纳米氧化铜对大鼠海马神经元Ik和PC12细胞活性的影响

用全细胞膜片钳方法研究纳米氧化铜(nanoCuO)颗粒对大鼠海马CA1区急性分离神经元延迟整流钾通道电流(Ik)的影响,并用MTT方法研究其对神经生长因子(NGF)诱导分化的PC12细胞活性的影响.结果显示5×10-5g/mL nanoCuO能够显著抑制海马CA1区神经元的Ik,使其失活曲线和对照组相比向左偏移,但对其激活过程无明显影响.MTT方法的实验结果显示不同浓度的nanoCuO(5×10-4、5×10-5、5×10-6g/mL)对NGF诱导分化的PC12细胞均有损伤作用,随着浓度的增加,损伤更加明显,呈量效和时效依赖关系.     

Demonstration and characterization of hippocampal cholinergic neurostimulating peptide (HCNP) processing enzyme activity in rat hippocampus

Hippocampal cholinergic neurostimulating peptide (HCNP) stimulates cholinergic activity of cultured medial septal nuclei explants. It consists of eleven amino acids that are located at the N-terminal region of its precursor protein. This report concerns the demonstration and characterization of an HCNP processing enzyme that cleaves the bioactive undecapeptide from the precursor. The enzyme was purified from the hippocampus of young Wistar rats. A synthetic deacetylated peptide (peptide_1–26) consisting of the N-terminal 26 amino acids of the HCNP precursor protein served as substrate. The product of the enzyme reaction was identified and quantitated by HPLC using deacetylated HCNP as standard. The amount of undecapeptide generated was directly proportional to the time of incubation of the enzyme reaction mixture. From molecular sieving chromatography it was estimated that the molecular mass of the enzyme is close to 68 kDa. The HCNP processing enzyme has a pH optimum of 6.0 and a K_m of 0.50 mM for peptide_1–26. Preincubation at 56°C causes rapid inactivation of the HCNP processing activity. Enzyme activity is enhanced by EDTA and 1,4-dithiothreitol, and inhibited by antipain, chymostatin and E-64. These findings suggest that the enzyme probably has a thiol group in its active site. This novel enzyme of the hippocampus may represent a valuable tool for further studies on the general protein metabolism in the central nervous system, as well as for elucidating the neurochemical aspects of neurodegenerative disorders.     

Enhanced Aspartate Release from Hippocampal Slices of Epileptic (El) Mice

The release of putative neurotransmitters [aspartate, glutamate, and gamma-aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 microns thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium-dependent and calcium-independent potassium-induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatography with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice +/- SEM. No significant differences were found between the El and B6 mice for the calcium-dependent potassium-evoked release of glutamate (18.20 +/- 2.62 and 15.41 +/- 3.56), or GABA (17.28 +/- 2.90 and 12.73 +/- 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 +/- 0.69) than in the B6 mice (3.31 +/- 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.     

Lysosomal vitamin E accumulation in Niemann-Pick type C disease

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.     

海马神经细胞胆固醇含量降低对其电压依赖钾电流的上调作用

胆固醇普遍存在于细胞膜中,其含量在细胞增殖、生长及各种疾病条件下会发生改变,这暗示胆固醇对细胞功能的调节起着重要的作用。运用全细胞膜片钳技术研究了胆固醇含量变化对海马神经细胞电压依赖钾电流的影响。实验观察到神经细胞经胆固醇去除剂β-甲基环化糊精(MβCD)处理后,胆固醇含量的减少促进了延迟整流钾电流IK的增加,且延缓了瞬间失活钾电流IA的失活。更进一步,延迟整流钾电流IK和瞬间失活钾电流IA分别经TEA和4-AP阻断后,MβCD对两种电流成分的影响显著降低。这一结果进一步表明胆固醇去除剂对电压依赖钾电流的上调是通过作用于IK和IA电流而共同实现的。基于电压依赖钾通道在神经细胞功能中的重要作用,实验结果暗示神经细胞胆固醇含量变化可对神经细胞的兴奋性起调节作用。     

Functional role of the activity of ATP-sensitive potassium channels in electrical behavior of hippocampal neurons: experimental and theoretical studies

ATP-sensitive K⁺ (K_ATP) channels are distributed in a variety of cell types, including hippocampal neurons. These channels provide a link between electrical activity of cell membranes and cellular metabolism. The activity of K_ATP channels in hippocampal H19-7 neurons treated with or without short interfering RNAs (siRNAs) directed against Kir6.2 mRNA was investigated in this study. In single-channel recordings, cell exposure to diazoxide (30 μM) significantly prolonged the mean open time of K_ATP channels; however, neither closed-time kinetics nor the single-channel conductance of the channel was altered by this compound. However, in cells transfected with Kir6.2 siRNAs, diazoxide-stimulated activity of K_ATP channels was abolished. Based on single-channel recordings, the activity of K_ATP channels was mathematically constructed in a Markovian manner. The simulated activity of single K_ATP channels was incorporated in a modeled hippocampal neuron to assess how any changes in K_ATP-channel activity affect burst firing of action potentials (APs). The modeled neuron was adopted from the model of Xu and Clancy (2008). Specifically, to mimic the action of diazoxide, the baseline value of open time (τ_bas) of K_ATP channels was arbitrarily elevated, while varying number of active channels (N_O) was set to simulate electrical behavior of Kir6.2 siRNAs-transfected cells. The increase of either N_O or τ_bas depressed membrane excitability of modeled neuron. Fast-slow analysis of AP bursting from this modeled neuron also revealed that the increased K_ATP-channel activity shifted the voltage nullcline in an upward direction, thereby leading to a reduction of the repetitive spike regime. Taken together, it is anticipated that the increased activity of K_ATP channels caused by increasing N_O or τ_bas contributes to or is responsible for burst firing of APs in hippocampal neurons if similar results occur in vivo.     

Aluminium interferes with hippocampal calcium signaling in a species-specific manner

Aluminium (Al) has been implicated in a number of neurodegenerative disorders and the disruption of calcium homeostasis has been proposed as a possible mechanism. To investigate ligand- and structure-specific effects of Al species, calcium imaging was used to probe the influence of five Al complexes - in comparison to inorganic Al (Al-S) - on N-methyl d-aspartate receptor (NMDAR) and voltage-dependent calcium channel (VDCC) function in hippocampal neurontos. The Al complexes utilized comprised three Al-citrate species (AlCit1-3), Al-quinate (AlQ) and Al-N-phosphonomethyliminodiacetate (AlNTAP). Our results suggest variable toxicity among the Al compounds tested: Al-S most potently affected neurons, with a full and irreversible inhibition of NMDAR and VDCC signaling at 500 μM. At all concentrations tested (10, 100, and 500 µM), all Al compounds investigated inhibited NMDA responses, however, no dose-dependency was evident. Furthermore, striking differences were noted with respect to calcium responses via VDCC activation. AlCit2 reduced calcium responses at all concentrations tested, AlQ at 10 and 100 µM, and AlNTAP at 500 µM only. In contrast, AlCit1 and AlCit3 had no significant effect. Collectively, diversely structured Al-ligand species selectively affect neuronal membrane channel function. The distinct chemical reactivity of the various Al forms reflects their unique interactions with neuronal structures and is poised to explain the diverse facets of Al toxicity.     

Combinatorial morphogenesis of dendritic spines and filopodia by SPAR and α-actinin2

Rap small GTPases regulate excitatory synaptic strength and morphological plasticity of dendritic spines. Changes in spine structure are mediated by the F-actin cytoskeleton, but the link between Rap activity and actin dynamics is unclear. Here, we report a novel interaction between SPAR, a postsynaptic inhibitor of Rap, and α-actinin, a family of actin-cross-linking proteins. SPAR and α-actinin engage in bidirectional structural plasticity of dendritic spines: SPAR promotes spine head enlargement, whereas increased α-actinin2 expression favors dendritic spine elongation and thinning. Surprisingly, SPAR and α-actinin2 can function in an additive rather than antagonistic fashion at the same dendritic spine, generating combination spine/filopodia hybrids. These data identify a molecular pathway bridging the actin cytoskeleton and Rap at synapses, and suggest that formation of spines and filopodia are not necessarily opposing forms of structural plasticity.     

丙二醛破坏大鼠海马神经元胞质钙离子稳态的信号机制

目的研究丙二醛（MDA）对原代培养的海马神经元胞质中钙离子稳态的破坏作用及可能的信号机制。方法以Fur2／AM为荧光指示剂,采用荧光分光光度法定量测定原代培养海马神经元胞质游离钙浓度变化。结果随着MDA浓度的升高和作用时间的延长,导致胞质中游离钙水平显著升高,破坏其钙稳态。MDA所导致的海马神经元胞质游离钙水平升高包括两个过程：100μmol／L的MDA可使胞质[Ca2＋]i水平在0—10min内的早期渐进升高过程,经历中间大约5min的平台期后,接下来15—30min的晚期显著升高。以细胞膜电压依赖的Ca2＋通道抑制剂nimodipine抑制外钙内流后,可显著抑制晚期胞质[Ca2＋]i水平的升高,以PLC的抑制剂U73122作用后,则可抑制早期胞质[Ca2＋]i水平的升高。结论100μmol／L的MDA作用下,海马神经元胞质中早期钙离子水平的升高和晚期钙离子水平的升高可能分别由不同的信号机制所介导。     

人（Homo sapiens）海马神经元microRNA调控网络的构建及其基本性质研究

目的：建立人海马神经元中的分子相互作用调控网络,研究miRNA在这个网络中是如何与其他信号通路相互作用并形成更复杂的生物网络,以及miRNA对网络中其靶点的调控如何影响生物网络的性质。方法：通过对已发表文献实验数据的挖掘分析,获得了哺乳动物海马神经元中主要信号通路的580个组分的一组相互作用数据,以及海马神经元中的miRNA表达谱。使用PITA,Miranda,TargetScan三个miRNA靶点预测软件计算出了这580个组分中的345个miRNA靶点。使用cytoscape对这些相互作用数据建立网络并对其性质进行计算分析。结果：建成了海马神经元中一个包含633个节点1653条边的miRNA调控网络,该网络中转录因子,adapter,酶更多的受到miRNA调控。结论：人海马神经元中,miRNA主要通过对转录因子,adapter和酶进行调控,与其他信号通路相互作用形成了一个更加复杂的网络,新形成的网络的集群系数,网络异质性,网络中心化程度,平均最短路径长度,平均邻点数都发生了变化。