龟龄集对 AD 小鼠皮层和海马 Fas/ FasL 表达及神经元凋亡的影响

目的 探究龟龄集对阿尔茨海默病 (Alzheimer’s disease, AD) 小鼠皮层和海马 Fas/ FasL 表达及 神经元凋亡的影响。 方法 构建 AD 小鼠模型, 小鼠随机分为对照组、 模型组、 多奈哌齐组及龟龄集低、 中、 高剂量组。 Morris 水迷宫检测干预前后小鼠的学习记忆能力; HE 染色检测小鼠皮层、 海马神经元病理 学改变情况; TUNEL 染色检测神经元凋亡情况; Western 印迹及 Real time PCR 分别检测 Fas、 FasL 的蛋白 表达水平及 mRNA 表达水平。 结果 与模型组比较, 龟龄集各组和多奈哌齐组小鼠学习记忆能力明显提高 (P< 0. 05), 皮层和海马神经元病理学损害及神经元凋亡改善 (P< 0. 05), Fas、 FasL 蛋白和 mRNA 水平下 降 (P< 0. 05)。 结论 龟龄集可能通过抑制 Fas/ FasL 表达抑制 AD 模型小鼠皮层、 海马神经元凋亡, 并改 善其学习记忆能力。     

Protective effect of chronic caffeine intake on gene expression of brain derived neurotrophic factor signaling and the immunoreactivity of glial fibrillary acidic protein and Ki-67 in Alzheimer’s disease

Alzheimer’s disease (AD) is a neurodegenerative disorder with progressive degeneration of the hippocampal and cortical neurons. This study was designed to demonstrate the protective effect of caffeine on gene expression of brain derived neurotrophic factor (BDNF) and its receptor neural receptor protein-tyrosine kinase-β (TrkB) as well as glial fibrillary acidic protein (GFAP) and Ki-67 immunoreactivity in Aluminum chloride (AlCl₃) induced animal model of AD. Fifty adult rats included in this study were classified into 5 group (10 rats each); negative and positive control groups (I&II), AD model group (III), group treated with caffeine from the start of AD induction (IV) and group treated with caffeine two weeks before AD induction (V). Hippocampal tissue BDNF and its receptor (TrkB) gene expression by real time RT-PCR in addition to immunohistochemical study of GFAP and Ki67 immunoreactivity were performed for all rats in the study. The results of this study revealed that caffeine has protective effect through improving the histological and immunohistochemical findings induced by AlCl₃ as well as BDNF and its receptor gene expression. It could be concluded from the current study, that chronic caffeine consumption in a dose of 1.5 mg/kg body weight daily has a potentially good protective effect against AD.     

Protective effects of lycopene against amyloid β-induced neurotoxicity in cultured rat cortical neurons

The neurotoxicity of amyloid β (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). Among antioxidant phytochemicals derived from fruit and vegetables, lycopene has recently received considerable attention for its potent protective properties already demonstrated in several models of oxidative damage. The present study aims to investigate whether lycopene could provide protective effects against Aβ-induced neurotoxicity in primary cultured rat cortical neurons. The cultured cortical neurons were pretreated with different dose of lycopene for 4 h, followed by the challenge with 25 μM Aβ_25–35 for 24 h. The results showed that pretreatment with lycopene efficiently attenuated Aβ_25–35-induced neurotoxicity, as evidenced by the improved cell viability and the decreased apoptotic rate. In addition, lycopene inhibited the reactive oxygen species generation and mitochondrial membrane potential depolarization caused by Aβ_25–35. Lycopene also restored the levels of proapoptotic Bax, antiapoptotic Bcl-2, and inhibited caspase-3 activation. These beneficial effects may contribute to the protection against Aβ-induced neurotoxicity. Together, our results suggest that the natural antioxidant lycopene has potential for neuroprotection and therefore, may be a promising candidate for AD treatment.     

Activation of nicotinic α7 acetylcholine receptor enhances long term potentation in wild type mice but not in APPswe/PS1ΔE9 mice

Amyloid β (Aβ) plays a central role in Alzheimer's disease (AD) and binds to the nicotinic α₇ receptor (α₇ nAChR). Little is known about the degree to which the binding of Aβ to the α₇ nAChR influences the role of this receptor in long-term potentiation (LTP), however. We have studied the effect of the partial α₇ nAChR agonist SSR180711 on hippocampal slice preparations from normal wild type (Wt) and APP_swe/PS1ΔE9 transgenic (Tg) mice. In the hippocampal slices from the 6 months old Wt mice, the application of both nicotine (5 μM) and SSR180711 (300 nM) resulted in a significant enhancement of LTP expressed in area CA1. However, in the Tg mice the application of SSR180711 did not result in an increase in LTP beyond control levels. The amount of binding of the α₇ nAChR ligand 125-I-α-bungarotoxin was not different between in Tg and Wt mice. These findings indicate that the α₇ nAChR is functionally blocked in the hippocampal neurons, downstream of the α₇ nAChR, and that this is likely due to an interaction between the receptor and Aβ, which leads to changes in LTP.     

灵芝孢子油与深海鱼油联合应用对小鼠学习记忆及海马神经元表达NOS的影响

目的探讨灵芝孢子油与深海鱼油联合应用对小鼠学习记忆及海马神经元表达NOS的影响。方法将受孕小鼠随机分为4组,自受孕第1天起分别胃饲生理盐水、灵芝孢子油、深海鱼油和灵芝孢子油加深海鱼油。在母鼠分娩21d后改为胃饲其幼鼠,然后将出生后45d的幼鼠处死。处死前进行Morris水迷宫行为学测试,处死后应用酶组织化学法检测海马神经元NOS的表达。结果在Morris水迷宫检测中,灵芝孢子油组、深海鱼油组和灵芝孢子油加深海鱼油组小鼠的逃逸潜伏期明显缩短。灵芝孢子油组和灵芝孢子油加深海鱼油组的小鼠平台象限游泳距离有增加。灵芝孢子油加深海鱼油组的小鼠大脑海马NOS阳性神经元与其它组的小鼠比较有显著性增加。结论灵芝孢子油和深海鱼油联合应用能够促进小鼠学习记忆能力及其大脑海马神经元表达NOS。     

Beta amyloid peptide plaques fail to alter evoked neuronal calcium signals in APP/PS1 Alzheimer's disease mice

Alzheimer's disease (AD) is a multifactorial disorder of unknown etiology. Mechanistically, beta amyloid peptides (Aβ) and elevated Ca²⁺ have been implicated as proximal and likely interactive features of the disease process. We tested the hypothesis that proximity to Aβ plaque might exacerbate activity-dependent neuronal Ca²⁺ signaling in hippocampal pyramidal neurons from APP_SWE/PS1_M146V mice. Using combined approaches of whole cell patch clamp recording and 2-photon imaging of neuronal Ca²⁺ signals with thioflavin-S plaque labeling in hippocampal slices, we found no correlation between thioflavin-S labeled Aβ plaque proximity and Ca²⁺ responses triggered by ryanodine receptor (RyR) activation or action potentials in either dendrites or somata of AD mice, regardless of age. Baseline and RyR-stimulated spontaneous excitatory postsynaptic potentials also showed little difference in relation to Aβ plaque proximity. Consistent with previous studies, RyR-evoked Ca²⁺ release in APP_SWE/PS1_M146V mice was greater than in nontransgenic controls. Within the soma, RyR-evoked Ca²⁺ release was elevated in older APP_SWE/PS1_M146V mice compared with younger APP_SWE/PS1_M146V mice, but was still independent of plaque proximity. The results indicate that early Ca²⁺ signaling disruptions can become yet more severe with age through mechanisms independent of Aβ plaques, suggesting that alternative pathogenic mechanisms might contribute to AD-associated dysfunction.     

Neuropep-1 ameliorates learning and memory deficits in an Alzheimer's disease mouse model,increases brain-derived neurotrophic factor expression in the brain,and causes reduction of amyloid beta plaques

Alzheimer's disease (AD) is a neurodegenerative disease characterized by amyloid beta (Aβ) deposits, hyperphosphorylated tau deposition, and cognitive dysfunction. Abnormalities in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in learning and memory formation, have been reported in the brains of AD patients. A BDNF modulating peptide (Neuropep-1) was previously identified by positional-scanning synthetic peptide combinatorial library. Here we examine the neuroprotective effects of Neuropep-1 on several in vitro neurotoxic insults, and triple-transgenic AD mouse model (3xTg-AD). Neuropep-1 protects cultured neurons against oligomeric Aβ_1–42, 1-methyl-4-phenylpyridinium, and glutamate-induced neuronal cell death. Neuropep-1 injection also significantly rescues the spatial learning and memory deficits of 3xTg-AD mice compared with vehicle-treated control group. Neuropep-1 treatment markedly increases hippocampal and cortical BDNF levels. Furthermore, we found that Neuropep-1-injected 3xTg-AD mice exhibit dramatically reduced Aβ plaque deposition and Aβ levels without affecting tau pathology. Neuropep-1 treatment does not alter the expression or activity of full-length amyloid precursor protein, α-, β-, or γ-secretase, but levels of insulin degrading enzyme, an Aβ degrading enzyme, were increased. These findings suggest Neuropep-1 may be a therapeutic candidate for the treatment of AD.     

The neuroprotective role of microglial cells against amyloid beta‐mediated toxicity in organotypic hippocampal slice cultures

During Alzheimer’s disease (AD) progression, microglial cells play complex roles and have potentially detrimental as well as beneficial effects. The use of appropriate model systems is essential for characterizing and understanding the roles of microglia in AD pathology. Here, we used organotypic hippocampal slice cultures (OHSCs) to investigate the impact of microglia on amyloid beta (Aβ)‐mediated toxicity. Neurons in OHSCs containing microglia were not vulnerable to cell death after 7 days of repeated treatment with Aβ_1‐42 oligomer‐enriched preparations. However, when clodronate was used to remove microglia, treatment with Aβ_1‐42 resulted in significant neuronal death. Further investigations indicated signs of endoplasmic reticulum stress and caspase activation after Aβ_1‐42 challenge only when microglia were absent. Interestingly, microglia provided protection without displaying any classic signs of activation, such as an amoeboid morphology or the release of pro‐inflammatory mediators (e.g., IL‐6, TNF‐α, NO). Furthermore, depleting microglia or inhibiting microglial uptake mechanisms resulted in significant more Aβ deposition compared to that observed in OHSCs containing functional microglia, suggesting that microglia efficiently cleared Aβ. Because inhibiting microglial uptake increased neuronal cell death, the ability of microglia to engulf Aβ is thought to contribute to its protective properties. Our study argues for a beneficial role of functional ramified microglia whereby they act against the accumulation of neurotoxic forms of Aβ and support neuronal resilience in an in situ model of AD pathology.     

Ablation of the microglial protein DOCK2 reduces amyloid burden in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) neuropathology is characterized by innate immune activation primarily through prostaglandin E2 (PGE₂) signaling. Dedicator of cytokinesis 2 (DOCK2) is a guanyl nucleotide exchange factor expressed exclusively in microglia in the brain and is regulated by PGE₂ receptor EP2. DOCK2 modulates microglia cytokine secretion, phagocytosis, and paracrine neurotoxicity. EP2 ablation in experimental AD results in reduced oxidative damage and amyloid beta (Aβ) burden. This discovery led us to hypothesize that genetic ablation of DOCK2 would replicate the anti-Aβ effects of loss of EP2 in experimental AD. To test this hypothesis, we crossed mice that lacked DOCK2 (DOCK2 −/−), were hemizygous for DOCK2 (DOCK2 +/−), or that expressed two DOCK2 genes (DOCK2 +/+) with APPswe-PS1Δe9 mice (a model of AD). While we found no DOCK2-dependent differences in cortex or in hippocampal microglia density or morphology in APPswe-PS1Δe9 mice, cerebral cortical and hippocampal Aβ plaque area and size were significantly reduced in 10-month-old APPswe-PS1Δe9/DOCK2 −/− mice compared with APPswe-PS1Δe9/DOCK2 +/+ controls. DOCK2 hemizygous APPswe-PS1Δe9 mice had intermediate Aβ plaque levels. Interestingly, soluble Aβ₄₂ was not significantly different among the three genotypes, suggesting the effects were mediated specifically in fibrillar Aβ. In combination with earlier cell culture results, our in vivo results presented here suggest DOCK2 contributes to Aβ plaque burden via regulation of microglial innate immune function and may represent a novel therapeutic target for AD.     

雷公藤内酯醇对AD细胞模型海马神经元凋亡的影响

目的:研究雷公藤内酯醇对阿尔茨海默病(AD)细胞模型海马神经元凋亡的影响,探讨雷公藤内酯醇治疗AD的可能机制。方法:用凝聚态Aβ1-40(20μg/ml)刺激的小胶质细胞条件培养液(MCM)作用于培养的大鼠海马神经元,建立AD细胞模型,应用MTT法和TUNEL染色,观察不同剂量的雷公藤内酯醇(5μg/ml和25μg/ml)在不同时程(2 h和24 h)内对AD细胞模型海马神经元凋亡的影响。结果:加药后2 h除模型MCM组海马神经元凋亡数高于正常对照组和正常MCM组(P<0.05)外,其余各组之间海马神经元凋亡数无明显差异。加药后24 h,模型MCM组海马神经元凋亡数较正常对照组和正常MCM组显著增多(P<0.01);低剂量用药MCM组和高剂量用药MCM组海马神经元凋亡数与模型MCM组比较显著降低(P<0.05,P<0.01);高剂量用药MCM组海马神经元凋亡数较低剂量用药MCM组明显降低(P<0.01)。结论:雷公藤内酯醇对AD细胞模型海马神经元的凋亡具有抑制作用。     

Analgesic effect of intrathecally γ-aminobutyric acid transporter-1 inhibitor NO-711 administrating on neuropathic pain in rats

Neuregulin-1 (NRG1) participates in numerous neurodevelopmental processes and plasticity of the brain. Despite this, little is known about its role in Alzheimer's disease (AD). Amyloid β (Aβ) peptide is generally believed to play a critical role in the pathogenesis of AD. The present study examined the effect of synthetic Aβ_1-42 peptides on long-term potentiation (LTP) in the CA1 region of mice hippocampal slices, a cellular model of learning and memory. We found that application of a test dose of Aβ_1-42 (200 nM) significantly inhibited the development of LTP without affecting basal synaptic transmission. Pretreatment with NRG1 effectively prevented Aβ_1-42-induced impairment of LTP, an effect that was dose-dependent. This LTP-restoring action of NRG1 was almost completely abolished by blocking ErbB4, a key NRG1 receptor, suggesting that NRG1 acts through ErbB4 to exert its protective action on LTP. The present study thus provides the first demonstration that NRG1/ErbB4 protects against Aβ-induced hippocampal LTP impairment, suggesting that NRG1 may be a promising candidate for the treatment of early-stage AD.     

L-type Ca currents at CA1 synapses,but not CA3 or dentate granule neuron synapses,are increased in 3xTgAD mice in an age-dependent manner

Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer's disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca²⁺ currents (L-VGCC), 4–aminopyridine-sensitive K⁺ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid β-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca²⁺ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K⁺ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca²⁺, K⁺, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients.     

Why do Alzheimer’s disease and Parkinson’s disease target the same neurons?

The puzzle is to explain how cerebral involvement in the sporadic forms of Alzheimer’s disease (AD) and Parkinson’s disease (PD) can target the same population of vulnerable neurons. These neurons are poorly-myelinated projection neurons, lack of myelin being associated with high metabolic demand, high oxygen consumption, and high baseline oxidative stress. Yet the two diseases are clearly separable, with different intracellular markers, different risk factors, and different patterns of subcortical involvement.A theory is developed to show how two different pathophysiologies can preferentially affect the same neurons. In the case of AD, the hypothesis is as follows: the so-called vascular risk factors of AD, which include hypertension, diabetes, hyperlipidemia, and smoking, are all associated with increased systemic extracellular oxidative stress. High extracellular oxidative stress synergizes with high baseline intracellular oxidative stress to cause the disease. In the case of PD, mitochondrial failure associated with normal aging leads to diminished energy production and increased leakage of reactive oxygen species from mitochondria, a process which preferentially targets neurons with high baseline oxidative stress. In one case, the extra oxidative stress comes from outside the cell and, in the other case, it comes from inside the cell, i.e. from mitochondria. There is also evidence that neurofibrillary tangles are a protective mechanism against extracellular oxidative stress and that α-synuclein is a marker for mitochondrial failure. The basic pathophysiological difference is that AD is caused by oxidative stress alone, whereas PD is caused by oxidative stress plus failure of energy production.     

Beneficial synergistic effects of microdose lithium with pyrroloquinoline quinone in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is a complicated, neurodegenerative disorder involving multifactorial pathogeneses and still lacks effective clinical treatment. Recent studies show that lithium exerts disease-modifying effects against AD. However, the intolerant side effects at conventional effective dosage limit the clinical use of lithium in treating AD. To explore a novel AD treatment strategy with microdose lithium, we designed and synthesized a new chemical, tri-lithium pyrroloquinoline quinone (Li₃PQQ), to study the synergistic effects of low-dose lithium and pyrroloquinoline quinone, a native compound with powerful antioxidation and mitochondrial amelioration. The results showed that Li₃PQQ at a relative low dose (6 and 12 mg/kg) exhibited more powerful effects in restoring the impairment of learning and memory, facilitating hippocampal long-term potentiation, and reducing cerebral amyloid deposition and phosphorylated tau level in APP/PS1 transgenic mice than that of lithium chloride at both low and high dose (5 and 100 mg/kg). We further found that Li₃PQQ inhibited the activity of glycogen synthase kinase-3 and increased the activity of β-amyloid-binding alcohol dehydrogenase, which might underlie the beneficial effects of Li₃PQQ on APP/PS1 transgenic mice. Our study demonstrated the efficacy of a novel AD therapeutic strategy targeting at multiple disease-causing mechanisms through the synergistic effects of microdose lithium and pyrroloquinoline quinone.     

Modelling of amyloid β-peptide induced lesions using roller-drum incubation of hippocampal slice cultures from neonatal rats

Pronounced neurodegeneration of hippocampal pyramidal neurons has been shown in Alzheimer’s disease. The aim of this study was to establish an organotypic in vitro model for investigating effects of the amyloid β (Aβ)-peptide on pyramidal neuron degeneration, glial cell activation and tau phosphorylation. Tissue cultures in a quasi-monolayer were obtained using roller-drum incubation of hippocampal slices from neonatal Sprague Dawley rats. Neuronal populations identified included N-methyl-D-aspartate (NMDA-R1) receptor immunoreactive pyramidal neurons, and neurons immunopositive for glutamic acid decarboxylase-65 (GAD65) or gamma amino butyric acid (GABA). Many neurons expressed phosphorylated tau as shown by pS³⁹⁶, AD2 and PHF-tau immunostaining. Astrocytes, microglial cells and macrophages were also identified. The Aβ_25–35 peptide formed fibrillar networks within 2 days as demonstrated by electron microscopy. In the presence of the neurotoxic Aβ_25–35 peptide, but not Aβ_35–25, deposits developed in the tissue that were stainable with Thioflavine T and Congo red and showed the characteristic birefringence of Aβ plaques. Following Aβ_25–35 exposure, neurodegenerative cells were observed with Fluoro-Jade B staining. Further characterization of pyramidal neurons immunopositive for NMDA-R1 showed a decrease of cell number in the immediate surrounding of Aβ_25–35 deposits in a time- and concentration-dependent fashion. Similar effects on pyramidal neurons were obtained following exposure to the full-length, Aβ_1–40 peptide. Also, a loss of neuronal processes was seen with GAD65, but not GABA, immunohistochemistry after exposure to Aβ_25–35. Aβ_25–35-exposed neurons immunopositive for phospho-tau showed degenerating, bent and often fragmented processes. Astrocytes showed increased GFAP-positive reactivity after Aβ_25–35 exposure and formation of large networks of processes. No obvious effect on microglial cells and macrophages could be seen after the Aβ_25–35 exposure. The developed in vitro system may constitute a useful tool for screening novel drugs against Aβ-induced alterations of tau and degeneration of hippocampal neurons.     

Mechanisms underlying basal and learning-related intrinsic excitability in a mouse model of Alzheimer's disease

Accumulations of β-amyloid (Aβ) contribute to neurological deficits associated with Alzheimer's disease (AD). The effects of Aβ on basal neuronal excitability and learning-related AHP plasticity were examined using whole-cell recordings from hippocampal neurons in the 5XFAD mouse model of AD. A robust increase in Aβ42 (and elevated levels of Aβ38-40) in naïve 5XFAD mice was associated with decreased basal neuronal excitability, evidenced by a select increase in Ca²⁺-sensitive afterhyperpolarization (AHP). Moreover, trace fear deficits observed in a subset of 5XFAD weak-learner mice were associated with a greater enhancement of the AHP in neurons, as compared to age-matched 5XFAD learner and 5XFAD naïve mice. Importantly, learning-related plasticity of the AHP remained intact in a subset of 5XFAD mice that learned trace fear conditioning to a set criterion. We show that APP-PS1 mutations enhance Aβ and disrupt basal excitability via a Ca²⁺-dependent enhancement of the AHP, and suggest disruption to learning-related modulation of intrinsic excitability resulted, in part, from altered cholinergic modulation of the AHP in the 5XFAD mouse model of AD (170 of 170).     

Immunohistochemical detection of the delayed formation of nonfibrillar large amyloid‐β aggregates

The occurrence of senile plaques consisting of amyloid‐β protein (Aβ) is a major neuropathological hallmark of Alzheimer's disease (AD). We previously developed and characterized monoclonal antibodies 31‐2 and 75‐2 that specifically bind to nonfibrillar Aβ_1–42 aggregates with diameters of more than 220 and 50 nm, respectively. Here, we report the use of these antibodies to examine the aggregation of exogenous Aβ_1–42 in cultured rat hippocampal neurons. From 6 to 24 h after transfection of Aβ_1–42, antibody 75‐2 immunolabeled almost all transfected neurons, whereas 31‐2‐positive cells were restricted to a part of the transfected neurons and gradually increased in number. Expression of the F19S/L34P‐mutant Aβ_1–42, which showed less of a tendency to aggregate, resulted in clearly reduced immunoreactivity to both antibodies. We also immunohistochemically investigated the temporal cortices of patients with AD and found that 31‐2 preferentially labeled the cores of a subpopulation of large amyloid plaques. The relative number of 31‐2‐immunoreactive plaques was found to correlate with the Braak stages of neurofibrillary tangles, but not with that of amyloid plaques. These results suggest that 31‐2‐reactive Aβ aggregates develop with a delayed time course in cultured neurons and amyloid plaques of AD brains.     

Amyloid-β25–35 induces impairment of cognitive function and long-term potentiation through phosphorylation of collapsin response mediator protein 2

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) protein and tau deposition in the brain. Numerous studies have reported a central role of Aβ in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of Aβ neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2^ki/ki) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of Aβ_25–35 peptide, a neurotoxic fragment of Aβ protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aβ_25–35 peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2^ki/ki mice. In electrophysiological recording, wt and crmp2^ki/ki mice have similar input–output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of Aβ_25–35 peptide-treated wt but not those of crmp2^ki/ki. Our findings indicate that CRMP2 phosphorylation is required for Aβ-induced impairment of cognitive memory and synaptic plasticity.     

神经鞘磷脂合成酶2基因敲除小鼠海马神经细胞自噬现象

目的 利用神经鞘磷脂合成酶2（SMS2）基因敲除小鼠探讨神经酰胺对海马神经细胞自噬现象的影响。方法 将SMS2杂合子（SMS2^+/- ）小鼠采用杂交、回交、互交的方法进行繁殖,用酚-氯仿提取法提取小鼠基因组DNA,PCR扩增目的基因,琼脂糖凝胶电泳对小鼠基因型作出鉴定,建立纯合子（SMS2^-/- ）小鼠模型;采用透射电子显微镜、免疫荧光染色及Western blotting技术观察生后第7天（P7）、P14和P30 SMS2^-/-小鼠海马CA1区神经细胞自噬的发生（每种方法每个时间点8只小鼠）。结果 1.SMS2^-/-小鼠海马CA1区神经细胞自噬现象：电子显微镜下,模型组海马神经元内出现较多自噬体或自噬溶酶体样结构。光镜下,模型组P7、P14和 P30 CA1区神经元自噬细胞数较对照组高(P ＜0.01）;2.自噬相关蛋白Beclin-1对于自噬的调节作用：Beclin-1在模型组与对照组间的表达规律与微管相关蛋白1轻链3（MAPLC3）基本一致,P7、P14、P30模型组Beclin-1阳性细胞数明显多于对照组(P ＜0.01）。Beclin-1与 MAPLC3两者在同一细胞中基本呈重叠表达; 3.Western blotting检测各组海马CA1区神经细胞MAPLC3蛋白的相对表达量与上述结果一致。结论SMS2-/-小鼠海马神经细胞内神经酰胺增高,神经酰胺不仅促进细胞凋亡,同时促进自噬,造成小鼠海马神经细胞自噬现象增强。