cAMP Response Element-binding Protein Signaling Research Articles

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.

Cilostazol attenuates ischemic brain injury and enhances neurogenesis in the subventricular zone of adult mice after transient focal cerebral ischemia

Evidence suggests that neurogenesis occurs in the adult mammalian brain, and that various stimuli, for example, ischemia/hypoxia, enhance the generation of neural progenitor cells in the subventricular zone (SVZ) and their migration into the olfactory bulb. In a mouse stroke model, focal ischemia results in activation of neural progenitor cells followed by their migration into the ischemic lesion. The present study assessed the in vivo effects of cilostazol, a type 3 phosphodiesterase inhibitor known to activate the cAMP-responsive element binding protein (CREB) signaling, on neurogenesis in the ipsilateral SVZ and peri-infarct area in a mouse model of transient middle cerebral artery occlusion. Mice were divided into sham operated ( n=12), vehicle- ( n=18) and cilostazol-treated ( n=18) groups. Sections stained for 5-bromodeoxyuridine (BrdU) and several neuronal and a glial markers were analyzed at post-ischemia days 1, 3 and 7. Cilostazol reduced brain ischemic volume ( P<0.05) and induced earlier recovery of neurologic deficit ( P<0.05). Cilostazol significantly increased the density of BrdU-positive newly-formed cells in the SVZ compared with the vehicle group without ischemia. Increased density of doublecortin (DCX)-positive and BrdU/DCX-double positive neural progenitor cells was noted in the ipsilateral SVZ and peri-infarct area at 3 and 7 days after focal ischemia compared with the vehicle group ( P<0.05). Cilostazol increased DCX-positive phosphorylated CREB (pCREB)-expressing neural progenitor cells, and increased brain derived neurotrophic factor (BDNF)-expressing astrocytes in the ipsilateral SVZ and peri-infarct area. The results indicated that cilostazol enhanced neural progenitor cell generation in both ipsilateral SVZ and peri-infarct area through CREB-mediated signaling pathway after focal ischemia.

Liver-Specific Overexpression of Pancreatic-Derived Factor (PANDER) Induces Fasting Hyperglycemia in Mice

The pancreas-derived hormones, insulin and glucagon, are the two main regulators of glucose homeostasis. However, their actions can be modulated by the presence of other circulating factors including cytokines. Pancreatic-derived factor (PANDER) is a novel cytokine-like molecule secreted from the endocrine pancreas, but its biological function is currently unknown. To address this, we employed adenoviral gene delivery to develop a novel murine model of PANDER overexpression, which we used to study PANDER's effect on glucose homeostasis. Although serum metabolites in fed mice were unaffected by PANDER overexpression, fasting glucose, insulin, and corticosterone levels were significantly elevated. Additionally, PANDER-overexpressing mice displayed elevated glucose and insulin levels during a glucose tolerance test, indicating that glucose tolerance was impaired. However, there were no defects in glucose-stimulated insulin secretion or peripheral insulin sensitivity. Elevated transcription of hepatic gluconeogenic genes, PEPCK and G6Pase accompanied the fasting hyperglycemia observed in PANDER-overexpressing animals. Similarly, treatment of primary hepatocytes with PANDER-expressing adenovirus or PANDER-enriched conditioned medium elevated gluconeogenic gene expression and glucose output. PANDER treatment also resulted in higher levels of Ser133-phosphorylated cAMP-response element-binding protein in hepatocytes stimulated with 8-bromo-cAMP and dexamethasone and higher levels of intracellular cAMP upon stimulation with forskolin. In summary, we provide the first report that identifies PANDER as a regulator of hepatic glucose metabolism, where it serves as a novel factor that amplifies hepatic cAMP and cAMP-response element-binding protein signaling to induce gluconeogenic gene expression and glucose output.

Inhibition of TRPC6 degradation suppresses ischemic brain damage in rats

Brain injury after focal cerebral ischemia, the most common cause of stroke, develops from a series of pathological processes, including excitotoxicity, inflammation, and apoptosis. While NMDA receptors have been implicated in excitotoxicity, attempts to prevent ischemic brain damage by blocking NMDA receptors have been disappointing. Disruption of neuroprotective pathways may be another avenue responsible for ischemic damage, and thus preservation of neuronal survival may be important for prevention of ischemic brain injury. Here, we report that suppression of proteolytic degradation of transient receptor potential canonical 6 (TRPC6) prevented ischemic neuronal cell death in a rat model of stroke. The TRPC6 protein level in neurons was greatly reduced in ischemia via NMDA receptor-dependent calpain proteolysis of the N-terminal domain of TRPC6 at Lys¹⁶. This downregulation was specific for TRPC6 and preceded neuronal death. In a rat model of ischemia, activating TRPC6 prevented neuronal death, while blocking TRPC6 increased sensitivity to ischemia. A fusion peptide derived from the calpain cleavage site in TRPC6 inhibited degradation of TRPC6, reduced infarct size, and improved behavioral performance measures via the cAMP response element-binding protein (CREB) signaling pathway. Thus, TRPC6 proteolysis contributed to ischemic neuronal cell death, and suppression of its degradation preserved neuronal survival and prevented ischemic brain damage.

Human Chorionic Gonadotropin Up-Regulates Expression of Myeloid Cell Leukemia-1 Protein in Human Granulosa-Lutein Cells: Implication of Corpus Luteum Rescue and Ovarian Hyperstimulation Syndrome

The corpus luteum is a dynamic endocrine structure with periodic development and regression during menstrual cycles. Its lifespan can be prolonged by human chorionic gonadotropin (hCG). However, the signal mechanisms of this phenomenon remain unclear. Our objective was to investigate the molecular mechanisms of hCG in the maintenance of the viability of granulosa-lutein cells. Granulosa-lutein cells were obtained from women undergoing in vitro fertilization. We examined the effects of hCG on the survival of cultured granulosa-lutein cells. The signal pathway inducing antiapoptotic protein was investigated. hCG enhanced viability of granulosa-lutein cells through antiapoptosis but not proliferation, because the apoptotic marker of annexin V was decreased, but the proliferative markers of Ki67 and proliferating cell nuclear antigen were not increased. Myeloid cell leukemia-1 (Mcl-1) protein, but not B-cell lymphoma protein-2 or B-cell lymphoma protein-xL, was significantly induced by hCG and LH. The granulosa-lutein cells secreted vascular endothelial growth factor that induced endothelial permeability. Mcl-1 small interfering RNA increased DNA fragmentation and diminished the antiapoptotic effect of hCG. hCG induced Mcl-1 expression through the LH/hCG receptor, adenylate cyclase, protein kinase A, and cAMP response element-binding protein signal pathway. Flavopiridol inhibited Mcl-1 production, released cytochrome c, and induced apoptosis of granulosa-lutein cells. We first demonstrate that hCG prevents apoptosis of granulosa-lutein cells through the induction of Mcl-1 protein via the LH/hCG receptor and a cAMP response element-binding protein-dependent pathway. We may have found the molecular mechanism for luteal rescue during early pregnancy. Mcl-1 prevents apoptosis and increases cell viability but not proliferation as mechanisms for luteal rescue. Mcl-1 is a key molecule of hCG signaling.

The novel squamosamide derivative FLZ enhances BDNF/TrkB/CREB signaling and inhibits neuronal apoptosis in APP/PS1 mice

The aim of this study was to study the effects of compound FLZ, a novel cyclic derivative of squamosamide from Annona glabra, on brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element-binding protein (CREB) signaling and neuronal apoptosis in the hippocampus of the amyloid precursor protein (APP)/presenilin-1 (PS1) double transgenic mice. APP/PS1 mice at the age of 5 months and age-matched wild-type mice (WT) were intragastrically administered FLZ (150 mg/kg) or vehicle [0.05% carboxymethyl cellulose sodium (CMC-Na)] daily for 20 weeks. The levels of BDNF in the hippocampus of WT and APP/PS1 mice were then measured by immunohistochemistry and Western blot analysis. Neuronal apoptosis in mouse hippocampus was detected by Nissl staining. Expression of NGF, NT3, pTrkB (Tyr515)/TrkB, pAkt (Ser473)/Akt, pERK/ERK, pCREB (Ser133)/CREB, Bcl-2/Bax, and active caspase-3 fragment/caspase-3 in the hippocampus of WT and APP/PS1 mice was detected by Western blot analysis. Compared with vehicle-treated APP/PS1 mice, FLZ (150 mg/kg) significantly increased BDNF and NT3 expression in the hippocampus of APP/PS1 mice. In addition, FLZ promoted BDNF high-affinity receptor TrkB phosphorylation and activated its downstream ERK, thus increasing phosphorylation of CREB at Ser133 in the hippocampus of APP/PS1 mice. Moreover, FLZ showed neuroprotective effects on neuronal apoptosis by increasing the Bcl-2/Bax ratio and decreasing the active caspase-3 fragment/caspase-3 ratio in the hippocampus of APP/PS1 mice. FLZ exerted neuroprotection at least partly through enhancing the BDNF/TrkB/CREB pathway and inhibiting neuronal apoptosis in APP/PS1 mice, which suggests that FLZ can be explored as a potential therapeutic agent in long-term Alzheimer's disease therapy.

Role of cGMP-dependent protein kinases for fear memory formation in the lateral amygdala

Fear memory formation critically relies on the function of the lateral amygdala (LA) and changes in synaptic transmission of its sensory inputs. There is evidence that signaling through NO/cGMP in the amygdala contributes to these processes [1-3]. The cGMP-dependent protein kinases (cGK) type I and II serve as universal effectors of the NO/cGMP signaling cascade. Recently, we established a functional role of cGKI for synaptic plasticity and fear memory consolidation in the amygdala. First, we demonstrated that the cGKIβ isoform is highly expressed in the LA. Furthermore, we showed that cGKI-deficient mice revealed deficits in amygdala-dependent fear memory consolidation. In contrast, short-term memory and hippocampus-dependent memory was intact in these mice. In line with the behavioral phenotype of impaired auditory long-term memory, we found reduced long-term potentiation (LTP) in the LA of cGKI-deficient mice, which is considered as a cellular mechanism for learning induced synaptic plasticity. Unlike cGKI-deficient mice, cGKII-knockout mice lack these phenotypes. These results clearly suggest a crucial role of cGKI for consolidation of amygdala-dependent fear memory, a process by which newly learned information is stabilized into long-term memory (LTM). It is a well known fact that memory consolidation strictly depends on transcriptional and translational processes which is also proved essential for amygdala-dependent fear memory consolidation [4,5]. A functional link between cGKI activity and gene transcription could already be demonstrated in vitro [6]. The upcoming question is if cGKI-induced transcription also takes place in amygdala neurons recruited during memory formation. Preliminary data indicate a role for cGKI in cAMP-response element binding protein (CREB) signaling in the LA, a prominent transcription factor involved in learning and memory.

Differential expression of mitogen-activated protein kinase signaling pathway in the hippocampus of rats exposed to chronic unpredictable stress

Much research has indicated that the mitogen-activated protein kinase (MAPK)-cAMP response element-binding protein (CREB) signal transduction pathway is involved in the pathophysiological mechanism of depression. But as to the question of which MAPKs are more relevant to stress effects, there is no definite answer. In the present study, 32 male Sprague–Dawley rats were divided into chronic unpredictable stress (CUS) and control groups, with 16 rats in each group. The CUS rats were exposed to 21-day chronic unpredictable stressors, and the controls were stress-free. After stress, 16 rats (8 in each group) were tested for spatial memory using Morris Water Maze, and 16 rats (8 from each group) were decapitated for detection of the three most extensively studied subgroups of MAPKs, ERK1/2, JNK and P38, and CREB in the hippocampus. The results showed that there was no statistical difference in the body weight between the two groups. The CUS rats showed impaired spatial memory in MWM. Western blot of hippocampus showed that CUS significantly decreased pCREB and pJNK levels, but there was no statistical difference between two groups in CREB, ERK1/2, pERK1/2, P38, pP38 and JNK levels. Immunohistochemistry showed that the reduced pCREB occurred in the dentate gyrus, not in the hippocampus proper. In conclusion, this study highlights that the JNK-CREB pathway, not the P38-CREB or ERK1/2-CREB pathway, in the hippocampus played an important role in the 21-day-CUS, and that the impaired spatial memory acquisition in the CUS rats can be restored to the level comparable to the pre-stressed state.

GABA-cAMP Response Element-Binding Protein Signaling Regulates Maturation and Survival of Newly Generated Neurons in the Adult Hippocampus

Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signaling pathways. Here, we investigate the role of cAMP response element-binding protein (CREB) signaling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous manner impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule-associated protein, doublecortin (DCX), and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects after loss of GABA-mediated excitation can be compensated by enhanced CREB signaling. These results indicate that CREB signaling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation.

Crucial role for Ser133-phosphorylated form of cyclic AMP-responsive element binding protein signaling in the differentiation and survival of neural progenitors under chronic cerebral hypoperfusion

Various stimuli, such as ischemia/hypoxia enhance newborn cell survival in the subventricular zone and their migration tangentially in chains toward the olfactory bulb. The present study assessed the fate of newborn neurons from subventricular zone to olfactory bulb under conditions of chronic cerebral hypoperfusion, and examined the role of cAMP-responsive element binding protein signaling on the survival of these neurons by using cilostazol, a potent inhibitor of type III phosphodiesterase. Rats underwent bilateral common carotid artery ligation. They were divided into sham-operated (n=70), vehicle- (n=70), and type III phosphodiesterase inhibitor–treated (n=70) groups. Immunohistochemically-stained section for 5-bromodeoxyuridine and a series of neuronal and glial markers were analyzed at days 7, 14, 21 and 28 after hypoperfusion. The reduction of olfactory bulb size gradually progressed in the vehicle group ( P<0.05), but not in the sham-operated and type III phosphodiesterase inhibitor-treated group. The subventricular zone of the vehicle-treated rats contained significantly larger numbers of newborn neuroblasts after hypoperfusion, compared with sham-operated rats ( P<0.05), but significantly lower numbers in the rostral migratory stream and olfactory bulb ( P<0.05). Treatment of rats with type III phosphodiesterase inhibitor increased the number of neuroblasts and enhanced the survival and differentiation of cells ( P<0.05). Phosphorylated cAMP-responsive element binding protein within neuroblasts was markedly decreased in the subventricular zone, rostral migratory stream, and olfactory bulb of vehicle-treated rats ( P<0.05), but treatment with type III phosphodiesterase inhibitor resulted in recovery of this expression throughout hypoperfusion, leading to enhanced neurogenesis ( P<0.05). These effects were abrogated by protein kinase A and C inhibitor. Our results indicated that cAMP-responsive element binding protein signaling is a key mediator of neurogenesis after prolonged hypoperfusion and provide the basis for new regenerative therapies for ischemic brain injury.

Rolipram, a Phosphodiesterase Type IV Inhibitor, Exacerbates Periventricular White Matter Lesions in Rat Pups

Periventricular white matter injury is the leading cause of cerebral palsy in premature infants for which no effective treatments are available. Our previous studies have demonstrated that pharmacological activation of the cAMP response element-binding protein (CREB) signaling pathway, before hypoxic-ischemia protected against neuronal injury in neonatal rats. We examined whether rolipram, a phosphodiesterase type IV inhibitor, treatment after hypoxic-ischemia is protective against white matter injury in neonatal rats. Rats were exposed to hypoxia-ischemia (HI) on P7 and then treated with daily injections of various doses of rolipram (P7-P11). Immunohistochemical staining for myelin basic protein, ED1, glial fibrillary acidic protein, CREB and O1 were examined on P11. We found that the periventricular white matter and deep cortical lesions were exacerbated by rolipram administration after HI injury. The lesions in the rolipram-treated group also showed increased astrogliosis and increased CREB phosphorylation in the activated microglia and astrocytes. Furthermore, the rolipram-posttreated HI group had markedly depleted preoligodendrocytes in the ipsilateral hemisphere, which may be related to decreased preoligodendrocytes proliferation after rolipram treatment per se. These data suggest that rolipram treatment after hypoxic-ischemia is not protective; in contrast, rolipram may exacerbate hypoxic-ischemic white matter injury in neonatal rat brains.

Steady‐state increase of cAMP‐response element binding protein, Rac, and PAK signaling in presenilin‐deficient neurons

Mutations in the genes encoding presenilins (PS1 and PS2) account for the majority of cases of early-onset Alzheimer's disease. PS1 and PS2 form the catalytic center of gamma-secretase, an enzyme responsible for intramembraneous proteolysis of several type I transmembrane proteins. Many gamma-secretase substrates are coupled to intracellular signaling events such as cAMP-response element binding protein and Rac1/p21-activated kinase pathways, which are associated with synaptic function. Here, we have examined the activation of these pathways in neurons lacking PS1 expression or gamma-secretase activity. We found evidence for heightened steady-state activation of cAMP-response element binding protein, Rac1, and p21-activated kinase signaling in PS-deficient neurons. Our study highlights the importance of PS-dependent proteolytic cleavage of gamma-secretase substrates in regulating neuronal signal transduction.

Deficits in amygdaloid cAMP-responsive element-binding protein signaling play a role in genetic predisposition to anxiety and alcoholism

Deficits in amygdaloid cAMP-responsive element-binding protein signaling play a role in genetic predisposition to anxiety and alcoholism

Intracerebral administration of protein kinase A or cAMP response element-binding protein antisense oligonucleotide can modulate amphetamine-mediated appetite suppression in free-moving rats

Although amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibitory action on neuropeptide Y (NPY), an appetite neurotransmitter abundant in the brain, molecular mechanisms underlying this effect are not well known. This study examined the possible role of protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling in this anorectic effect, and the results showed that both PKA and CREB mRNA levels in hypothalamus were increased following AMPH treatment, which was relevant to a reduction of NPY mRNA level. To determine whether PKA or CREB was involved in the anorectic response, intracerebroventricular infusions of antisense oligonucleotide (or missense control) were performed 60 min before daily AMPH treatment in conscious rats, and results showed that either PKA or CREB knockdown could block AMPH-induced anorexia as well as restore NPY mRNA level, indicating the respective involvement of PKA and CREB signaling in the regulation of NPY gene expression. It is suggested that hypothalamic PKA and CREB signaling may involve the central regulation of AMPH-mediated feeding suppression via the modulation of NPY gene expression.

Scientists Home In on Link Between Anxiety, Alcohol Use

Scientists Home In on Link Between Anxiety, Alcohol Use

Deficits in amygdaloid cAMP-responsive element–binding protein signaling play a role in genetic predisposition to anxiety and alcoholism

We investigated the role of cAMP-responsive element-binding protein (CREB) in genetic predisposition to anxiety and alcohol-drinking behaviors using alcohol-preferring (P) and -nonpreferring (NP) rats. The levels of CREB, phosphorylated CREB, and neuropeptide Y (NPY) were innately lower in the central amygdala (CeA) and medial amygdala (MeA), but not in the basolateral amygdala (BLA), of P rats compared with NP rats. P rats displayed higher baseline anxiety-like behaviors and consumed higher amounts of alcohol compared with NP rats. Ethanol injection or voluntary intake reduced the higher anxiety levels in P rats. Ethanol also increased CREB function in the CeA and MeA, but not in the BLA, of P rats. Infusion of the PKA activator Sp-cAMP or NPY into the CeA decreased the alcohol intake and anxiety-like behaviors of P rats. PKA activator infusion also increased CREB function in the CeA of P rats. On the other hand, ethanol injection or voluntary intake did not produce any changes either in anxiety levels or on CREB function in the amygdaloid structures of NP rats. Interestingly, infusion of the PKA inhibitor Rp-cAMP into the CeA provoked anxiety-like behaviors and increased alcohol intake in NP rats. PKA inhibitor decreased CREB function in the CeA of NP rats. These novel results provide the first evidence to our knowledge that decreased CREB function in the CeA may be operative in maintaining the high anxiety and excessive alcohol-drinking behaviors of P rats.

Association of CaV1.3 L-Type Calcium Channels with Shank

Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. Both CaV1.2 and CaV1.3 subunits contain class I PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-binding consensus at their C termini. In yeast two-hybrid screen of rat brain cDNA library with the C-terminal bait of CaV1.3a (long C-terminal splice variant) L-type Ca2+ channel subunit, we isolated multiple clones of postsynaptic adaptor protein Shank. We demonstrated a specific association of CaV1.3a C termini, but not of CaV1.2 C termini, with Shank PDZ domain in vitro. We further demonstrated that the proline-rich region present in C termini of CaV1.3a subunit binds to Shank Src homology 3 domain. We established that CaV1.3a and Shank localized to postsynaptic locations in cultured rat hippocampal neurons. By expressing epitope-tagged recombinant CaV1.3 subunits in rat hippocampal neuronal cultures, we demonstrated that the presence of Shank-binding motifs in CaV1.3a sequence is both necessary and sufficient for synaptic clustering of CaV1.3 L-type Ca2+ channels. In experiments with dominant-negative peptides and dihydropyridine-resistant CaV1.3a mutants, we demonstrated an importance of Shank-binding motif in CaV1.3a sequence for phosphorylated cAMP response element-binding protein (pCREB) signaling in cultured hippocampal neurons. Our results directly link CaV1.3 neuronal L-type Ca2+ channels to macromolecular signaling complex formed by Shank and other modular adaptor proteins at postsynaptic density and provide novel information about the role played by CaV1.3 L-type Ca2+ channels in pCREB signaling.

Effects of nefiracetam on the levels of brain-derived neurotrophic factor and synapsin I mRNA and protein in the hippocampus of microsphere-embolized rats

Our recent study demonstrated that nefiracetam, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, prevented impairment of the cyclic AMP (cAMP)/cAMP-responsive element binding (CREB) protein signaling pathway in sustained cerebral ischemia. The purpose of the present study was to determine whether nefiracetam has an effect on the expression of brain-derived neurotrophic factor (BDNF) and synapsin I mRNAs that are believed to be produced via CREB, and the alteration in their protein contents in the hippocampus after cerebral ischemia. Sustained cerebral ischemia was induced by injection of 700 microspheres into the right hemisphere of each rat. The rats were treated once daily with 10 mg/kg nefiracetam, p.o., from 15 h after the operation. Treatment with nefiracetam reduced the prolongation of the escape latency in the water maze test on days 7–9 after microsphere embolism-induced sustained cerebral ischemia, suggesting an improvement in the spatial learning function. Microsphere-embolized rats on day 5 showed decreases in BDNF and synapsin I mRNA levels and their protein contents in the ipsilateral hippocampus. Treatment with nefiracetam partially attenuated the decreases. These results suggest that enhancement of BDNF and synapsin I expression by nefiracetam treatment may be, at least in part, due to the improvement in the CREB binding activity, contributing to the prevention of learning and memory dysfunction after sustained cerebral ischemia.

Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2.

GABA-mediated fast-hyperpolarizing inhibition depends on extrusion of chloride by the neuron-specific K-Cl cotransporter, KCC2. Here we show that sustained interictal-like activity in hippocampal slices downregulates KCC2 mRNA and protein expression in CA1 pyramidal neurons, which leads to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF acting on tyrosine receptor kinase B (TrkB), with down-stream cascades involving both Shc/FRS-2 (src homology 2 domain containing transforming protein/FGF receptor substrate 2) and PLCgamma (phospholipase Cgamma)-cAMP response element-binding protein signaling. The plasmalemmal KCC2 has a very high rate of turnover, with a time frame that suggests a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. A downregulation of KCC2 may be a general early response involved in various kinds of neuronal trauma.

Loss of Kv3.1 tonotopicity and alterations in cAMP response element-binding protein signaling in central auditory neurons of hearing impaired mice.

The promoter for the kv3.1 potassium channel gene is regulated by a Ca2+-cAMP responsive element, which binds the transcription factor cAMP response element-binding protein (CREB). Kv3.1 is expressed in a tonotopic gradient within the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem, where Kv3.1 levels are highest at the medial end, which corresponds to high auditory frequencies. We have compared the levels of Kv3.1, CREB, and the phosphorylated form of CREB (pCREB) in a mouse strain that maintains good hearing throughout life, CBA/J (CBA), with one that suffers early cochlear hair cell loss, C57BL/6 (BL/6). A gradient of Kv3.1 immunoreactivity in the MNTB was detected in both young (6 week) and older (8 month) CBA mice. Although no gradient of CREB was detected, pCREB-immunopositive cells were grouped together in distinct clusters along the tonotopic axis. The same pattern of Kv3.1, CREB, and pCREB localization was also found in young BL/6 mice at a time (6 weeks) when hearing is normal. In contrast, at 8 months, when hearing is impaired, the gradient of Kv3.1 was abolished. Moreover, in the older BL/6 mice there was a decrease in CREB expression along the tonotopic axis, and the pattern of pCREB labeling appeared random, with no discrete clusters of pCREB-positive cells along the tonotopic axis. Our findings are consistent with the hypothesis that ongoing activity in auditory brainstem neurons is necessary for the maintenance of Kv3.1 tonotopicity through the CREB pathway.