沙棘油对高脂小鼠诱发阿尔兹海默症的预防作用

目的:通过检测沙棘油作用高脂小鼠海马神经元内微管相关蛋白(Tau)及脑源性神经营养因子(BDNF)的表达水平,探讨沙棘油对高脂小鼠并发阿尔兹海默综合征的预防作用。方法:40只KM小鼠,随机取10只为正常对照组;30只以高脂饲料喂养建立高脂模型(HF),按10 mg/kg以生理盐水(阴性对照)、沙棘油(实验)、辛伐他丁(阳性对照)灌胃3 w。取小鼠海马组织进行HE染色、免疫组织化学检测和蛋白印迹分析,检测不同组别小鼠海马神经元内Tau蛋白及BDNF表达的变化。结果:高脂模型组与正常组比较,海马神经元结构在光镜下有明显差别;阴性对照组小鼠海马神经细胞数目减少,神经元内有黄色颗粒样沉淀;实验及阳性组海马损伤有改善,斑块状淀粉样蛋白减少;免疫组化及蛋白印迹显示各组间两种蛋白表达水平不同。结论:沙棘油对高脂小鼠海马体内Tau蛋白表达有抑制作用,加速淀粉样前体蛋白的代谢,降低了由β-淀粉样蛋白沉积诱发阿尔茨海默病的风险;而对BDNF表达有促进作用,能防止神经元受损伤死亡、改善神经元的病理状态、促进受损伤神经元再生。即沙棘油能有效预防高脂人群并发阿尔兹海默综合征。     

An ion-pair reversed-phase HPLC method for determination of fresh tissue adenine nucleotides avoiding freeze-thaw degradation of ATP

Knowledge of the energetic state of tissue is required in a wide range of experimental studies, particularly those investigating the decline and recovery of cellular metabolism after metabolic stress. Such information can be obtained from high-performance liquid chromatography (HPLC) determination of tissue levels of adenine nucleotides (ATP, ADP, and AMP) and their interrelationship in the tissue energy charge (EC). Accordingly, a large range of techniques with which to measure these molecules and their downstream metabolites have been reported. However, the accurate determination of the tissue EC also depends on the nucleotide extraction procedure given that changes in adenine nucleotide levels take place very quickly when ATPases are not inactivated immediately. In this article, we describe an ion-pair reversed-phase HPLC method by which separation of adenine nucleotides can be performed rapidly, allowing multiple analyses in 1 day, with both high sensitivity and extraction efficiency and using fresh samples, thereby avoiding freeze-thaw degradation of nucleotides. We applied this method to hippocampal brain slice extracts and show that same-day extraction and analysis results in a more accurate determination of the in situ energetic state than does the commonly used snap-freezing in liquid nitrogen.     

Sexual dimorphism in the induction of LTP: critical role of tetanizing stimulation

Numerous studies have suggested that sexual dimorphism may exist in learning and memory, particularly in types involving the hippocampus. In the present study, we examined the effects of two different tetani on the induction of long-term potentiation in the CA1 region of hippocampal slices from adult female and male rats to determine the sexual differences in their responses to tetanizing stimulation. We found that the induction of LTP is sex-dependent, and that there were clear sexual differences in the responses to different tetanus patterns, but not impulse number or stimulation frequency. Multiple trains of tetani were more effective in the indution of LTP in male rats than in female ones. These findings suggest that male rats can react to a broader range of tetanizing stimulation compared with female rats. Based on our results and the findings of other studies, we propose that the interaction of gonadal hormones with Ca2+/NMDAR and the subsequent regulation of the ERK/MAP kinase pathway are critical mechanisms for sexual dimorphism in the induction of LTP.     

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.     

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.     

Effects of Glucose, Insulin, and Supernatant from Pancreatic β-cells on Brain–Pancreas Relative Protein in Rat Hippocampus

Brain–pancreas relative protein (BPRP) is a novel protein that mainly expresses in brain and pancreas. In our previous study, we found that various stressors significantly decreased the expression of BPRP in pancreas in vivo, accompanied by changes in insulin and glucose levels, and that expression of BPRP in pancreas also decreased significantly in diabetic rats induced by Streptozocin (STZ). All these findings suggest that BPRP may be a glucose or insulin-sensitive protein. However, how the changes in insulin or glucose levels influence the expression of BPRP in hippocampus requires further study. Here, we investigated the effects of insulin or glucose on the expression of BPRP in primary cultured hippocampal neurons. We supplied hippocampal neurons with glucose, insulin, or supernatant from pancreatic β-cells, which secrete insulin into the supernatant. Our data showed that insulin had beneficial effect on the viability while no significant effect on the expression of BPRP in hippocampal neurons. On the contrary, 40 mM glucose or free glucose culture significantly decreased the expression of BPRP, while had no significant effect on the viability and apoptosis of hippocampal neurons. Further study showed that levels of insulin in the supernatant collected from pancreatic β-cells medium changed over days, and that supernatant increased the viability of hippocampal neurons, while it had no obvious effect on the expression of BPRP in hippocampal neurons. These results suggest that BPRP may be a glucose-sensitive protein.     

An interaction between benzodiazepines and neuroactive steroids at GABA A receptors in cultured hippocampal neurons

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA_A receptors, accumulates in the brains of HE patients where it can potentiate GABA_A receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA_A mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03–0.3 μM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 μM). In contrast, flumazenil (10 μM) had no effect on the ability of allopregnanolone to potentiate GABA_A currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 μM) from 1.5 ± 0.7 to 0.1 ± 0.04 Hz. This action was partially reversed by Ro15-4513 (10 μM) but was not significantly influenced by flumazenil (10 μM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA_A receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.     

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K⁺ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A₁ receptor antagonist 8-cyclo-pentyltheophylline. In the same synaptosomal preparation, K⁺ (30 mM) strongly stimulated the release of the preloaded [³H]adenosine in a partially Ca²⁺-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both l -glutamate and l -aspartate enhanced the release of [³H]adenosine derivatives ([³H]ADD). The gluta-mate-evoked release was dose dependent and appeared to be Ca²⁺ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d -glutamate and d -aspartate were able to stimulate [³H]ADD release. In contrast, the specific glutamate agonists N-methyl-d -aspartate, kainate, and quisqualate failed to stimulate [³H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg²⁺ and l -glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6–7-dinitroquinoxaline-2, 3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ-anunobutyric acid stimulated [³H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.     

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons

Abstract: The ability of various stimuli to convert Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) into a Ca²⁺-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca²⁺] ([Ca²⁺]_o) (～50 nM) and then shifted to normal [Ca²⁺]_o (～1.26 mM) by addition of CaCl₂ (referred to as “Ca²⁺ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K⁺] or treatment with glutamate or a Ca²⁺ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca²⁺-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca²⁺ stimulation, ³²P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr²⁸⁶Thr²⁸⁷ of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca²⁺ stimulation was accompanied by only a small rise in intracellular [Ca²⁺]. Inhibitor studies showed that Na⁺-dependent action potentials and Ca²⁺ influx through glutamate receptors or voltage-sensitive Ca²⁺ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn²⁺, Mg²⁺, Co²⁺, or Gd³⁺. Although the mechanism of Ca²⁺ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca²⁺ selectivity of the response suggest a direct and specific relationship between [Ca²⁺]_o and the state of activation of the kinase in intact neurons.