Early-onset cognitive deficits and axonal transport dysfunction in P301S mutant tau transgenic mice

Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD) are neurodegenerative “tauopathies” characterized by hyperphosphorylated tau accumulation and neurofibrillary tangles. The P301S mutation of tau, a causal mutation of a familial type of FTLD, is believed to be involved in neurodegenerative progression. We developed a transgenic mouse, named TPR50, harboring human P301S tau. Tau phosphorylation in the hippocampus of TPR50 mice increased with age, particularly at S202/T205. Insolubilization and intracellular accumulation of tau were detected in the hippocampus by 9 months of age. Expression of calbindin was significantly reduced in 6- and 9-month-old TPR50 mice but not in 3-month-old mice. TPR50 mice demonstrated cognitive dysfunction at 5 months. At this age or earlier, although no intracellular tau accumulation was observed in the hippocampus, abnormally increased microtubule (MT)-related proteins and MT hyperdynamics in the hippocampus, and impaired axonal transport in the septo-hippocampal pathway were already observed. Therefore, cognitive dysfunction in TPR50 mice may result from early MT dysfunction and impaired axonal transport rather than accumulation of insoluble tau and neurodegeneration. TPR50 mice are a valuable new model to study progression of tauopathies at both the behavioral and neurocellular levels and may also prove useful for testing new therapies for neurodegenerative diseases.     

Neurofibrillary tangle-related synaptic alterations of spinal motor neurons of P301L tau transgenic mice

We investigated axosomatic synapses of anterior horn cells of transgenic (TG) mice expressing mutant P301L human tau and non-transgenic (NTG) mice using electron microscopic methods to demonstrate the relationship between neurofibrillary tangles (NFTs) and synaptic alterations. Animals aged 3.5-8.5 months were used because at this age many motor neurons in TG mice have NFTs. We measured the perimeter of anterior horn cell perikarya, the number of boutons and total length of boutons in contact with the neuronal perikarya from the micrographs of NFT and non-NFT-bearing neurons. We also calculated the proportion of the perimeter covered by boutons, density of boutons and mean size of boutons. The density of synaptic boutons in contact with NFT-bearing neurons was significantly decreased compared to non-NFT-bearing neurons. These findings suggest that synaptic reduction occurs during neurofibrillary degeneration and is probably associated with NFT. In addition, synaptic boutons were detached from NFT-bearing neurons with the resulting space occupied by astrocytic processes, suggesting that astrocytes may be involved in the observed synaptic alterations.     

Tau inclusions in retinal ganglion cells of human P301S tau transgenic mice: Effects on axonal viability

Tau inclusions play a key role in the pathogenesis of tauopathies. Altered tau levels have been detected in retina and optic nerve of patients with glaucoma, suggesting the possibility of shared pathogenic mechanisms with tauopathies. Here we report that hyperphosphorylated transgenic tau accumulates in the nerve fibre layer and, from 2 months of age, aggregates into filamentous inclusions in retinal ganglion cells of human P301S tau transgenic mice. Axonopathy and accumulation of hyperphosphorylated tau in the nerve fibre layer preceded inclusion formation. Hyperphosphorylated tau and tau inclusions were also detected in cultured retinal explants from 5-month-old transgenic mice. Axonal outgrowth was similar in transgenic and wild-type retinal explants under basal conditions. However, when exposed to growth-promoting stimuli, axon elongation was enhanced in explants from wild-type but not transgenic mice, indicating that the presence of abnormal tau can impair stimulated axonal outgrowth. These findings suggest that the retina is a good model system for investigating tau-driven neurodegeneration and for assessing potential pharmacological modifiers for tauopathies.     

NMDA受体亚单位在海马脑片和在体发育海马结构中表达变化的比较

目的：对比长期培养大鼠海马脑片与在体生后发育海马结构中N-甲基-D-天冬氨酸(NMDA)受体亚单位NR1、NR2A和NR2B表达变化的异同,为利用海马脑片研究NMDA受体亚单位在生理和病理状态中的作用提供资料。方法：免疫组织化学染色、海马脑片培养技术及图像分析。结果：NRI、NR2B在海马脑片各区和生后大鼠海马结构各区表达模式相似,均呈“先升、后降”的趋势,但升降幅度不同;NR2A在海马脑片各区是“先升、后降”的趋势,在生后海马结构各区则呈现“先降后升”的模式。NRI、NR2A、NR2B在脑片和在体发育海马的相似期间,在CA1、CA3、PG区表达均无显著性差异。结论：P3w／C2w时NRI和NR2B在海马脑片各区的表达强度,和其在生后大鼠海马结构中的表达较相似。     

雄激素对快速老化小鼠SAMP8海马神经元突触蛋白的快速影响

目的:研究去势以及去势后雄激素补充治疗对快速老化小鼠(SAMP8)海马神经元突触蛋白的快速影响。方法:7月龄健康雄性SAMP8小鼠,随机分为假手术对照组(对照组)、去势组、去势+睾酮补充治疗组(T组)和去势+双氢睾酮补充治疗组(DHT组)。通过免疫组织化学显色和免疫印迹研究去势及雄激素补充治疗对其海马突触蛋白SNAP25、Syt1、SYN、PSDD95、NR1和Drebrin表达的快速影响。结果:免疫组织化学显色结果显示,与SAMP8小鼠假手术组相比,去势组CA1区和CA3区各突触蛋白的OD值均明显降低。T组和DHT组CA1区和CA3区各突触蛋白的OD值较去势组明显增加。免疫印迹结果显示,与SAMP8小鼠假手术组相比,去势组海马结构各突触蛋白条带光密度的相对值均明显降低。T组和DHT组海马结构各突触蛋白条带光密度的相对值较去势组明显增加。结论:SAMP8小鼠去势后,雄激素缺乏导致海马结构内突触蛋白表达减少;去势后雄激素补充治疗能快速增加SAMP8小鼠海马结构内突触蛋白的表达。     

NMDA受体亚单位NR1、NR2A和NR2B在大鼠海马的免疫组织化学表达

目的 :观察N 甲基 D 门冬氨酸受体亚单位 1 (N methyl D aspartatereceptorsubunit 1 ,NR1 )、亚单位 2A(NR2A)和亚单位 2B(NR2B)在成年大鼠海马结构各区的表达特点 ,为研究三者在海马生理和病理过程中的作用提供形态学资料。方法 :大鼠脑 2 0 μm厚冰冻切片 ,免疫组织化学ABC法显色 ,图像分析。结果 :NR1、NR2A和NR2B在海马CA1～CA3区锥体细胞以及齿状回颗粒细胞普遍表达 ,三者中以NR1免疫组织化学反应最强 ,NR2A最弱 ,NR2B居中。NR1与NR2A在海马各区间的表达水平都无显著差异 ;NR2B在海马CA1区的表达明显强于其在CA3区及齿状回的表达 ,尤其是CA1区锥体细胞的顶树突在贯穿辐射层及腔隙分子层的全长中都呈高表达。结论 :NR1、NR2A和NR2B在正常海马结构各区的表达强度和形式存在差异 ,提示各区间天然N 甲基 D 门冬氨酸(N methyl D aspartate ,NMDA)受体的亚单位构成比例可能有所不同     

侧脑室注射NMDA对精神分裂症小鼠海马颗粒细胞层NMDA受体NR1亚基表达的调控

目的:阐明N-甲基-D-天冬氨酸受体亚单位1(NR1)在精神分裂症小鼠海马CA1、CA3、DG区颗粒细胞层的表达及NMDA对其表达的调控作用。方法:将小鼠随机分为实验组、对照组及空白组,实验组每日连续腹腔注射MK-801(0.6 mg/kg)14 d后侧脑室注射NMDA 1次,72 h后取材,应用免疫荧光标记法检测各组小鼠海马CA1、CA3、DG区颗粒细胞层NR1阳性细胞的表达。结果:对照组小鼠DG区NR1阳性细胞的表达比实验组和空白组明显升高(P0.05);在CA1区,实验组NR1的表达与对照组及空白组相比显著降低(P0.05);在CA3区,对照组NR1的表达与空白组相比显著降低(P0.05)。结论:(1)精神分裂症小鼠海马颗粒细胞层NR1阳性细胞的表达在DG区和CA1区显著升高,CA3区显著降低;(2)NMDA可调节精神分裂症小鼠海马结构颗粒细胞层NR1阳性细胞的表达趋于正常水平。     

Induction of inflammatory mediators and microglial activation in mice transgenic for mutant human P301S tau protein

Mice transgenic for human P301S tau protein exhibit many characteristics of the human tauopathies, including the formation of abundant filaments made of hyperphosphorylated tau protein and neurodegeneration leading to nerve cell loss. At 5 months of age, the pathological changes are most marked in brainstem and spinal cord. Here we show that these changes are accompanied by marked neuroinflammation. Many tau-positive nerve cells in brainstem and spinal cord were strongly immunoreactive for interleukin-1beta and cyclooxygenase-2, indicating induction and overproduction of proinflammatory cytokines and enzymes. In parallel, numerous activated microglial cells were present throughout brain and spinal cord of transgenic mice, where they concentrated around tau-positive nerve cells. These findings suggest that inflammation may play a significant role in the events leading to neurodegeneration in the tauopathies and that anti-inflammatory compounds may have therapeutic potential.     

Filamentous tau in oligodendrocytes and astrocytes of transgenic mice expressing the human tau isoform with the P301L mutation

We recently reported a transgenic mouse line (JNPL3) that expresses mutant (P301L) tau and develops neurofibrillary tangles composed of filamentous tau aggregates. Here we show that these mice have abnormal tau filaments not only in neurons, but also in oligodendrocytes and astrocytes. Similar results were detected in another transgenic line (JNPL2+3+) that expresses the longest human tau isoform with the P301L mutation. The ultrastructure of the tau filaments and immunoreactivity with tau and ubiquitin antibodies were similar in glia and neurons. Given similarities of the lesions in the mice to human neuronal and glial inclusions, these transgenic mice appear to be a valuable model to study pathogenesis of the neurodegenerative tauopathies.     

转录因子EB及下游自噬相关基因Beclin1在肌萎缩侧索硬化症转基因鼠海马中的表达变化

目的:探讨TFEB及其下游自噬相关基因Beclin1在95、108 d和122 d肌萎缩侧索硬化症(ALS)转基因鼠大脑海马中的表达情况。方法:分别取95、108 d和122 d ALS转基因鼠和野生型鼠的海马,应用RT-PCR、免疫印迹和免疫荧光,检测TFEB和Beclin1在大脑海马区的表达变化。结果:与同窝野生型鼠比较,ALS转基因鼠TFEBmRNA和蛋白水平在95d升高,108 d和122 d均降低。免疫荧光结果显示,与同窝野生型鼠比较,95 d TFEB免疫阳性反应增强,108 d和122 d减弱。自噬相关基因Beclin1表达情况与TFEB一致。结论:TFEB和Beclin1在ALS转基因鼠海马中表达异常,表明TFEB和Beclin1调节的自噬与ALS发生密切相关。     

小鼠生后不同发育阶段海马和丘脑组织中三叶因子3的表达

目的:了解小鼠生后不同时相海马以及丘脑组织中三叶因子3(TFF3)的表达。方法:取生后P0、P1、P3、P5、P7、P9、P11、P13、P15、P17、P19、P21、P23、P25、P27及成年昆明种小鼠全脑,参照小鼠脑图谱,进行石蜡包埋、切片厚6μm、H-E染色和TFF3免疫组织化学显色。另取不同时相小鼠海马组织液氮冻存,提取组织总RNA,RT-PCR检测TFF3 mRNA的转录水平。结果:TFF3在海马CA1、CA2、CA3、CA4及齿状回神经元的胞质与胞核中均有表达,随生后发育,胞质阳性信号增强,P15开始胞核呈阴性表达,且CA1~CA4区阳性神经元减少;丘脑内侧背核、外侧背核、缰内侧核、外侧膝状体背核、丘脑后核、丘脑腹后内侧核、外侧核、中央内侧核等也呈TFF3免疫反应阳性,且表达趋势与海马相似。随着个体的发育,P15后海马组织中TFF3 mRNA的表达量与P1~P13相比逐渐减少;海马CA2、CA3区TFF3平均光密度在P1~P15水平较高,P15最高,P15之后逐渐下降,成年最低。结论:小鼠海马和丘脑TFF3生后发育的变化,提示TFF3可能参与海马和丘脑各核团的发育。     

APP/PS1转基因小鼠海马内少突胶质细胞变化的体视学研究

目的:定量研究APP/PS1转基因小鼠海马内少突胶质细胞(OLG)的改变,探讨β-淀粉样蛋白(Aβ)对OLG的影响。方法:随机选取10月龄雄性APP/PS1转基因小鼠(AD组)和10月龄同窝生雄性野生型小鼠(WT组)各13只,运用Morris水迷宫检测各组小鼠的空间学习和记忆能力;运用体视学方法计数各组小鼠海马CA1、CA2-3和齿状回(DG)内Olig2+细胞和2',3'-环核苷酸3'-磷酸二酯酶(CNPase)阳性细胞总数;体外培养小鼠少突胶质前体细胞(MOPC),给予Aβ1-42,运用real time RT-PCR和Western Blot检测OLG相关蛋白的表达水平和含量。结果:AD小鼠逃避潜伏期显著性长于WT小鼠(P<0.05),穿台次数显著性少于WT小鼠(P<0.05);AD小鼠海马各区Olig2+细胞总数均较WT小鼠显著性增加(P<0.05),且与逃避潜伏期呈正相关,与穿台次数呈负相关,而各区CNPase+细胞总数均较WT小鼠显著性减少(P<0.05),且与逃避潜伏期呈负相关;Aβ1-42干预后MOPC的NG2及CNPase的mRNA水平显著性降低(P<0.05),Olig2含量也显著性降低(P<0.05)。结论:APP/PS1转基因小鼠海马内存在成熟OLG丢失、少突胶质细胞系异常增殖;Aβ可能引起OLG损伤和发育异常;保护海马成熟OLG以及调控OLG发育可能是防治AD的有效策略。     

Cyclin D1在肌萎缩侧索硬化症转基因小鼠大脑皮层和海马中的表达

目的:通过检测细胞周期相关蛋白D1(Cyclin D1)在肌萎缩侧索硬化症(ALS)转基因小鼠大脑皮层和海马中的表达变化,探讨Cyclin D1表达改变与ALS发病的关系。方法:选取成年ALS小鼠和同窝野生型小鼠,于发病早、中、晚期(95 d,108 d,122 d)取材,应用免疫荧光技术检测Cyclin D1在大脑皮层和海马的表达规律及与神经元和星形胶质细胞的共定位关系,应用RT-PCR检测Cyclin D1 mRNA表达情况,应用免疫印迹法检测蛋白表达量的改变。结果:ALS小鼠和野生型小鼠大脑皮层和海马中均可检测到Cyclin D1阳性细胞,且与神经元共表达。在发病早、中、晚期,ALS小鼠大脑皮层中Cyclin D1 mRNA和蛋白表达较野生型鼠增多(P0.05,P0.01,P0.001);与同窝野生型小鼠相比,ALS小鼠海马中Cyclin D1 mRNA和蛋白在发病的早、中、晚期表达均降低(P0.05,P0.01,P0.001)。Cyclin D1阳性细胞主要分布在海马CA区(包括CA1、CA2和CA3),DG区仅散在表达。结论:Cyclin D1在ALS转基因小鼠大脑皮层和海马中表达异常,表明Cyclin D1调节的细胞周期改变与ALS大脑皮层和海马区病变密切相关。     

谷氨酸受体与γ-氨基丁酸_A受体在APPswe转基因小鼠海马结构中的表达

目的探讨谷氨酸受体和γ-氨基丁酸A(GABAA)受体在阿尔茨海默病(AD)发生、发展中的作用。方法利用免疫荧光技术标记正常及APPswe转基因小鼠P0至P360各年龄段海马CA3区N-甲基-D-天门冬氨酸受体亚单体1(NMDAR1)、GluR2/3和GABAARα1-6阳性细胞,并利用免疫印迹法对海马组织内NMDAR1和GABAARα1-6的活化片段进行半定量分析。结果在模型组与对照组小鼠海马CA3区,NMDAR1、GluR2/3和GABAARα1-6在出生后P7时明显增加,P30达到高峰,以后逐渐下降至成年水平;与同龄对照组相比,老龄(P360)模型鼠NMDAR1和GluR2/3阳性细胞密度明显减少(P0.05),而GABAARα1-6阳性细胞密度变化不明显(P0.05)。免疫印迹法检测结果与免疫荧光技术统计结果吻合。结论 NMDAR1和GluR2/3表达下降与AD相关的神经退行性疾病有关;而GABAARα1-6在AD发病中的作用尚不能确定。     

APP/PS1转基因AD鼠海马CA1区神经元突触超微结构

目的:探讨携带5个家族性基因突变的APP/PS1转基因阿尔茨海默病(AD)(5×FAD)模型小鼠海马CA1区神经元突触超微结构改变。方法:应用透射电镜观察和形态计量学分析5×FAD转基因AD鼠海马CA1区GrayⅠ型突触界面结构参数,包括突触间隙长度、突触间隙面积、突触后致密物浓度和突触界面曲率的变化。结果:5×FAD转基因AD鼠海马CA1区神经元突触活性区长度显著小于对照组,差异有统计学意义;突触后致密物厚度、突触界面曲率及宽度与对照组差异无统计学意义。结论:5个家族性突变基因导致小鼠海马CA1区神经元突触可塑性的改变,这可能是该突变基因导致的发病机制之一。     

Zinc,magnesium and NMDA receptor alterations in the hippocampus of suicide victims

Background

There is evidence for an association between suicidal behavior and depression. Accumulating data suggests that depression is related to a dysfunction of the brain's glutamatergic system, and that the N-methyl-d-aspartate (NMDA) receptor plays an important role in antidepressant activity. Zinc and magnesium, the potent antagonists of the NMDA receptor complex, are involved in the pathophysiology of depression and exhibit antidepressant activity.

Methods

The present study investigated the potency of Zn²⁺ and Mg²⁺ to [³H] MK-801, which binds to the NMDA receptor channel in the hippocampus of suicide victims (n=17) and sudden death controls (n=6). Moreover, the concentrations of zinc and magnesium (by flame atomic absorption spectrometry) and levels of NMDA subunits (NR2A and NR2B) and PSD-95 protein (by Western blotting) were determined.

Results

Our results revealed that there was a statistically significant decrease (by 29% and 40%) in the potency of zinc and magnesium (respectively) to inhibit [³H] MK-801 binding to NMDA receptors in the hippocampus in suicide tissue relative to the controls. These alterations were associated with increased NR2A (+68%) and decreases in both the NR2B (−46%) and PSD-95 (−35%) levels. Furthermore, lower concentrations (−9%) of magnesium (although not of zinc) were demonstrated in suicide tissue.

Conclusions

Our findings indicate that alterations in the zinc, magnesium and NMDA receptor complex in the hippocampus are potentially involved in the pathophysiology of suicide-related disorders (depression), which may lead to functional NMDA receptor hyperactivity.     

海马Cajal-Retzius细胞的正常发育及在APPswe转基因小鼠中的改变

目的 观察reelin阳性Cajal-Retzius细胞（CR细胞）在正常小鼠及APPswe转基因小鼠海马发育中的变化,探讨CR细胞在阿尔茨海默病（AD）发生发展过程中所起的作用,为研究AD发病机制和临床治疗提供新的思路和方法。 方法 80只实验小鼠分为APPswe转基因模型组和对照组,每一组内分E16、P0、P7 、P15 、P30、P90、P180和P360 8个年龄段,每一年龄段小鼠各取5只。另取12月龄模型组和对照组小鼠各3只,用硫黄素S染色技术检测APPswe转基因小鼠脑内沉积的老年斑;免疫荧光技术标记正常及模型组小鼠齿状回分子层内reelin阳性CR细胞,同时采用谷氨酸、γ氨基丁酸(GABA)、活化型Caspase-3分别与reelin双重标记以研究CR细胞的组织化学特点及凋亡情况;最后利用免疫印迹方法对海马组织内reelin的活化片段进行半定量分析。 结果 随着海马发育CR细胞逐渐减少,不同时期的reelin阳性CR细胞可以分别被谷氨酸、GABA、Caspase-3标记;APPswe转基因小鼠海马内CR细胞的数量明显少于正常对照组,免疫印迹法结果与免疫细胞化学统计结果吻合。 结论 出生后CR细胞的丢失是由于凋亡所致,在海马发育的不同时期CR细胞分泌兴奋性或抑制性神经递质,以此来调节突触间的信息传递与突触可塑性。APPswe转基因小鼠海马内CR细胞低于正常对照组,提示CR细胞丢失可能与AD相关的神经元退行性病变有关。     

Colocalization and alteration of estrogen receptor-alpha and -beta in the hippocampus in Alzheimer's disease

The human hippocampus is severely affected in Alzheimer's disease (AD). Because postmenopausal estrogen use may decrease the risk and delay the onset and progression of AD, possibly by a direct action on the hippocampal neurons, we used fluorescence immunocytochemistry to examine the colocalization of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) in the hippocampus of elderly human controls and AD patients. Double-labeling cells (DLCs) of ERalpha and ERbeta can be divided into 3 types: double-cytoplasm-staining cells (DCCs), double-nucleus-staining cells (DNCs), and ERalpha nucleus-staining and ERbeta cytoplasm-staining cells (NCCs). There was no difference in the percentage of DLCs in total ERalpha-positive cells or in total ERbeta-positive cells in the CA1 to CA4 subfields of the hippocampus between controls and AD patients. Interestingly, the ratio of DNCs to the total ERalpha-positive cells (2.6% +/- 0.5%) or to the total ERbeta-positive cells (1.8% +/- 0.3%) in the CA1 subfield of the AD hippocampus was significantly decreased in comparison with controls (5.0% +/- 0.7% and 3.9% +/- 0.6%, respectively; P<0.001), suggesting that changes in the compartmentalization of these receptors could play a role in the pathogenesis of AD.     

Distribution of AMPA receptor subunits and TARPs in synaptic and extrasynaptic membranes of the adult rat nucleus accumbens

We characterized the distribution of AMPA receptor (AMPAR) subunits and the transmembrane AMPA receptor regulatory proteins (TARPs) γ-2 and γ-4 in adult rat nucleus accumbens (NAc) using a method that separates plasma membranes into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions. We also measured GluA1 phosphorylated at serine 845 (pS845 GluA1) and serine 831 (pS831 GluA1). GluA1-3 protein levels and pS831 GluA1/total GluA1 were higher in synaptic membranes. However, pS845 GluA1/total GluA1 was higher in extrasynaptic membranes, consistent with a role for S845 phosphorylation in GluA1 insertion at extrasynaptic sites. Homeric GluA1 receptors were detected in extrasynaptic membranes, consistent with evidence for extrasynaptic Ca(2+)-permeable AMPARs in other systems. The TARP γ-2 was enriched in synaptic membranes, whereas γ-4 was mainly found in extrasynaptic membranes, suggesting distinct roles for these proteins in the NAc. These experiments provide fundamental information that will aid in the interpretation of studies on AMPAR-related plasticity in the NAc.