金雀异黄酮通过调控Ca^(2+)-CaMKIV通路对Aβ_(25-35)诱导海马神经元损伤的保护作用

目的:观察金雀异黄酮通过Ca^(2+)-钙调蛋白依赖性蛋白激酶IV(calmodulin-dependent protein kinase IV,CaMKIV)通路对Aβ_(25-35)诱导海马神经元损伤的保护作用。方法:取24 h内新生SD乳鼠的海马组织,进行神经元的分离纯化和培养,并用免疫荧光染色进行鉴定。神经元细胞随机分为空白对照组、模型组、金雀异黄酮组(50μmol/L)和阳性对照戊酸雌二醇组(10μmol/L),金雀异黄酮组和戊酸雌二醇组预处理3 h后,除空白对照组外,其他各组采用Aβ_(25-35)诱导海马神经元构建细胞损伤模型。利用噻唑蓝法检测细胞存活率,荧光探针检测神经元细胞内Ca^(2+)荧光强度,Western blot检测钙调蛋白(calmodulin,CaM)、钙调蛋白依赖性蛋白激酶激酶(calcium/calmodulin dependent protein kinase kinase,CaMKK)、磷酸化钙调蛋白依赖性蛋白激酶IV(p-calmodulin-dependent protein kinase,p-CaMKIV)和p-Tau蛋白的相对表达量。结果:免疫荧光分析结果显示大鼠海马神经元分离成功。与空白对照组比较,模型组海马神经元细胞存活率极显著下降(P<0.01),细胞Ca^(2+)荧光强度极显著升高(P<0.01),CaM、CaMKK、p-CaMKIV和p-Tau蛋白相对表达量极显著提高(P<0.01);与模型组比较,金雀异黄酮极显著提高了Aβ_(25-35)所致海马神经元损伤模型中细胞的存活率(P<0.01),降低了细胞Ca^(2+)荧光强度(P<0.01),下调了CaM、CaMKK、p-CaMKIV和p-Tau蛋白相对表达量(P<0.01)。结论:金雀异黄酮对Aβ25-35诱导的海马神经元损伤具有明显的神经保护作用,其作用可能是通过Ca^(2+)-CaMKIV通路介导的。

脊髓缺血再灌注损伤对大鼠CaMKIV基因及蛋白表达的影响

目的分析大鼠脊髓缺血再灌注损伤(SCII)后钙调蛋白依赖性蛋白激酶4(Ca MKIV)基因及蛋白表达的变化。方法雄性SD大鼠84只,按随机数字表法分为假手术组、SCII 0 d组、SCII 1 d组、SCII 3 d组、SCII 7 d组、SCII 14 d组、SCII 28 d组,每组12只。假手术组仅暴露肾动脉下腹主动脉,不阻断。SCII各组采用Naslund法阻断腹主动脉,复制SCII模型。采用免疫组织化学法、Western blot、实时荧光定量聚合酶链反应(q RT-PCR)检测各组大鼠Ca MKIV蛋白及基因的表达,BBB评分评价大鼠后肢运动功能。结果免疫组织化学法结果显示,假手术组Ca MKIV蛋白表达很少,SCII组Ca MKIV蛋白高表达于神经元的细胞浆和细胞核;在伤后3 d,Ca MKIV阳性细胞数最多,且高于假手术组(P<0.05)。Western blot结果提示,Ca MKIV蛋白表达在再灌注0 d即高于假手术组(P<0.05),于再灌注3 d达峰值后逐渐降低;至28 d,Ca MKIV蛋白表达量与假手术组比较,差异无统计学意义(P>0.05)。q RT-PCR结果表明,Ca MKIV基因表达在再灌注0 d开始增加,与假手术组比较,差异有统计学意义(P<0.05),于3 d达峰值后降低,至28 d仍高于假手术组(P<0.05)。BBB评分显示,SCII各组大鼠后肢功能随再灌注时间延长有所改善,但至28 d时仍差于假手术组。结论 SCII早期可快速诱导Ca MKIV基因表达上调和蛋白合成,Ca MKIV在SCII中可能发挥重要作用。

002C-3 protects the brain against ischemia-reperfusion injury by inhibiting autophagy and stimulating CaMKK/CaMKIV/HDAC4 pathways in mice

This study was designed to investigate the effect of 002C-3, a derivative of magnolol, on transient cerebral middle occlusion （tMCAO） in a mice model and to identify the underlying mechanisms. 002C-3 （100 and 150 pg/kg, i.v. after ending occlusion） significantly reduced neurological deficit scores, infarct volumes, and brain water contents after 1.5 h MCAO and 24 h reperfusion. 002C-3 （75 150μg/kg） decreased the exudation of Evans blue from brain capillaries. 002C-3 （100 μg/kg） significantly inhibited the activity of MMP-9 and MMP-2 in the injured hemisphere. 002C-3 decreased the expression of autophagy-associated proteins, Beclin-1 and LC3B-Ⅱ, and increased the level of p62 in injured hemisphere. 002C-3 （100 pg/kg） significantly increased the expression of p-CaMKIV and p-HDAC4 in injured hemisphere. In conclusion, 002C-3 shows a neuroprotective effect on tMCAO injury in mice, and its mechanisms may be associated with alleviation of blood-brain barrier damage caused by the activation of MMPs, inhibition of autophagy, and stimulation of calcium signals related to cell survival. These findings suggest that 002C-3 is a neuroprotective agent that acts on multiple pathways.

朊病毒感染中钙调蛋白相关激酶含量异常变化的研究

朊病毒病是一类具有致死性、传染性和进行性的神经退行性疾病。目前研究发现许多因子都参与了疾病的发生发展过程,包括细胞因子、激酶和一些离子,其中钙离子及相关激酶在朊病毒病致病机制中的研究报道较少,为了探究朊病毒感染中钙调蛋白相关下游激酶的含量变化情况,本研究利用多种检测方法对朊病毒感染细胞系及小鼠脑组织进行了分析。结果显示朊病毒感染后,钙离子和钙调蛋白(CaM)的表达水平升高,下游Ca^(2+)/CaM复合物依赖性激酶CaMKIα和CaMKIV表达水平下降,同时这些激酶的上游激酶CaMKKα含量降低,提示朊病毒感染后神经元中钙离子和相关激酶稳态失衡,这种异常变化很可能影响下游多种转录因子合成,这些结果为解释朊病毒感染后神经元大量丢失提供了科学依据。

Tau-Induced Ca^(2+)/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer’s disease(AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells.Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca2+concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca2+concentration with a simultaneous increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV(Ca MKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca2+/Ca MKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation ofthe Ca2+/calmodulin complex abolished the okadaic acidinduced tau hyperphosphorylation in the nuclear fraction.We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca2+/Ca MKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca2+concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.