GAP‐43 mRNA in growth cones is associated with HuD and ribosomes

The neuron‐specific ELAV/Hu family member, HuD, interacts with and stabilizes GAP‐43 mRNA in developing neurons, and leads to increased levels of GAP‐43 protein. As GAP‐43 protein is enriched in growth cones, it is of interest to determine if HuD and GAP‐43 mRNA are associated in developing growth cones. HuD granules in growth cones are found in the central domain that is rich in microtubules and ribosomes, in the peripheral domain with its actin network, and in filopodia. This distribution of HuD granules in growth cones is dependent on actin filaments but not on microtubules. GAP‐43 mRNA is localized in granules found in both the central and peripheral domains, but not in filopodia. Ribosomes were extensively colocalized with HuD and GAP‐43 mRNA granules in the central domain, consistent with a role in the control of GAP‐43 mRNA stability in the growth cone. Together, these results demonstrate that many of the components necessary for GAP‐43 mRNA translation/stabilization are present within growth cones. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

阻断NMDA受体可增强皮质酮对海马脑源性神经营养因子表达的抑制:cAMP反应元件结合蛋白的作用

高浓度的皮质酮可引起海马形态与功能的损伤,其中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) 表达的改变在海马形态与功能损伤中扮演重要角色。本实验的目的是观察单次皮下注射皮质酮后海马内BDNF-mRNA、前 体蛋白及成熟型蛋白表达的改变,并观察N-甲基-D-天冬氨酸(N-methyl-D-aspartate NMDA)受体阻滞剂MK801对皮质酮 作用的影响。实验结果显示,单次皮下注射皮质酮2 mg／kg,3 h后海马内BDNF mRNA、前体蛋白及成熟型蛋白的表达 均降低;MK801(0．1 mg／kg)对皮质酮的这一作用有增强效果。单独给予皮质酮或注射MK801 30 min后再给予皮质酮, 均能明显降低海马中cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)的磷酸化水平,MK801与 皮质酮联用时CREB的磷酸化水平降低更为显著(与单独给予皮质酮相比,P<0．05)。实验结果提示,CREB磷酸化水平降 低可能是皮质酮引起海马BDNF表达减少的重要中间环节,阻断NMDA受体可加强皮质酮降低BDNF表达的效应。     

Activity‐dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live‐cell imaging

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning‐related synaptic plasticity and potentiating N‐methyl‐D ‐aspartate (NMDA) receptor‐dependent signaling. These findings suggest that tPA may localize to, and undergo activity‐dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization‐induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense‐core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca⁺² concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse‐pinch‐linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA‐type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Cardiac β‐adrenergic receptor activation mediates distinct and cell type‐dependent changes in the expression and distribution of connexin 43

Activation of the sympatho‐β‐adrenergic receptors (β‐ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type‐dependent changes in Cx43 remodelling due to β‐AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β₂‐AR were used, which exhibited cardiac fibrosis and up‐regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β₂‐AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β₂‐antagonist ICI‐118551 or protein kinase A inhibitor H‐89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β‐AR activation‐induced production of IL‐18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β‐AR in mediating distinct and cell type‐dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.     

CSPGs inhibit axon branching by impairing mitochondria‐dependent regulation of actin dynamics and axonal translation

Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin‐dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria‐dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl‐l ‐carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

原位杂交检测大鼠前庭代偿中GAP-43 mRNA水平

用ＤＩＧ标记的ＧＡＰ－４３ｃＤＮＡ为探针,以大鼠海马切片作阳性对照,使用原位杂交方法检测了大鼠迷路损毁５,１２,２０和３０ｄ后前庭核区ＧＡＰ－４３ｍＲＮＡ水平的变化,结果表明,迷路损毁后前庭核区ｍＲＮＡ水平升高,原位杂交的应用,为前庭代偿中轴突发芽,突触重组的神经可塑性研究打下了方法学基础。     

The effect of postnatal manganese exposure on the NMDA receptor signaling pathway in rat hippocampus

Overexposure to manganese (Mn) is associated with neurological disorders in children. Evidence indicated that N‐methyl‐d ‐aspartate (NMDA) receptor signaling pathway was critical for neurobehavioral function. However, whether NMDA receptor signaling pathway contributes to Mn‐induced neurotoxicity remains unknown. In this study, newborn Sprague–Dawley rats were randomly assigned to four groups exposed to 0, 10, 20, and 30 mg/kg of Mn²⁺ by intraperitoneal injection (n = 10/group: five males and five females). After 3 weeks of Mn exposure, messenger RNA (mRNA) and protein expression of NMDA receptor subunits (NR1, NR2A, and NR2B), cAMP‐response element binding protein (CREB), and brain‐derived neurotrophic factor (BDNF) in hippocampus were measured by real‐time quantitative RT‐PCR and Western blot. In Mn‐exposed rats, decreased mRNA and protein expression of NR1, NR2A, and NR2B, CREB, and BDNF was observed. The results imply that downregulated NMDA receptor signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity.     

Differential regulation of NMDA receptor function by DJ‐1 and PINK1

Dysfunction of PTEN‐induced kinase 1 (PINK1) or DJ‐1 promotes neuronal death and is implicated in the pathogenesis of Parkinson’s disease, but the underlying mechanisms remain unclear. Given the roles of N‐methyl‐d‐ aspartate receptor (NMDAr)‐mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ‐1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr‐mediated whole‐cell currents by enhancing the function of NR2A‐containing NMDAr subtype (NR2ACNR). However, DJ‐1 decreased NMDAr‐mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B‐containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ‐1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ‐1‐dependent signal pathways DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ‐1, while promoted NMDA‐induced neuronal death through the overactivation of PTEN/NR2BCNR‐dependent cell death pathway, induced a neuroprotective effect to counteract DJ‐1 dysfunction‐mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival–promoting pathway. Thus, PINK1 acts with DJ‐1 in a common pathway to regulate NMDAr‐mediated neuronal death. This study suggests that the DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson’s disease.     

Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus

It has been proposed that assembly of the final NMDA receptor complex may be modified by prenatal ethanol exposure, resulting in long-term alterations of NMDA receptor pharmacology. We investigated the effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus. Female Sprague-Dawley rats were chronically intoxicated for 4 weeks with a 10% (v/v) ethanol solution administered throughout pregnancy and lactation. Hippocampus and cerebellum were isolated from pups (postnatal days 1-28) of the ethanol-exposed and ad libitum groups. Our results, using a semiquantitative RT-PCR technique, showed a selective effect of ethanol exposure on the various NMDA receptor subunits. Ethanol exposure significantly increased the levels of NR1(1XX), NR1(X11) and NR2(D) mRNAs on postnatal days 7 and 14 and decreased the level of NR2(C) on postnatal day 1. Immunoblot analyses demonstrated that NR2(D) protein levels were increased on postnatal day 7 after ethanol exposure. However, the developmental profile of mRNAs encoding for NR2(A-B), NR3(L/S), GBP and Gly/TCP-BP subunits were not affected. Moreover, no significant effects of ethanol exposure were observed on the developmental transition from expression of NR1(0XX) to NR(1XX) splice variants occurring in the cerebellum on postnatal day 19. Unexpectedly, [(3) H]MK-801 binding experiments showed that ethanol exposure increased the B (max) values of high-affinity sites on postnatal days 14 and 28, with no change of K (d) values. These findings indicate that prenatal and/or postnatal ethanol exposure alters the hippocampal levels of mRNAs encoding for certain subunits and the density of high-affinity [(3) H]MK-801 binding sites. As these subunits have been shown to modulate the functional properties of NMDA receptors, these results suggest that this altered expression could be involved in the neurodevelopmental disorders associated with fetal ethanol exposure.     

Tissue‐specific and light‐dependent regulation of phytochrome gene expression in rice

Phytochromes are red‐ and far red light photoreceptors in higher plants. Rice (Oryza sativa L.) has three phytochromes (phyA, phyB and phyC), which play distinct as well as cooperative roles in light perception. To gain a better understanding of individual phytochrome functions in rice, expression patterns of three phytochrome genes were characterized using promoter‐GUS fusion constructs. The phytochrome genes PHYA and PHYB showed distinct patterns of tissue‐ and developmental stage‐specific expression in rice. The PHYA promoter‐GUS was expressed in all leaf tissues in etiolated seedlings, while its expression was restricted to vascular bundles in expanded leaves of light‐grown seedlings. These observations suggest that light represses the expression of the PHYA gene in all cells except vascular bundle cells in rice seedlings. Red light was effective, but far red light was ineffective in gene repression, and red light‐induced repression was not observed in phyB mutants. These results indicate that phyB is involved in light‐dependent and tissue‐specific repression of the PHYA gene in rice.     

Profiling the genes affected by pathogenic TDP‐43 in astrocytes

Mutation in TAR DNA binding protein 43 (TDP‐43) is a causative factor of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurodegeneration may not require the presence of pathogenic TDP‐43 in all types of relevant cells. Rather, expression of pathogenic TDP‐43 in neurons or astrocytes alone is sufficient to cause cell‐autonomous or non‐cell‐autonomous neuron death in transgenic rats. How pathogenic TDP‐43 in astrocytes causes non‐cell‐autonomous neuron death, however, is not clear. Here, we examined the effect of pathogenic TDP‐43 on gene expression in astrocytes. Microarray assay revealed that pathogenic TDP‐43 in astrocytes preferentially altered expression of the genes encoding secretory proteins. Whereas neurotrophic genes were down‐regulated, neurotoxic genes were up‐regulated. Representative genes Lcn2 and chitinase‐3‐like protein 1 were markedly up‐regulated in astrocytes from primary culture and intact transgenic rats. Furthermore, synthetic chitinase‐3‐like protein 1 induced neuron death in a dose‐dependent manner. Our results suggest that TDP‐43 pathogenesis is associated with the simultaneous induction of multiple neurotoxic genes in astrocytes, which may synergistically produce adverse effects on neuronal survival and contribute to non‐cell‐autonomous neuron death.

     

Age‐dependent alterations of the NMDA receptor developmental profile and adult behavior in postnatally ketamine‐treated mice

Ketamine is a NMDA receptor (NMDAR) antagonist used in pediatric anesthesia. Given the role of glutamatergic signaling during brain maturation, we studied the effects of a single ketamine injection (40 mg/kg s.c) in mouse neonates depending on postnatal age at injection (P2, P5, or P10) on cortical NMDAR subunits expression and association with Membrane‐Associated Guanylate Kinases PSD95 and SAP102. The effects of ketamine injection at P2, P5, or P10 on motor activity were compared in adulthood. Ketamine increased GluN2A and GluN2B mRNA levels in P2‐treated mice without change in proteins, while it decreased GluN2B protein in P10‐treated mice without change in mRNA. Ketamine reduced GluN2A mRNA and protein levels in P5‐treated mice without change in GluN2B and GluN1. Ketamine affected the GluN2A/PSD95 association regardless of the age at injection, while GluN2B/PSD95 association was enhanced only in P5‐treated mice. Microdissection of ketamine‐treated mouse cortex showed a decrease in GluN2A mRNA level in superficial layers (I–IV) and an increase in all subunit expressions in deep layers (V–VI) in P5‐ and P10‐treated mice, respectively. Our data suggest that ketamine impairs cortical NMDAR subunit developmental profile and delays the synaptic targeting of GluN2A‐enriched NMDAR. Ketamine injection at P2 or P10 resulted in hyperlocomotion in adult male mice in an open field, without change in females. Voluntary running‐wheel exercise showed age‐ and sex‐dependent alterations of the mouse activity, especially during the dark phase. Overall, a single neonatal ketamine exposure led to short‐term NMDAR cortical developmental profile impairments and long‐term motor activity alterations persisting in adulthood. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 315–333, 2015     

Sociability development in mice with cell‐specific deletion of the NMDA receptor NR1 subunit gene

Social affiliative behavior is an important component of everyday life in many species and is likely to be disrupted in disabling ways in various neurodevelopmental and neuropsychiatric disorders. Therefore, determining the mechanisms involved in these processes is crucial. A link between N‐methyl‐d ‐aspartate (NMDA) receptor function and social behaviors has been clearly established. The cell types in which NMDA receptors are critical for social affiliative behavior, however, remain unclear. Here, we use mice carrying a conditional allele of the NMDA R1 subunit to address this question. Mice bearing a floxed NMDAR1 (NR1) allele were crossed with transgenic calcium/calmodulin‐dependent kinase IIα (CaMKIIα)‐Cre mice or parvalbumin (PV)‐Cre mice targeting postnatal excitatory forebrain or PV‐expressing interneurons, respectively, and assessed using the three‐chambered Social Approach Test. We found that deletion of NR1 in PV‐positive interneurons had no effect on social sniffing, but deletion of NR1 in glutamatergic pyramidal cells resulted in a significant increase in social approach behavior, regardless of age or sex. Therefore, forebrain excitatory neurons expressing NR1 play an important role in regulating social affiliative behavior.