained 45-52% less BDNF than the lesioned young hippocampus. NGF and NT-3 levels after injury were comparable across the three age groups, however. Furthermore, lower BDNF concentration in the injured aging hippocampus was associated with normal astrocytic response but significantly diminished microglial reaction. Thus, in comparison with the injured young hippocampus, the injured aging hippocampus contains considerably less BDNF but similar levels of NGF and NT-3. Lower BDNF levels in the injured aging hippocampus might underlie the diminished spontaneous healing response observed in the aging hippocampus after injury, particularly in terms of synaptic reorganization and dentate neurogenesis.     

Amyloid beta up-regulates brain-derived neurotrophic factor production from astrocytes: rescue from amyloid beta-related neuritic degeneration

Astrocytes, the most abundant type of glia in the brain, are considered to play a key role in Alzheimer's disease (AD) pathologies. In a cell culture study, we have previously shown that astroglial responses against amyloid beta (Abeta) occur before obvious neuronal damage could be detected, suggesting the possibility that astrocytes might be an attractive therapeutic target for treating AD. In the present study, we investigated astroglial gene expression changes in response to Abeta to elucidate further the role of astrocytes in Abeta toxicity. By using real-time PCR and ELISA analyses, we found that Abeta rapidly induced astrocytes to produce brain-derived neurotrophic factor (BDNF). Abeta42 was more effective than Abeta40 in increasing astroglial BDNF production. Moreover, BDNF treatment rescued the neuronally differentiated human neuroblastoma cells from neuritic degeneration caused by Abeta toxicity. This is the first study to demonstrate that astrocytes are capable of increasing the production of a particular neurotrophic factor in response to Abeta. Our findings also identify BDNF as a potential therapeutic agent for preventing Abeta-related neuritic degeneration.     

The role of neurotrophins in muscle under physiological and pathological conditions

This review summarizes the various effects of neurotrophins in skeletal muscle and how these proteins act as potential regulators of development, maintenance, function, and regeneration of skeletal muscle fibers. Increasing evidence suggests that this family of neurotrophic factors not only modulates survival and function of innervating motoneurons and proprioceptive neurons but also development and differentiation of myoblasts and muscle fibers. Neurotrophins and neurotrophin receptors play a role in the coordination of muscle innervation and functional differentiation of neuromuscular junctions. However, neurotrophin receptors are also expressed in differentiating muscle cells, in particular at early developmental stages in myoblasts before they fuse. In adults with pathological conditions such as human degenerative and inflammatory muscle disorders, variations of neurotrophin expression are found, but the role of neurotrophins under such conditions is still not clear. The goal of this review is to provide a basis for a better understanding and future studies on the role of these factors under such pathological conditions and for treatment of human muscle diseases.     

Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes - A preliminary study

Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for the treatment of epilepsy and bipolar disorder. However, the underlying therapeutic mechanisms of the treatment of each disease remain unclear. Recently, the anti-epileptic effect of VPA has been found to lead to modulation of the synaptic excitatory/inhibitory balance. In addition, the therapeutic action of VPA has been linked to its effect on astrocytes by regulating gene expression at the molecular level, perhaps through an epigenetic mechanism as a histone deacetylase (HDAC) inhibitor. To provide insight into the mechanisms underlying the actions of VPA, this study investigated whether the synaptic excitatory/inhibitory (E/I) balance could be mediated by VPA through astrocytes. First, using the primary rat neuronal, astroglial, and neuro-glial mixed culture systems, we demonstrated that VPA treatment could regulate the mRNA levels of two post-synaptic cell adhesion molecules(neuroligin-1 and neuregulin-1) and two extracellular matrices (neuronal pentraxin-1and thrombospondin-3) in primary rat astrocyte cultures in a time- and concentration-dependent manner. Moreover, the up-regulation effect of VPA was noted in astrocytes, but not in neurons. In addition, these regulatory effects could be mimicked by sodium butyrate, a HDAC inhibitor, but not by lithium or two other glycogen synthase kinase-3 beta inhibitors. With the known role of these four proteins in regulating the synaptic E/I balance, we further demonstrated that VPA increased excitatory post-synaptic protein (postsynaptic density 95) and inhibitory post-synaptic protein (Gephyrin) in cortical neuro-glial mixed cultures. Our results suggested that VPA might affect the synaptic excitatory/inhibitory balance through its effect on astrocytes. This work provides the basis for future evaluation of the role of astroglial cell adhesion molecules and the extracellular matrix on the control of excitatory and inhibitory synapse formation.     

Activation of Akt signaling in rat brain by intracerebroventricular injection of ouabain: A rat model for mania

Intracerebroventricular (ICV) injection of ouabain, a specific Na–K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na–K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1 mM of ouabain. Phosphorylation of GSK-3β (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8 h after a single 1 mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3β (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania.