Corticotropin‐releasing hormone‐binding protein is up‐regulated by brain‐derived neurotrophic factor and is secreted in an activity‐dependent manner in rat cerebral cortical neurons

A recent study revealed that corticotropin‐releasing hormone (CRH) in the cerebral cortex (CTX) plays a regulatory role in emotional behaviors in rodents. Given the functional interaction between brain‐derived neurotrophic factor (BDNF) and the CRH‐signaling pathway in the hypothalamic‐pituitary‐adrenal axis, we hypothesized that BDNF may regulate gene expression of CRH and its related molecules in the CTX. Findings of real‐time quantitative PCR (RT‐qPCR) indicated that stimulation of cultured rat cortical neurons with BDNF led to marked elevations in the mRNA levels of CRH and CRH‐binding protein (CRH‐BP). The BDNF‐induced up‐regulation of CRH‐BP mRNA was attenuated by inhibitors of tropomyosin related kinase (Trk) and MEK, but not by an inhibitor for PI3K and Phospholipase C gamma (PLCγ). The up‐regulation was partially blocked by an inhibitor of lysine‐specific demethylase (KDM) 6B. Fluorescent imaging identified the vesicular pattern of pH‐sensitive green fluorescent protein‐fused CRH‐BP (CRH‐BP‐pHluorin), which co‐localized with mCherry‐tagged BDNF in cortical neurons. In addition, live‐cell imaging detected drastic increases of pHluorin fluorescence in neurites upon membrane depolarization. Finally, we confirmed that tetrodotoxin partially attenuated the BDNF‐induced up‐regulation of CRH‐BP mRNA, but not that of the protein. These observations indicate the following: In cortical neurons, BDNF led to gene expression of CRH‐BP and CRH. TrkB, MEK, presumably ERK, and KDM6B are involved in the BDNF‐induced gene expression of CRH‐BP, and BDNF is able to induce the up‐regulation in a neuronal activity‐independent manner. It is suggested that CRH‐BP is stored into BDNF‐containing secretory granules in cortical neurons, and is secreted in response to membrane depolarization.

     

脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

目的：探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法：健康雄性SD大鼠60只,随机分为：假手术（Sham）组（n=10）,脑缺血和缺血/再灌注模型组（n=50）.模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果：脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组（P<0.01）;缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组（P<0.05）;缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组（P<0.01）。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异（P<0.01）。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组（P<0.05）。HE染色显示：仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论：局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。     

Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology

Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology. About 131 spots were recognized as phosphorylated proteins as they showed different migration behaviour after phosphatase treatment. The analysis of 42 selected spots was carried out by LC/MS/MS technology resulting in the identification of two new phosphoproteins.     

Caspase-dependent and -independent cell death induced by 3-nitropropionic acid in rat cortical neurons

Mitochondria play a critical role in cell death by releasing apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), from the intermembrane space into the cytoplasm. Because mitochondrial dysfunction has been shown to be involved in several neurodegenerative diseases, mitochondrial toxins are largely used to model these disorders. These include 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, which has been used to model Huntington's disease and was previously reported by us to induce apoptotic cell death through caspase activation. In the present study, we evaluated the involvement of caspase-independent neuronal cell death induced by 3-NP (1 mM) and the effect of z-VDVAD-fmk, an inhibitor of caspase-2, using cortical neurons in culture. Our results highly suggest that 3-NP induces both caspase-dependent and -independent cell death. We showed that z-VDVAD-fmk prevented both caspase-2 and -3-like activities evoked by 3-NP, but only partly prevented chromatin fragmentation/condensation. However, z-VDVAD-fmk did not avoid 3-NP-induced release of cytochrome c or AIF from mitochondria nor did it affect the levels of mitochondrial Bax. Furthermore, 3-NP-mediated decrease in plasma membrane integrity was not affected by z-VDVAD-fmk. Under these conditions, the inhibitor prevented the caspase-dependent cell death.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.     

Dopamine D1 receptor signaling system regulates ryanodine receptor expression after intermittent exposure to methamphetamine in primary cultures of midbrain and cerebral cortical neurons

Regulatory mechanisms of ryanodine receptor (RyR) expression are not well known, although methamphetamine (METH) has been reported to up-regulate RyRs in mouse brain. This study investigate regulatory mechanisms of RyR expression by dopaminergic system using the midbrain and cerebral cortical neurons in primary culture intermittently exposed to METH and dopamine receptor (DR) agonists (1 h/day, for 3 days). Intermittent METH (10 μM) exposure enhanced RyR-1 and -2 proteins and their mRNA, but not RyR-3 expression in the both types of the neurons. These METH-induced increases of RyR proteins and their mRNA were dose-dependently blocked by SCH23390 (a selective D(1) DR antagonist), but not a D(2)DR antagonist sulpiride, suggesting a regulatory role of D(1)DRs in RyR expression by METH in these neurons. In cerebral cortical neurons, intermittent SKF82958 (a selective D(1)DR agonist) exposure increased RyR-1 and -2 proteins and their mRNA, whereas quinpirole (a selective D(2)DR agonist) showed no effects. KT5720, a protein kinase A inhibitor, dose-dependently attenuated the METH-stimulated RyR-1 and -2 expressions in cerebral cortical neurons. METH significantly increased phosphorylation of cAMP-response element-binding protein, which was completely suppressed by SCH23390. These results indicate that RyR-1 and -2 expressions are regulated by D(1)DRs via the signal transduction linked to D(1)DRs.     

Co-localization and interaction of DPYSL3 and GAP43 in primary cortical neurons

Dihydropyrimidinase-like 3 (DPYSL3) and GAP43 are both involved in neurite outgrowth, a crucial process for the differentiation of neurons. The present study shows for the first time that DPYSL3 co-localizes with GAP43 in primary cortical neurons. Further co-immunoprecipitation and overlay assay showed the ability of both recombinant and endogenous DPYSL3 to bind GAP43, indicating a specific interaction between DPYSL3 and GAP43 in primary cortical neurons.     

Angiotensin II regulates tyrosine hydroxylase activity and mRNA expression in rat mediobasal hypothalamic cultures: the role of specific protein kinases

Dopamine secreted by hypothalamic neurons is crucial in regulating prolactin secretion from the pituitary. We have examined the ability of angiotensin II (AngII) to regulate the activity of these dopaminergic neurons and thus act as a potential physiological regulator of prolactin secretion. Using a hypothalamic cell culture preparation we determined the effect of AngII on tyrosine hydroxylase activity and expression (TOH). This is important because TOH is the rate-limiting enzyme in dopamine biosynthesis. AngII stimulated a time- and concentration-dependent increase in TOH activity which was suppressed by inhibitors able to act on protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMPKII). An inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD 98059, reduced basal TOH activity but the AngII response was still detectable. AngII stimulation enhanced the phosphorylation of TOH at Ser19, Ser31 and Ser40. AngII also induced a time-dependent increase in TOH mRNA expression which was unaffected by inhibitors able to act on PKA and CaMPKII, but was abolished by inhibitors able to act on ERK and PKC. AngII responses were very much larger in cultures prepared from female when compared to male rat pups. Data from adult hypothalamic slices confirmed this sexual dimorphism and supported the role of the protein kinases noted above. Therefore AngII can regulate both the activity and expression of TOH in hypothalamic neurons employing multiple, but only partially overlapping, signaling pathways.     

Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: Involvement of PI3K/Akt and GSK-3 beta pathway

Erythropoietin (EPO) prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the neuroprotective effect of EPO in ketamine-induced neurotoxicity in primary cortical neurons. EPO in combination with ketamine greatly increased the cell viability and reduced the number of TUNEL-positive cells. To elucidate a possible mechanism by which EPO exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase pathway using LY294002. The neuroprotection of EPO was prevented by LY294002. Immunoblotting revealed that EPO induced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3β). Moreover, the caspase-3-like activity was increased by addition of ketamine, and decreased by administration of ketamine with EPO. Decreased caspase-3-like activity by administration of ketamine with EPO was restored by LY294002. Our results suggest that PI3K/Akt and GSK-3β pathway are involved in the neuroprotective effect of EPO. You Shang and Yan Wu have contributed equally to this work.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles

Little is known about the direction and specificity of protein adsorption to solid surfaces, a knowledge that is of great importance in many biotechnological applications. To resolve the direction in which a protein with known structure and surface potentials binds to negatively charged silica nanoparticles, fluorescent probes were attached to different areas on the surface of the protein human carbonic anhydrase II. By this approach it was clearly demonstrated that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein. Furthermore, the adsorption direction is strongly pH-dependent. At pH 6.3, a histidine-rich area around position 10 is the dominating adsorption region. At higher pH values, when the histidines in this area are deprotonated, the protein is also adsorbed by a region close to position 37, which contains several lysines and arginines. Clearly the adsorption is directed by positively charged areas on the protein surface toward the negatively charged silica surface at conditions when specific binding occurs.     

Serotonin 5-HT receptor blockade enhances Ca(2+)/calmodulin-dependent protein kinase II function and membrane expression of AMPA receptor subunits in the rat hippocampus: implications for memory formation

Stimulation of hippocampal 5-HT(1A) receptors impairs memory retention. The highly selective 5-HT(1A) antagonist, WAY-100635, prevents the cognitive deficits induced not only by 5-HT(1A) stimulation but also by cholinergic or NMDA receptor blockade. On this basis, the effects of WAY-100635 on molecular events associated with memory storage were explored. In rat hippocampus, WAY-100635 produced a rapid increase in phosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and in Ca(2+)-independent CaMKII and protein kinase A (PKA) enzyme activity. This increase was followed a few hours later by an enhanced membrane expression of AMPA receptor subunits, especially of the GluR1 subunit phosphorylated at the CaMKII site, pGluR1(Ser831). The same qualitative effects were found with the weaker 5-HT(1A) antagonist NAN-190. The effects of both antagonists were no longer apparent in rats with a previous 5-HT depletion induced by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), suggesting that 5-HT(1A) receptor blockade removes the tonic inhibition of 5-HT through 5-HT(1A) receptor stimulation on excitatory hippocampal neurons, with the consequent increase in PKA activity. In addition, administration of WAY-100635 potentiated the learning-specific increase in the hippocampus of phospho-CaMKII, Ca(2+)-independent CaMKII activity, as well as the phosphorylation of either the CaMKII or the PKA site on the AMPA receptor GluR1 subunit. This study suggests that blockade of hippocampal 5-HT(1A) receptors favours molecular events critically involved in memory formation, and provides an in vivo molecular basis for the proposed utility of 5-HT(1A) receptor antagonists in the treatment of cognitive disorders.     

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT2 receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

Alterations in protein and gene expression within the barrel cortices of ZnT3 knockout mice: experience-independent and dependent changes

Much evidence exists for the involvement of vesicular zinc in neurotransmission and cortical plasticity. Recent studies have reported that mice deficient in zinc transporter-3 protein (ZnT3) and thus, vesicular zinc, have significant behavioural and biochemical deficits. Here, we examined whether phenotypic differences existed in the barrel cortices of ZnT3 KO mice using functional proteomics and quantitative PCR. Additionally, by manipulating whisker input, we also investigated experience-dependent changes in protein and gene expression, thereby assaying how cortical plasticity is different in the absence of vesicular zinc. The GABA metabolizing protein ABAT was observed in lower abundances consistently in KO mice. Several presynaptic proteins were identified that were abundant in differing amounts between the WT and KO groups in an experience-dependent manner. At baseline, we observed a decrease in the relative expression of Dlg4, Grin2a, Mt3, and Ntrkb genes in KO mice. The reduced expression of Nrtkb persisted with whisker plucking. These data demonstrate that fundamental changes in the expression of proteins and genes important in neurotransmission occur in the absence of vesicular zinc. Furthermore, the complement of experience-dependent changes were different between WT and KO mice, indicating that the lack of vesicular zinc affects the process of cortical plasticity.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr_Z were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr_Z/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr_Z by P₆₈₀^+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn₄CaO₅ cluster in the S₂ and S₃ states could weaken the strength of the H-bond interaction between Tyr_Z^● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr_Z.