Synaptic strength regulated by palmitate cycling on PSD-95

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.     

Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95

The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Here we report that phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. We present evidence that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibited NMDA-induced AMPA receptor internalization and blocked the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295.     

Spatiotemporal Expression of PSD-95 and nNOS After Rat Sciatic Nerve Injury

Neuronal nitric oxide synthase (nNOS) has been implicated to influence peripheral nerve lesion and regeneration. Post-synaptic density-95 (PSD-95) is one of nNOS-anchoring proteins and plays an important role in specifying the sites of reaction of NO in nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of PSD-95 and nNOS. At gene levels, PSD-95 mRNA diminished shortly after crush, and significantly elevated from 2 days to 2 weeks, whereas nNOS decreased progressively post-operation, reached the valley at 1 day, and markedly up-regulated from 1 to 2 weeks after SNC. The expression of both molecules returned to the control level at 4 weeks post-injury. At protein levels, PSD-95 and nNOS underwent the similar changes as their gene expression except for a time lag during up-regulating. At their peak expression, PSD-95 co-labeled with nNOS in Schwann cells (SCs) of sciatic nerve within 0.5 mm from the lesion site, but had few colocalization in axons. In addition, the interaction between PSD-95 and nNOS enhanced significantly at 2 weeks after SNC. These results suggest a correlation of PSD-95 up-regulation with nNOS in reactive SCs of crushed sciatic nerve, which may lead to understanding the function of PSD-95 during peripheral nerve regeneration. Shangfeng Gao and Min Fei contributed equally to this work.     

Lentiviral Expression of Rabies Virus Glycoprotein in the Rat Hippocampus Strengthens Synaptic Plasticity

Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two μl (10⁸ T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat’s brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.

     

Effects of a Cardiotonic Medicine,Danshen Pills,on Cognitive Ability and Expression of PSD-95 in a Vascular Dementia Rat Model

A widely used Chinese cardiotonic proprietary medicine, compound Danshen dripping pills (CDDP, Fufang Danshen Diwan) has also begun to be used for treatment of vascular dementia (VaD). We tried to explore the mechanism of CDDP action in this case. A VaD experimental model was built in rats by bilateral ligation of the common carotid arteries. The cognitive ability of experimental animals was evaluated in the Morris water maze test. Synaptic ultrastructural changes in the hippocampus were detected by transmission electron microscopy; expression of PSD-95 mRNA in the hippocampus was examined using hybridization in situ. The latter index (mRNA expression) in the VaD group was significantly lower than those in the CDDP and sham-operated groups (P < 0.05). CDDP treatment considerably improved disturbed ultrastructural synaptic characteristics in the hippocampus of VaD rats. The mean escape latency in the Morris water maze test was significantly shorter in CDDP-treated VaD rats, compared with that those of the VaD group (P < 0.05). In the CDDP group compared to the VaD one, escape strategies improved from edge and random searches to more linear swim pathway (P < 0.05). Thus, decreasing expression of PSD-95 plays an important role in the pathogenesis of VaD. CDDP treatment improves the learning and memory ability of VaD rats by improving neural synaptic ultrastructural characteristics and increasing expression of PSD-95 mRNA in the hippocampus.     

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.     

小鼠海马内HDAC2的表达及其与NMDA受体、PSD-95的共定位

目的探讨组蛋白去乙酰化酶2（HDAC2）在成年C57BL/6小鼠海马内的分布及其与突触后致密区（PSD）蛋白成员的共定位,为揭示HDAC2与PSD蛋白复合物之间的内在联系及在海马相关的学习记忆过程中可能起到的调控作用提供形态学依据。方法应用免疫组化方法观察HDAC2在C57BL/6小鼠海马各区的表达分布。应用免疫荧光双标技术研究HDAC2与PSD蛋白成员N-甲基-D-天冬氨酸（NMDA）受体亚单位1（NR1）、PSD-95之间是否存在共定位。结果 HDAC2在小鼠海马CA1～CA3区锥体细胞和齿状回颗粒细胞均具有明显表达,而在各区的始层、辐射层、腔隙-分子层以及齿状回多形细胞层表达均较少。免疫荧光双标染色图片的重叠表明,HDAC2与NR1、PSD-95在小鼠海马CA1～CA3区锥体细胞层和齿状回颗粒细胞层内均可见显著共表达现象,其他区域偶见散在分布的双染神经元。结论 HDAC2在小鼠海马锥体细胞层和颗粒细胞层表达丰富,并与PSD蛋白成员间存在共定位现象。本实验结果为探讨HDAC2对谷氨酸能突触后神经元依赖的突触可塑性的调节机制提供了形态学依据。     

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by a tyrosine-based trafficking signal

Synaptic function requires proper localization of proteins at synaptic sites. Targeting of the postsynaptic density protein 95 (PSD-95) relies on multiple signals within the protein, including twelve C-terminal amino acids. We now show that this C-terminal targeting domain of PSD-95 mediates postsynaptic localization through a short tyrosine-based motif followed by a pair of hydrophobic amino acids. Consistent with a role in cellular trafficking, the tyrosine motif resembles the canonical motif for interactions with clathrin adaptor proteins. In fact, we find that the C-terminal targeting domain of PSD-95 is sufficient to mediate clathrin-dependent endocytosis when appended to a transmembrane protein. Furthermore, systematic mutagenesis reveals that endocytosis mediated by this domain depends on both the tyrosine motif and the dihydrophobic amino acid pair. Thus, postsynaptic targeting of PSD-95 requires a tyrosine-based signal that can mediate clathrin-coated vesicle formation.     

Effects of Fluoride on Expression of P450, CREM and ACT Proteins in Rat Testes

Fluoride (F) is an essential trace element that humans and animals ingest from water, air, and fluoride-containing products; however, excessive fluoride absorption can damage a variety of organs and tissues, including the male reproductive system. Our previous studies found that fluoride exposure lowered sperm quality and interfered with spermatogenesis; however, the exact mechanism remained unclear. Proteins cytochrome P450 (P450), cAMP-responsive element modulator (CREM), and activator of CREM in testis (ACT) play the key roles in spermatogenesis and sperm motility. To investigate whether fluoride affects the expression of P450, CREM, and ACT, we used immunohistochemical techniques to determine expression levels of these proteins in testes of rats administered 100 mg NaF/L for 2 weeks via drinking water. The results showed that P450 expression was decreased while CREM and ACT expression was increased in the fluoride group, compared to the control. These data suggest that fluoride can impair male reproduction by affecting expression of P450, CREM, and ACT in the testes.     

Synaptic Membrane Antigens in Developing Rat Brain Cerebral Cortex and Cerebellum

Abstract: The contents of five synaptic membrane antigens (56K, 58K, 62K, 63K, and 64K) were determined in rat cerebral cortex and cerebellum at eight developmental time points: E9, E14, P < 1, P5, P14, P28, P60, and P180 (E, embryonic; P, postnatal). In cerebral cortex, the five antigens showed five different developmental patterns with respect both to specific content (i.e., quantity per unit of membrane) and total content (i.e., quantity per cortex). The 56K, 58K, and 62K polypeptides were first detected at E14, increased slightly to P5, then increased rapidly from P5 to P28 by 14-, 11-, and 18-fold, respectively. From P28 to PI80, the patterns of these antigens showed very large differences. The 63K and 64K antigens were first detected at P14 and P28, respectively. The specific content of 63K antigen continued to increase steadily throughout adult life; in contrast, the specific content of the 64K antigen did not change appreciably. In cerebellum only three antigens (56K, 58K, and 62K) were detected. These three antigens showed different developmental patterns. The 56K polypeptide was first detected at E14; its specific content increased very rapidly to a maximum at P < 1; it then decreased, first slowly, and then more rapidly, disappearing at P60. The 58K polypeptide also was detectable at E14 and increased very rapidly to a maximum at P < 1. It then decreased markedly to P5, followed by an increase, returning almost to its maximum level at P14. It then slowly decreased disappearing at P180. The 62K antigen was first detected at P14 and then it slowly decreased with disappearance at P60. The patterns with respect to total contents per cerebellum were similar for the three antigens, with a maximum at P28. We conclude that the highest increase in the contents of these antigens roughly corresponds to the period of maximal synaptogenesis (P9 to P28) in both regions. Differences among developmental patterns probably reflect changing molecular machinery required for development and functional differentiation of synapses in different brain regions. The fine structure of these patterns suggests that the quantitative measurement of synaptic membrane antigens will be useful for delineating complex processes occurring during synaptogenesis.     

大鼠脑突触质膜糖皮质激素受体的纯化

本文利用抗大鼠肝细胞内糖皮质激素受体的单克隆抗体制备的免疫亲和层析柱,将大鼠脑突触质膜糖皮质激素受体纯化了约1150倍,SDS聚丙烯酰胺簿层梯度凝胶电泳显示,在约67kD处有一较明显的染色条带。     

Membrane Fluidity in Senescing Apples: Effects of Temperature and Calcium

Fluorescence polarization values of 1,6-diphenyl-2,3,5-hexatriene(DPH) in microsomal membranes from cold stored (2?C) untreatedand calcium-treated apples were lower than those obtained inmembranes from apples stored at room temperature. Calcium treatmentof apples appeared to reduce the increase in polarization ofDPH in membranes which occurred after storing apples at roomtemperature. ¹Scientific Paper No. SP 6785, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ²Supported by Washington State Tree Fruit Research CommissionGrant to B.W.P. ³Calcium infusion process has been patented by B. W. Poovaiahand G. A. Moulton and assigned to Washington State UniversityResearch Foundation. United States Patent Number 4,331,691,issued May 25, 1982. (Received February 6, 1984; Accepted May 25, 1984)     

Neurotoxic Effects of Methamphetamine on Rat Hippocampus Pyramidal Neurons

Methamphetamine (MAP) is known to alter behavior and cause deficits in learning and memory. While the major site of action of MAP is on mesolimbic dopaminergic pathways, the effects on learning and memory raise the possibility of important actions in the hippocampus. We have studied electrophysiologic and morphologic effects of MAP in the CA1 region of hippocampus from young male rats chronically exposed to MAP, male rats exposed during gestation only and the effects of bath perfusion of MAP onto brain slices from control rats. Pyramidal neurons in brain slices from chronically exposed rats had reduced membrane potential and membrane resistance. Long-term potentiation (LTP) was reduced as compared to control, but when MAP was acutely perfused over control slices the amplitude of LTP was increased. LTP in young adult animals that had been gestationally exposed to MAP showed reduced LTP as compared to controls. Morphologically CA1 pyramidal neurons in chronically exposed animals showed a high prevalence of extensive blebbing of dendrites. We conclude that the NMDA receptor and the process of LTP are also targets of MAP dysfunction, at least in the hippocampus.