APP／PS1阿尔茨海默病转基因动物模型胼胝体损害研究

目的探讨APP／PS1阿尔茨海默病转基因动物模型胼胝体病理学改变。方法取胼胝体作为白质代表区域,采用氯化金髓鞘染色和轴突免疫组织化学染色方法,利用光密度方法对胼胝体轴突密度和髓鞘化程度的染色进行ROD定量分析。结果年龄相关性分析表明,老年组小鼠（包括APP／PS1和PS1）的轴突密度较年轻组小鼠显著下降,然而老年组小鼠的髓鞘化程度较年轻组有所增高。不同表型分析显示,年轻组APP／PS1小鼠的轴突密度和髓鞘化程度和年轻组PS1小鼠一致,然而老年组APP／PS1小鼠轴突密度和髓鞘化程度较老年组PSI小鼠显著下降。结论在APP／PS1阿尔茨海默病转基因动物模型中胼胝体存在着轴突的丢失和脱髓鞘改变,与β淀粉样蛋白对白质的毒性作用有关。     

Expression of DeltaNp73 in hippocampus of APP/PS1 transgenic mice following GFP-BMSCs transplantation

Abstract

Objective: To study the effect of hippocampal bone marrow stromal cells (GFP-BMSCs) transplantation on spatial memory and DeltaNp73 expression in APP/PS1 transgenic mice.

Methods: Twelve APP/PS1 transgenic mice randomly received either 10 μl GFP-BMSCs suspension in medium (GFP-BMSCs transplantation group) or 10 μl complete medium (sham-operated group). Learning and memory function of mice in both groups were observed and tested in Morris water maze experiment at 2 weeks after surgery. Senile plaques and DeltaNp73 protein in hippocampuses were determined by immunohistochemistry and western blot at 3 weeks after surgery, respectively.

Results: APP/PS1 mice treated with BMSCs performed significantly better on the water maze test than those in sham-operated group (P<0·05). Immunohistochemistry showed that GFP-BMSCs distributed uniformly and the number of Alzheimer’s senile plaques reduced after transplantation. Western blot showed that quantified DeltaNp73 protein expression was significantly higher in BMSCs transplantation group when compared with sham-operated group (P<0·01).

Conclusions: Our results suggest that BMSCs transplatation could retard Alzheimer’s disease (AD) like pathology and upregulate DeltaNp73 expression in hippocampuses of APP/PS1 transgenic mice. GFP-BMSCs transplantation will be a potential treatment for AD.     

Characterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease

Transgenic mice carrying disease-linked forms of genes associated with Alzheimer disease often demonstrate deposition of the beta-amyloid as senile plaques and cerebral amyloid angiopathy. We have characterized the natural history of beta-amyloid deposition in APPswe/PS1dE9 mice, a particularly aggressive transgenic mouse model generated with mutant transgenes for APP (APPswe: KM594/5NL) and PS1 (dE9: deletion of exon 9). Ex vivo histochemistry showed Abeta deposition by 4 months with a progressive increase in plaque number up to 12 months and a similar increase of Abeta levels. In vivo multiphoton microscopy at weekly intervals showed increasing beta-amyloid deposition as CAA and plaques. Although first appearing at an early age, CAA progressed at a significantly slower rate than in the Tg2576 mice. The consistent and early onset of beta-amyloid accumulation in the APPswe/PS1dE9 model confirms its utility for studies of biochemical and pathological mechanisms underlying beta-amyloid deposition, as well as exploring new therapeutic treatments.     

Alternations of central insulin‐like growth factor‐1 sensitivity in APP/PS1 transgenic mice and neuronal models

Although many post‐mortem studies have found evidence of central insulin resistance in Alzheimer's disease (AD) patients, results on changes of central insulin‐like growth factor‐1 (IGF‐1) signaling in the pathological process of AD remain controversial. In the present study, we observed the activation states of IGF‐1 downstream signaling in brain slices of transgenic mice carrying APPswe/PS1dE9 mutations (APP/PS1 mice) at both early and late stages (ex vivo) and further investigated the involvement of oligomeric β‐amyloid (Aβ) and Aβ‐enriched culture medium (CM) on IGF‐1 sensitivity employing neuronal models (in vitro). In 6‐ and 18‐month‐old APP/PS1 mice, the phosphorylations of IGF‐1 receptor (IGF‐1R) and Akt in response to IGF‐1 stimulation were significantly reduced in the hippocampal and cortical slices, whereas IGF‐1R protein expression and mRNA levels of IGF‐1 and IGF‐1R in the hippocampal slices were significantly higher than that in wild‐type mice. In agreement with these results, reduced IGF‐1 sensitivity was verified in APP and PS1 double stably transfected CHO cells; moreover, IGF‐1 stimulated phosphorylations of IGF‐1R and Akt were also markedly weakened by oligomeric Aβ or Aβ‐enriched CM posttreatment in CHO cells without APP/PS1‐transfected (K1 cells) and primary hippocampal neurons. These observations indicate that the impaired central IGF‐1 sensitivity at early and late stages of APP/PS1 transgenic mice might be attributable, at least partially, to the overproduced Aβ, especially the oligomeric Aβ. These findings may shed new light on the mechanisms underlying the defective IGF‐1 signaling in AD pathogenesis and provide important clues for AD drug discovery. © 2013 Wiley Periodicals, Inc.     

姜黄素对APP/PS1双转基因小鼠认知功能、炎症反应及海马区突触素表达的影响

目的 探讨姜黄素对APP/PS1双转基因小鼠认知功能、炎症反应及海马区突触素表达的影响。方法 60只6月龄APP/PS1双转基因雄性小鼠随机分为A组、B组和C组,各20只;A组采用姜黄素100mg/kg/d加入小鼠饲料喂养,B组采用姜黄素300mg/kg/d喂养,C组采用姜黄素600mg/kg/d喂养;3组均喂养6个月。治疗前、治疗3、6个月,采用Morris水迷宫实验评估小鼠认知功能,采用免疫吸附试验法检测尾静脉血血清白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）等炎症因子水平;治疗6个月,采用免疫组化染色法检测小鼠海马 CA1 区突触素表达情况。结果 姜黄素治疗3、6个月,3组小鼠认知功能明显改善（P<0.05）,血清IL-6、TNF-α水平明显降低（P<0.05）。与A组比较,B组和C组治疗3、6个月认知功能明显改善（P<0.05）,血清IL-6、TNF-α水平均明显降低（P<0.05）,海马CA1区突触素表达水平明显升高（P<0.05）。而B组与C组均无统计学差异（P>0.05）。结论 姜黄素有助于改善APP/PS1双转基因小鼠海马突触素表达,抑制小鼠炎症反应,改善小鼠认知功能。     

Aged wild-type and APP, PS1, and APP + PS1 mice present similar deficits in associative learning and synaptic plasticity independent of amyloid load

Wild-type and single-transgenic (APP, PS1) and double-transgenic (APP + PS1) mice were studied at three different (3-, 12-, and 18-month-old) age periods. Transgenic mice had reflex eyelid responses like those of controls, but only 3-month-old mice were able to fully acquire conditioned eyeblinks, using a trace paradigm, whilst 12-month-old wild-type and transgenic mice presented intermediate values, and 18-month-old wild-type and transgenic mice were unable to acquire this type of associative learning. 18-month-old wild-type and transgenic mice presented a normal synaptic activation of CA1 pyramidal cells by the stimulation of Schaffer collaterals, but they did not show any activity-dependent potentiation of the CA3–CA1 synapse across conditioning sessions, as was shown by 3-month-old wild-type mice. Moreover, 18-month-old wild-type and transgenic mice presented a noticeable deficit in long-term potentiation evoked in vivo at the hippocampal CA3–CA1 synapse. The 18-month-old wild-type and transgenic mice also presented a significant deficit in prepulse inhibition as compared with 3-month-old controls. Except for results collected by prepulse inhibition, the above-mentioned deficits were not related with the presence of amyloid β deposits. Thus, learning and memory deficits observed in aged wild-type and transgenic mice are not directly related to the genetic manipulations or to the presence of amyloid plaques.     

Glutathione peroxidase 4 protects cortical neurons from oxidative injury and amyloid toxicity

Polyunsaturated fatty acids (PUFA) in membrane lipids are prone to attack by reactive oxygen species (ROS), and the resulting lipid peroxidation can cause injury and death of cells. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme that can directly detoxify lipid hydroperoxides generated by ROS. Overexpression of Gpx4 has been shown to be protective against oxidative damage in several cell lines. We examined in this study the stress response of neurons with increased expression of Gpx4, because neurons are especially vulnerable to oxidative injury as a result of their high content of PUFA. Our results show that primary culture cortical neurons derived from Gpx4 transgenic mice, which had increased expression of Gpx4, had increased cell survival and reduced level of apoptosis after exposure to t-butyl hydroperoxide and hydrogen peroxide. We also studied the protective role of Gpx4 against beta-amyloid toxicity, because beta-amyloid-induced neural toxicity is believed to be mediated through lipid peroxidation. Primary culture cortical neurons from Gpx4 transgenic mice had significantly less cell toxicity than their wild-type counterparts after exposure to Abeta25-35 and Abeta1-40 peptides, and apoptosis induced by Abeta25-35 was attenuated in neurons from Gpx4 transgenic mice. Our data demonstrate that overexpression of Gpx4 protects neurons against oxidative injury and beta-amyloid-induced cytotoxicity.     

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)

Cumulative evidence indicates that aberrant re‐expression of many cell cycle‐related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer’s disease (AD) pathogenesis. Evidence of cell cycle activation in post‐mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway‐specific RT²Profiler? PCR Arrays, we detected changes in a number of cell cycle‐related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5′‐bromo‐2′‐deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin‐dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle‐related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.     

Fimbria-fornix lesion does not affect APP levels and amyloid deposition in the hippocampus of APP+PS1 double transgenic mice

The deposition of amyloid beta peptides (Abeta) and cholinergic dysfunction are two characteristic features of Alzheimer's disease. Several studies have suggested that a compromised cholinergic transmission can increase the amount of amyloid precursor protein (APP) in the denervated cortex (or hippocampus); however, whether this will increase Abeta production is unknown. To investigate the relation between cholinergic neurotransmission and APP metabolism, and the possible role of cholinergic dysfunction in the development of amyloid neuropathology, we lesioned the fimbria-fornix pathway in APP+PS1 double transgenic mice, at 5 and 7 months of age. Three months and 11 months postlesion, the mice were sacrificed for biochemical and histopathological analyses. The fimbria-fornix transection resulted in a substantial depletion of cholinergic markers in the hippocampus at both time points. Three months postlesion, hippocampal APP and Abeta levels were not significantly changed. At 11 months postlesion, the fimbria-fornix lesion did not result in an alteration in either the hippocampal Abeta levels or the extent of Abeta deposition, as assessed by amyloid plaque counts and image analysis of Abeta load in the 18-month-old APP+PS1 mice. Our findings indicate that APP metabolism in mice may be dissociated from cholinergic neurotransmission rather than related as previously suggested in other mammalian species.     

小檗碱对三转基因阿尔茨海默病小鼠的学习记忆和海马PSD95突触相关蛋白表达水平的影响

目的 探讨小檗碱(BBR)对三转基因阿尔茨海默病(AD)小鼠的学习记忆及海马组织PSD95突触蛋白表达水平的影响。方法 将30只三转基因(APP/Tau/PS1)AD小鼠按随机数字表法分成3组,即AD对照组、AD+25 mgBBR组、AD+50 mgBBR,每组各10只,后2组以灌胃方式且剂量分别为25 mg·kg^-1·d^-1、50 mg·kg^-1·d^-1,对照组给予等剂量生理盐水连续3个月灌胃处理; 采用Morris水迷宫方法探测各组AD小鼠行为学改变、空间记忆及探索情况; 免疫荧光染色检测各组小鼠海马组织突触后致密蛋白95(PSD95)阳性表达水平; Western blotting(WB)法检测各组三转基因AD小鼠海马脑组织PSD95蛋白、磷酸化蛋白激酶B(p-Akt)和磷酸化雷帕霉素靶蛋白(p-mTOR)表达水平及微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)自噬水平。结果 AD+25 mgBBR组的逃避潜伏期的学习记忆能力、免疫荧光PSD95表达水平以及PSD995、LC3-Ⅱ、p-Akt、p-mTOR蛋白表达水平与AD对照组比较均有明显差异(P<0.05); AD+50 mgBBR组逃避潜伏期的学习记忆能力、免疫荧光PSD95表达水平以及LC3-Ⅱ、p-Akt、p-mTOR表达水平与AD对照组比较差异均更明显(P<0.05,P<0.01)。结论 应用50 mg小檗碱能较好改善三转基因AD小鼠的学习记忆、空间探索能力,其机制可能是通过增加自噬水平LC3-Ⅱ调控Akt/mTOR信号通路,增加突触蛋白PSD95的表达水平及突触数量,以改善AD相关临床症状。     

Potential in vivo amelioration by N‐acetyl‐L‐cysteine of oxidative stress in brain in human double mutant APP/PS‐1 knock‐in mice: Toward therapeutic modulation of mild cognitive impairment

Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly. Although the underlying cause has yet to be established, numerous data have shown that oxidative stress is implicated in AD as well as in preclinical stages of AD, such as mild cognitive impairment (MCI). The oxidative stress observed in brains of subjects with AD and MCI may be due, either fully or in part, to increased free radicals mediated by amyloid‐β peptide (Aβ). By using double human mutant APP/PS‐1 knock‐in mice as the AD model, the present work demonstrates that the APP/PS‐1 double mutation results in elevated protein oxidation (as indexed by protein carbonyls), protein nitration (as indexed by 3‐nitrotyrosine), as well as lipid peroxidation (as indexed by protein‐bound 4‐hydroxy‐2‐nonenal) in brains of mice aged 9 months and 12 months. APP/PS‐1 mice also exhibited lower levels of brain glutathione peroxidase (GPx) in both age groups studied, whereas glutathione reductase (GR) levels in brain were unaffected by the mutation. The activities of both of these antioxidant enzymes were significantly decreased in APP/PS‐1 mouse brains, whereas the activity of glucose‐6‐phosphate dehydrogenase (G6PDH) was increased relative to controls in both age groups. Levels of peptidyl prolyl isomerase 1 (Pin1) were significantly decreased in APP/PS‐1 mouse brain aged 9 and 12 months. Administration of N‐acetyl‐L‐cysteine (NAC), a glutathione precursor, to APP/PS‐1 mice via drinking water suppressed increased protein oxidation and nitration and also significantly augmented levels and activity of GPx in brain from both age groups. Oral administration of NAC also increased the diminished activity of GR and protected against lipid peroxidation in brains of 9‐month‐old APP/PS‐1 mice only. Pin1 levels, GR levels, and G6PDH activity in brain were unaffected by oral administration of NAC in both age groups. These results are discussed with reference to the therapeutic potential of this brain‐accessible glutathione precursor in the treatment of MCI and AD. © 2010 Wiley‐Liss, Inc.     

Deposition of mouse amyloid beta in human APP/PS1 double and single AD model transgenic mice

The deposition of amyloid beta (Abeta) peptides and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). To investigate the relation between amyloid precursor protein (APP) production, amyloid beta deposition and the type of Abeta in deposits, i.e., human and/or mouse, we performed a histopathological analysis, using mouse and human specific antibodies, of the neocortex and hippocampus in 6, 12 and 19 months old APP/PS1 double and APP and PS1 single transgenic mice. There was a significant correlation between the human amyloid beta deposits and the intrinsic rodent amyloid beta deposits, that is, all plaques contained both human and mouse Abeta, and the diffuse amyloid beta deposits also colocalized human and mouse Abeta. Furthermore, some blood vessels (mainly leptomeningeal vessels) show labeling with human Abeta, and most of these vessels also label with mouse Abeta. Our findings demonstrate that the human amyloid deposits in APP/PS1 transgenic mice are closely associated with mouse Abeta, however, they do not precisely overlap. For instance, the core of plaques consists of primarily human Abeta, whereas the rim of the plaque contains both human and mouse amyloid beta, similarly, human and mouse Abeta are differentially localized in the blood vessel wall. Finally, as early as amyloid beta deposits can be detected, they show the presence of both human and mouse Abeta. Together, these data indicate that mouse Abeta is formed and deposited in significant amounts in the AD mouse brain and that it is deposited together with the human Abeta.     

Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology

Summary. The cleavage of APP by BACE initiates the amyloidogenic process in Alzheimers disease (AD). We have generated transgenic mice expressing BACE and double transgenic mice expressing BACE and the Swedish mutations of APP (SwAPP) in neurons. BACE transgenic mice did not develop -amyloid plaques by age of 14 months, but showed intracellular -amyloid immunoreactivity that was co-localized with transgenic BACE in neurons. A levels were increased and AD-like pathology was accelerated in double transgenic mice expressing both BACE and SwAPP. At two months of age, early signs of extracellular A deposition and reactive astrocytes were found in double transgenic, but not in single transgenic mice. Furthermore, at four months, well defined -amyloid deposits surrounded by activated astrocytes could be detected in the double transgenic mice. We suggest that BACE overexpression is not sufficient to produce -amyloid plaques, but simultaneous expression of BACE and its substrate (SwAPP) leads to an accelerated amyloid plaque formation.     

Regulation of astrocyte pathology by fluoxetine prevents the deterioration of Alzheimer phenotypes in an APP/PS1 mouse model

Studies have implicated astrocytic dysfunction in Alzheimer's disease (AD). However, the role of astrocytes in the pathophysiology and treatment of the disease is poorly characterized. Here, we identified astrocytes as independent key factors involved in several Alzheimer‐like phenotypes in an APP/PS1 mouse model, including amyloid pathology, altered neuronal and synaptic properties, and impaired cognition. In vitro astrocytes from APP/PS1 mice induced synaptotoxicity as well as reduced dendritic complexity and axonal branching of hippocampal neurons. These astrocytes produced high levels of soluble β‐amyloid (Aβ) which could be significantly inhibited by fluoxetine (FLX) via activating serotonin 5‐HT₂ receptors. FLX could also protect hippocampal neurons against astrocyte‐induced neuronal damage in vitro. In the same APP/PS1 mice, FLX inhibited activation of astrocytes, lowered Aβ products, ameliorated neurotoxicity, and improved behavioral performance. These findings may provide a basis for the clinical application of FLX in patients, and may also lay the groundwork for exploration of other novel astrocyte‐based therapies of AD. GLIA 2016;64:240–254     

Olfactory dysfunction in the APP/PS1 transgenic mouse model of Alzheimer's disease: Morphological evaluations from the nose to the brain

Olfactory dysfunction is among the signs of Alzheimer's disease (AD) and cognitive impairment. It has been demonstrated Aβ was associated with olfactory impairment observed in both transgenic mice and in AD patients. In this study, we evaluated amyloid deposition in the olfactory circuit of APP/PS1 transgenic mouse model of AD, which showed olfactory dysfunction in olfactory behavior tests. We found amyloid depositions were widely distributed in the whole olfactory circuit. Moreover, we think these amyloid depositions contribute to neuronal atrophy, dendritic abnormalities, synapse loss and axonal degeneration. Therefore, there was a correlation between olfactory deficits and amyloid deposition. Our findings provide initial insights into the pathological basis of AD‐related olfactory dysfunction.     

Temporal memory deficits in Alzheimer's mouse models: rescue by genetic deletion of BACE1

Transgenic mouse models of Alzheimer's disease (AD) exhibit amyloid-beta (Abeta) accumulation and related cognitive impairments. Although deficits in hippocampus-dependent place learning have been well characterized in Alzheimer's transgenic mice, little is known about temporal memory function in these AD models. Here, we applied trace fear conditioning to two different Alzheimer's mouse models and investigated the relationship between pathogenic Abeta and temporal memory deficits. This behavioral test requires hippocampus-dependent temporal memory processing as the conditioned and unconditioned stimuli are separated by a trace interval of 30 s. We found that both amyloid precursor protein (APP) transgenic (Tg2576) and APP/presenilin (PS)1 transgenic (Tg6799) mice were impaired in memorizing this association across the time gap. Both transgenic groups performed as well as wild-type control mice in delay fear conditioning when the trace interval was removed, indicating that the trace conditioning deficits are hippocampus-specific. Importantly, Tg6799 mice engineered to lack the major Alzheimer's beta-secretase (beta-site APP-cleaving enzyme 1: BACE1) showed behavioral rescue from temporal memory deficits. Elevated levels of soluble Abeta oligomers found in Tg6799+ mouse brains returned to wild-type control levels without changes in APP/PS1 transgene expression in BACE1-/- * Tg6799+ bigenic mouse brains, suggesting Abeta oligomers as potential mediators of memory loss. Thus, trace fear conditioning is a useful assay to test the mechanisms and therapeutic interventions for Abeta-dependent deficits in temporal associative memory. Our gene-based approach suggests that lowering soluble Abeta oligomers by inhibiting BACE1 may be beneficial for alleviating cognitive disorders in AD.