ZnT3与Aβ在APP/PS1转基因小鼠脑血管及脉络丛的一致性分布

目的研究锌转运蛋白3(Zinc Transporter 3,ZnT3)与β-淀粉样蛋白(β-amyloid,Aβ)在APP/PS1转基因小鼠大脑血管壁及脉络丛上皮的定位分布,探讨ZnT3影响脑锌平衡从而参与AD发病的可能机制。方法应用免疫荧光技术和共聚焦激光扫描显微镜观察ZnT3和Aβ在APP/PS1转基因小鼠大脑血管壁及脉络丛上皮的共存情况。结果APP/PS1转基因小鼠侧脑室及第三、四脑室的脉络丛上皮细胞均呈ZnT3和Aβ染色阳性,二者共同表达于上皮细胞的胞质内,而细胞核未见任何着色。在APP/PS1转基因小鼠大脑皮层中,几乎所有Aβ阳性的血管壁上均有ZnT3的表达,二者的分布同样具有一致性。结论ZnT3与Aβ在APP/PS1转基因小鼠大脑血管及脉络丛上皮的一致性分布,提示ZnT3可能参与Aβ在大脑血管及脉络丛上皮的沉积。     

APP/PS1双转基因小鼠大脑皮质老年斑的病变对其学习记忆能力的影响

β-淀粉样蛋白(Aβ)沉积形成的老年斑是Alzheimer’s disease(AD)的主要病理特点,学习记忆功能障碍是其行为学的重要变化。为探讨老年斑对学习记忆的影响,本实验用17、28、44周C57系APP/PS1双转基因小鼠各4只,同龄野生型C57小鼠做对照,用Y-型迷宫方法比较不同年龄的AD模型鼠和C57鼠学习记忆能力的差异,利用免疫组织化学方法检测小鼠大脑皮质老年斑的病理变化,并比较AD模型鼠脑内老年斑的病变与其学习记忆能力改变之间的关系。结果显示:17周和28周AD模型鼠的学习记忆能力的各测试指标与对照组相应指标间均相差不显著(P>0.05),而44周AD模型鼠的学习记忆能力明显下降,各项指标与对照组相比具有显著性(P<0.01)。17周AD模型鼠脑切片上,可见大脑皮质内有少量老年斑的形成,而44周AD鼠皮质内斑块数量明显增多,体积明显增大。本结果提示老年斑的病变在一定程度上影响了其行为学的改变,这可能与β-淀粉样蛋白沉积继发的神经元缺失密切相关。     

β-淀粉样蛋白和drebrin在培养的APP/PS1转基因小鼠神经元中的表达

目的: 探讨阿尔茨海默病(AD)脑内β-淀粉样蛋白(Aβ)积聚与树突棘蛋白drebrin表达变化之间的关系.方法: 采用免疫细胞化学、ELISA和免疫印迹法等方法检测大脑皮层原代培养的APP/PS1转基因小鼠的神经元和同种野生型(WT)小鼠的神经元Aβ和drebrin的表达.结果: 培养至12d (days in vitro)时,可见Aβ在APP/PS1神经元胞体内聚集,培养基内的Aβ水平也同时升高;此期神经元内drebrin斑点状免疫反应产物分布较为稀疏,drebrin的表达水平下降.18d时,Aβ在胞体内聚集的基础上,向突起内延伸,培养基内的Aβ水平进一步升高;drebrin的表达则明显下降.结论: 原代培养的APP/PS1小鼠神经元内Aβ积聚的同时,树突棘蛋白drebrin的表达下降.提示AD脑内Aβ积聚可能是影响树突棘蛋白drebrin表达的因素之一.     

HuD在N2aAPP细胞和APP/PS1转基因小鼠中的表达

目的研究RNA结合蛋白HuD在AD细胞和动物模型中的表达水平,探讨HuD与AD的关系;明确PI3K/AKT通路对神经细胞HuD表达的调控作用。方法免疫荧光检测HuD在C57BL/6小鼠脑组织中的表达水平;构建过表达APP的N2a APP细胞模型,Western blot检测HuD的表达情况;购买APP/PS1转基因小鼠,Western blot检测HuD在脑组织中的表达情况;应用LY294002处理3种神经细胞(N2a、SH-SY5Y和HT22),阻断PI3K/AKT通路,观察该通路对HuD的调控作用。结果 HuD在整个C57BL/6小鼠脑组织中均有表达,且在海马体的颗粒细胞尤为明显;在N2a APP细胞,HuD的表达水平明显低于对照组;APP/PS1转基因小鼠脑组织中的HuD水平也低于野生对照鼠;LY294002处理下调了HuD在神经细胞中的表达水平。结论 HuD在N2a APP细胞和APP/PS1转基因小鼠中均呈低表达;HuD在神经细胞中受PI3K/AKT通路的调控,为研究HuD与AD的关系及寻找新的AD诊断和治疗靶点提供了理论基础。     

外源性NGF对APP/PS1转基因小鼠学习记忆能力的影响

目的观察外源性神经生长因子(nerve growth factor,NGF)对APP/PS1转基因小鼠学习记忆能力的影响和环磷酸腺苷反应元件结合蛋白(cAMP response element bling protein,CREB)蛋白的调节作用。方法实验分为野生组(雄性5月龄C57BL/6小鼠6只,鼻腔给予生理盐水),AD组(雄性5月龄APP/PS1小鼠6只)和AD+NGF组(雄性5月龄APP/PS1小鼠6只,鼻腔给予小鼠NGF治疗14w),应用Morris水迷宫检测小鼠的学习记忆能力变化,应用Western blot检测小鼠脑内CREB蛋白、p-CREB蛋白的表达。结果 AD+NGF组小鼠的潜伏期和找到平台前所经过的总路程均显著低于AD组小鼠,经过目标平台位置的次数明显高于AD组小鼠,游泳总路程和平均游泳速度均明显低于AD组小鼠。外源性NGF上调APP/PS1转基因小鼠脑内CREB蛋白和p-CREB蛋白的表达。结论外源性NGF改善APP/PS1转基因小鼠的学习记忆能力可能与上调CREB和p-CREB蛋白的表达相关。     

10月龄APP/PS1双转基因AD小鼠海马超微结构特征的观察

目的:观察APP/PS1双转基因小鼠海马内Aβ沉积的形态特征。方法:以10月龄雄性APP/PS1双转基因小鼠和C57BL/6小鼠为研究对象,采用Aβ免疫组化和透射电镜技术,观察海马部位Aβ的沉积情况及相关的病理特征。结果:在双转基因小鼠海马内存在致密性神经炎性斑块及营养障碍性突起,但脑微血管壁的Aβ沉积不甚明显;营养障碍性突起及神经细胞、微血管内皮细胞、周细胞内存在大量的自噬泡及次级溶酶体,提示该模型存在自噬溶酶体途径功能紊乱。结论:该鼠模拟了AD的老年斑及相关退行性变等病理改变,其中可能伴有细胞多种代谢的变化及血脑屏障功能的变化。从老年斑角度而言,该动物是成功的阿尔茨海默病动物模型,可用于AD的生化、病理研究及新治疗方法的探索。     

APP/PS1双转基因AD小鼠早期内质网应激诱导的凋亡

目的观察APP/PS1双转基因AD小鼠神经细胞凋亡,及内质网分子伴侣葡萄糖调节蛋白（GRP78）和内质网促凋亡因子半胱氨酸蛋白酶-12（Caspase-12）表达的改变,探讨APP/PS1双转基因AD小鼠早期内质网应激诱导的凋亡。方法选取5、7月龄的APP/PS1双转基因小鼠和同月龄同背景的野生型小鼠（WT）,分为5月龄WT组、5月龄APP/PS1组、7月龄WT组和7月龄APP/PS1组,每组6只,应用原位细胞凋亡检测法（TUNEL）检测凋亡细胞,免疫组织化学方法检测其脑内GRP78和Caspase-12的表达水平。结果 TUNEL检测凋亡率分别为7月龄APP/PS1鼠（35.0±6.31）%、5月龄APP/PS1鼠（9.0±2.78）%、7月龄WT鼠（4.0±1.89）%、5月龄WT鼠（4.0±1.83）%,其中7月龄APP/PS1鼠凋亡率显著升高（P〈0.05）;免疫组织化学检测GRP78阳性率分别为7月龄APP/PS1鼠（30.0±5.43）%、5月龄APP/PS1鼠（10.0±2.12）%、7月龄WT鼠（2.0±1.71）%、5月龄WT鼠（3.0±1.41）%,7月龄APP/PS1鼠GRP78表达明显升高（P〈0.05）;免疫组织化学检测Caspase-12阳性率分别为7月龄APP/PS1鼠（33.0±5.98）%、5月龄APP/PS1鼠（12.0±2.60）%、7月龄WT鼠（4.0±2.56）%、5月龄WT鼠（2.0±1.79）%,7月龄APP/PS1鼠Caspase-12表达明显升高（P〈0.05）。结论 7月龄的APP/PS1双转基因小鼠出现了内质网应激诱导的凋亡。本实验结果为临床AD早期预防和治疗提供了重要依据。     

雷公藤甲素抑制APP/PS1双转基因AD模型小鼠海马内Aβ沉积和老年斑形成

目的 研究雷公藤甲素(T10)对APP/PS1双转基因AD模型小鼠(APP/PS1dtg)β-淀粉样蛋白(Aβ)沉积与老年斑(SP)形成的影响。 方法 取18只45月龄健康雄性APP/PS1dtg, 随机分为3组,分别以T10灌胃：5μg/(kg&#8226;d) (T10 H组)、1μg/(kg&#8226;d) (T10 L组)和等容量的溶媒灌胃（PLC组）,共计45d。45d后取材,左侧半大脑切片,6E10免疫组织化学方法染色和刚果红染色结合无偏性体视学定量分析,研究T10对海马Aβ沉积和SP形成的影响;分离右侧半海马,免疫印迹法分析海马Aβ蛋白水平的变化。 结果 与PLC组比较, T10 H组海马6E10阳性的Aβ沉积总面积减少了35％(P<0.001),SP总面积减少了32％(P<0.001); T10 L组海马Aβ斑总面积减少了18％(P<0.05), SP总面积减了21％(P<0.05); T10呈剂量依赖性抑制Aβ在APP/PS1dtg 海马的沉积和SP的形成,减少海马内Aβ蛋白水平; 免疫印迹分析也显示,T10 H组海马Aβ蛋白水平最低,PLC组Aβ蛋白水平最高,T10 L组Aβ蛋白水平介于两者之间。     

Drebrin和SYP在APP/PS1转基因AD小鼠海马内的表达与认知障碍的关系

已知Alzheimer病(AD)患者脑内的主要病理变化之一是树突棘与突触的退化,而树突棘与突触的正常发育及病理改变均与树突棘肌动蛋白结合蛋白drebrin和突触前成分内突触素(synaptophysin,SYP)的表达水平密切相关。为探讨drebrin和SYP的表达与AD认知障碍之间的关系,本实验先采用Morris水迷宫法观测了6、9、12月龄APP/PS1转基因小鼠和同种野生型小鼠的学习记忆能力;再用Western blot法检测了其海马内drebrin和SYP蛋白表达水平。结果显示:9月龄APP/PS1转基因小鼠海马内drebrin蛋白的表达水平已有下降,其学习记忆能力也相应地降低,12月龄时二者下降明显;但作为突触前成分中的SYP蛋白表达水平的下降则较前二者的出现为迟。本结果提示,drebrin的表达下降在AD早期行为学改变的分子机制中发挥重要的作用,而SYP的表达下降则在drebrin下降之后参与AD的进一步发展。     

游离锌离子在APP/PS1转基因小鼠嗅球内的定位

目的研究锌离子在APP/PSI转基因小鼠嗅球内的定位,为锌离子参与阿尔茨海默病发病的相关性研究提供重要的形态学依据。方法应用浸入式金属自显影技术(AMG)检测锌离子在野生型小鼠和APP/PSI转基因小鼠嗅球内的分布。结果在野生型小鼠和APP/PSI转基因小鼠嗅球内,AMG反应产物呈棕黑色,嗅球的5层结构清晰可见,其中颗粒层锌离子含量最高。在APP/PSI转基因小鼠嗅球内,还可见锌离子聚集在老年斑内,AMG阳性的老年斑主要分布于含锌量较高的颗粒层,其它各层仅见散在分布。野生型小鼠嗅球内未见AMG阳性的老年斑。结论APP/PSI转基因小鼠嗅球的老年斑内含有大量的锌离子,提示锌离子在嗅球Aβ老年斑的形成过程中发挥重要作用,参与AD的发病和进展。     

MyD88 Is Dispensable for Cerebral Amyloidosis and Neuroinflammation in APP/PS1 Transgenic Mice

Activated microglia are associated with amyloid plaques in transgenic mouse models of cerebral amyloidosis and in human Alzheimer disease; yet, their implication in Alzheimer disease pathogenesis remains unclear. It has been suggested that microglia play dual roles depending on the context of activation, contributing negatively to disease pathogenesis by secreting proinflammatory innate cytokines or performing a beneficial role via phagocytosis of amyloid beta (Aβ) deposits. Toll-like receptors, most of which signal through the adaptor protein myeloid differentiation factor 88 (MyD88), have been suggested as candidate Aβ innate pattern recognition receptors. It was recently reported that MyD88 deficiency reduced brain amyloid pathology and microglial activation. To assess a putative role of MyD88 in cerebral amyloidosis and glial activation in APPswe/PS1ΔE9 (APP/PS1) mice, we crossed MyD88-deficient (MyD88^−/−) mice with APP/PS1 mice, interbred first filial offspring, and studied APP/PS1 MyD88^+/+, APP/PS1 MyD88^+/−, and APP/PS1 MyD88^−/− cohorts. Biochemical analysis of detergent-soluble and detergent-insoluble Aβ_1-40 or Aβ_1-42 in brain homogenates did not reveal significant between-group differences. Furthermore, no significant differences were observed on amyloid plaque load or soluble fibrillar Aβ by quantitative immunohistochemical analysis. In addition, neither activated microglia nor astrocytes differed among the three groups. These data suggest that MyD88 signaling is dispensable for Aβ-induced glial activation and does not significantly affect the nature or extent of cerebral β-amyloidosis in APP/PS1 mice.Alzheimer disease (AD) is an insidious public health threat characterized by deposition of β-amyloid as senile plaques, formation of neurofibrillary tangles, and large-scale cortical neuronal loss leading to dementia. In addition to these pathognomonic features of the disease, AD patients exhibit low-level chronic neuroinflammation. This is hallmarked by the spatial and temporal occurrence of activated microglia with amyloid beta (Aβ) deposits. Yet, the mechanisms by which microglia recognize and respond to Aβ accumulation remain unclear. Current evidence suggests that there are varied forms of activated microglia in AD, some of which are detrimental and others beneficial.¹ Because microglial activation is a complex continuum of varied responses,² it stands to reason that a wide array of immune molecules may orchestrate microglial responses to Aβ. Ultimately, a clearer understanding of the pathways leading to beneficial microglial responses and clearance of misfolded proteins could open new avenues for AD treatment.Numerous recent studies have proposed that Toll-like receptors (TLRs) play a role in the microglial response to Aβ and, more specifically, that aggregated Aβ can activate microglia via TLRs.^3–11 Most TLRs (except TLR3) signal through the adaptor protein myeloid differentiation factor 88 (MyD88), suggesting that it may play an important role in microglial activation in response to cerebral amyloid accumulation. To test this possibility, two recent studies crossed MyD88 knockout mice with APP/PS1 mouse models of cerebral amyloid deposition and examined effects on cognitive deficits and AD-like pathology. In one study, it was reported that MyD88 deficiency of the doubly transgenic APPswe/PS1dE9 mouse reduced cerebral amyloid pathology and microglial activation and decreased expression of CX3CR1 in 10-month-old animals.¹² Lim and coworkers¹² suggested that inhibiting MyD88-associated TLR signaling would alter the microglial activation state, and they reported less cerebral amyloid deposition in this cross. However, their findings were perplexing given previous reports showing that activation of TLRs leads to decreased amyloid load and increased Aβ phagocytosis, leading to the hypothesis that MyD88 deficiency would either cause buildup of amyloid or have no effect on amyloid levels in APP/PS1 mice.^{4,6,11,13–15} Another recent study published findings more consistent with this hypothesis, demonstrating that APPswe/PS1A246E mice heterozygous for MyD88 had accelerated spatial learning and memory deficits and increased levels of soluble Aβ oligomers. These results led the authors to conclude that MyD88-mediated activation of microglia was protective in the context of cerebral amyloid deposition.¹⁶ In an attempt to clarify the uncertainty surrounding this critical question, we crossed APPswe/PS1dE9 (APP/PS1) mice with MyD88 knockout (MyD88^−/−) mice (both on a C57BL/6 background) and analyzed APP/PS1 MyD88^+/+, APP/PS1 MyD88^+/−, and APP/PS1 MyD88^−/− cohorts for Alzheimer-like pathology at 15 months of age.     

Dinucleotide repeat polymorphism in the third intron of the NRAMP2/DMT1 gene

Nramp1 (natural resistance-associated macrophage protein 1), encoding a polytropic integral membrane protein, was isolated as a candidate of the mouse Lsh/Ity/Bcg locus, which regulates macrophage activation for antimicrobial activity against intracellular pathogens. The NRAMP2 gene was cloned from human genome as a homologue of NRAMP1. We found a polymorphic dinucleotide repeat in the third intron of the NRAMP2 gene. This polymorphism will be a useful genetic marker to study disease associated with susceptibility to infection with intracellular pathogens. Received: August 13, 1999 / Accepted: August 19, 1999     

Curcumin Ameliorates Memory Deficits by Enhancing Lactate Content and MCT2 Expression in APP/PS1 Transgenic Mouse Model of Alzheimer's Disease

Curcumin is a natural product with several anti-Alzheimer's disease (AD) neuroprotective properties. This study aimed to investigate the effects of curcumin on memory deficits, lactate content, and monocarboxylate transporter 2 (MCT2) in APP/PS1 mouse model of AD. APP/PS1 transgenic mice and wild-type (WT) C57BL/6J mice were used in the present study. Spatial learning and memory of the mice was detected using Morris water-maze test. Cerebral cortex and hippocampus lactate contents were detected using lactate assay. MCT2 expression in the cerebral cortex and hippocampus was examined by immunohistochemistry and Western blotting. Results showed that spatial learning and memory deficits were improved in curcumin-treated APP/PS1 mouse group compared with those in APP/PS1 mice group. Brain lactate content and MCT2 protein level were increased in curcumin-treated APP/PS1 mice than in APP/PS1 mice. In summary, our findings indicate that curcumin could ameliorate memory impairments in APP/PS1 mouse model of AD. This phenomenon may be at least partially due to its improving effect on the lactate content and MCT2 protein expression in the brain. Anat Rec, 302:332–338, 2019. © 2018 Wiley Periodicals, Inc.     

Increased regional cerebral glucose uptake in an APP/PS1 model of Alzheimer's disease

Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is characterized by the invariant cerebral accumulation of β-amyloid peptide. This event occurs early in the disease process. In humans, [18F]-fluoro-2-deoxy-D-glucose ([18F]-FDG) positron emission tomography (PET) is largely used to follow-up in vivo cerebral glucose utilization (CGU) and brain metabolism modifications associated with the Alzheimer's disease pathology. Here, [18F]-FDG positron emission tomography was used to study age-related changes of cerebral glucose utilization under resting conditions in 3-, 6-, and 12-month-old APP(SweLon)/PS1(M146L), a mouse model of amyloidosis. We showed an age-dependent increase of glucose uptake in several brain regions of APP/PS1 mice but not in control animals and a higher [18F]-FDG uptake in the cortex and the hippocampus of 12-month-old APP/PS1 mice as compared with age-matched control mice. We then developed a method of 3-D microscopic autoradiography to evaluate glucose uptake at the level of amyloid plaques and showed an increased glucose uptake close to the plaques rather than in amyloid-free cerebral tissues. These data suggest a macroscopic and microscopic reorganization of glucose uptake in relation to cerebral amyloidosis.     

APP/PS1转基因小鼠发育过程中神经细胞增殖变化

目的:探讨β-淀粉样前体蛋白基因(β-amyloid precusor protein,APP)和突变早老素1基因(presinilin,PS1)转基因小鼠(APP/PS1)发育过程中齿状回神经细胞增殖规律。方法:取不同发育时间(P14、P30、P180、P360)APP/PS1转基因模型鼠与同时间点对照鼠,用二等分Y型迷宫箱测试小鼠学习记忆能力,BrdU免疫细胞化学观察齿状回内神经细胞增殖变化。结果:随着小鼠的生长发育,BrdU阳性细胞密度逐渐降低,在P360时间点,模型组齿状回BrdU阳性细胞密度比对照组低(P<0.05);其空间识别以及记忆能力明显低于对照组(P<0.01)。结论:发育中的APP/PS1转基因小鼠齿状回内神经细胞增殖减少可能与阿尔茨海默病的学习、记忆能力下降有关。     

Locus coeruleus degeneration exacerbates olfactory deficits in APP/PS1 transgenic mice

Neuronal loss in the locus coeruleus (LC) is 1 of the early pathological events in Alzheimer's disease (AD). Projections of noradrenergic neurons of the LC innervate the olfactory bulb (OB). Because olfactory deficits have been reported in early AD, we investigated the effect of induced LC degeneration on olfactory memory and discrimination in an AD mouse model. LC degeneration was induced by treating APP/PS1 mice with N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (DSP4) repeatedly between 3 and 12 months of age. Short term odor retention, ability for spontaneous habituation to an odor, and spontaneous odor discrimination were assessed by behavioral tests. DSP4 treatment in APP/PS1 mice resulted in an exacerbation of short term olfactory memory deficits and more discrete weakening of olfactory discrimination abilities, suggesting that LC degeneration contributes to olfactory deficits observed in AD. Importantly, DSP4 treatment also increased amyloid β (Aβ) deposition in the olfactory bulb of APP/PS1 mice, which correlated with olfactory memory, not with discrimination deficits.     

Early temporal short-term memory deficits in double transgenic APP/PS1 mice

We tested single APP (Tg2576) transgenic, PS1 (PS1dE9) transgenic, and double APP/PS1 transgenic mice at 3 and 6 months of age on the acquisition of a hippocampal-dependent operant “differential reinforcement of low rate schedule” (DRL) paradigm. In this task mice are required to wait for at least 10 seconds (DRL-10s) between 2 consecutive nose poke responses. Our data showed that while single APP and PS1 transgene expression did not affect DRL learning and performance, mice expressing double APP/PS1 transgenes were impaired in the acquisition of DRL-10s at 6 months, but not at 3 months of age. The same impaired double transgenic mice, however, were perfectly capable of normal acquisition of signaled DRL-10s (SDRL-10s) task, a hippocampal-independent task, wherein mice were required to emit responses when the end of the 10-second delay was signaled by a lighting of the chamber. The age-dependent and early deficits of APP/PS1 mice suggest that the appetitive DRL paradigm is sensitive to the amyloid pathology present in double APP/PS1 mice, and that this mouse line represents a good model with which to study the efficacy of therapeutic strategies against Alzheimer's disease.     

Caloric restriction attenuates amyloid deposition in middle-aged dtg APP/PS1 mice

Caloric restriction (CR) mitigates neurological damage arising from aging and a variety of other sources, including neuropathology in young adult mice that express single and double transgenic (tg) mutations associated with Alzheimer disease (AD). To evaluate the potential of CR to protect against relatively heavy AD-type pathology, middle-aged (13–14-month-old) mice that co-express two mutations related to familial AD, amyloid precursor protein (APP) and presenilin 1 (PS1), were fed balanced diets with 40% fewer calories than ad libitum-fed controls. Following 18 weeks of treatment, mice were killed and brains were processed for quantification of total volume of amyloid-beta (Aβ) in the hippocampal formation and the overlying neocortex. Computerized stereology confirmed that CR reduced the total Aβ volume by about one-third compared to that in age-matched controls. Thus, CR appears to attenuate the accumulation of AD-type neuropathology in two cortical brain regions of middle-aged dtg APP/PS1 mice. These findings support the view that CR could be a potentially effective, non-pharmacology strategy for reducing relatively heavy Aβ deposition in older adult dtg APP/PS1 mice, and possibly afford similar protection against the onset and progression of AD in older adult humans.