| Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is characterized by cognitive impairment. The neuropathological hallmarks of AD include intracellular neurofibrillary tangles, senile plaques (SP) accumulated by-amyloid peptide (Aβ) and neuron loss. Aβ can selectively activate N-methy-D-aspartate receptor (NMDAr) to enhance the NMDAr-mediated Ca2+influx, eventually inducing intracellular calcium overload and neuronal death.Sigma-1receptor (σ1R) is a G protein-coupled receptor containing223amino acid residues. The σ1R is mainly distributed in hippocampus and other limbic systems. Numerous studies have demonstrated that the aiR agonist can enhance the activation of NMDAr. Our previous research showed that the Ca2+influx across NMDAr was enhanced by the activation of σ1R to trigger phosphorylation. of NR2B in hippocampual CA1pyramidal neurons. Meanwhile, either σ1R deficiency or σ1R antagonists can arrest the NMDAr function through reducing the NR2B phosphorylation. Positron emission tomography (PET) has revealed that the densities of cerebral and cerebellar σ1R are reduced in early AD brains. The recent clinical results show that the polymorphism of σ1R is associated with complete linkage disequilibrium in AD patients. Further study has found that the reduced σ1R density occurred mainly in the late-onset AD patients. Based on the clinical data and preliminary physiological study of σ1R, we proposed that reduced σ1R exerts a neuroprotective effect in AD brain through depressing the Aβ-enhanced Ca2+influx across NMDAr. To dermine whether reduced σ1R could influence Aβ-neurotoxicity, we employed Aβ25-35-injected heterozygous σ1R knockout (σ1R+/-) mice to explore the role of σ1R in the pathological process of AD.Objective1. To determine whethre σ1R deficiency could influence Aβ-induced neuronal death and cognitive deficits.2. To explore the molecular mechanisms underlying the effects of σ1R deficiency on Aβ-neurotoxicity.—The First PartMethods1. Genotypic identification:The genotype of mice was identified by PCR using DNA from mice tails, including homozygous (-/-), heterozygous (+/-) and wild-type (WT). Male σ1R+/-and WT littermates at12weeks age were used at the beginning of all experiments.2. Preparation of AD mouse model:Male mice were injected into ventricles (i.c.v.) with "aggregated" Aβ25-35(6nmol/3μl/mouse). Aβ25-35-treated mice contain three experimental groups:Aβ25-35-treated WT mice (AP25-35-WT mice), Aβ25-35-treated σ1R+/-mice (Aβ25-35-σ1R+/-mice) and Aβ25-35-treated σ1R-/-mice (Aβ25-35-σ1R-/-mice). The control mice were injected with the same volume of dissolvent.3. Behavioral tests:Morris water maze task and Y-maze task were used to examine the cognitive performance.4. Histological examination:The pyramidal cells in hippocampal CA1and CA3regions were assessed by Toluidine blue staining and stereological analysis.5. Apoptosis examination:TUNEL and Hoechst staining were used to examine the number of apoptotic cells in hippocampal CA1region.6. Drugs administration:NE100(σ1R antagonist, lmg/kg) or PRE084(σ1R agonist,1mg/kg) were injected intraperitoneally once daily on days1-4after AP25-35-injection.Results1. Cognitive function:No significant difference in the escape-latency and alternation rate was noted between WT mice and σ1+/-mice. Aβ25-35-WT mice spent longer escape-latency to reach the hidden platform and showed less alternation rate compared to WT mice. By contrast, Aβ25-3s-σ1R+/-mice showed nearly the same time in the target quadrant and alternation rate as that in WT mice or σ1R+/-mice.2. Number of pyramidal cells in hippocampal CA1and CA3regions:Compared with WT mice, the number of pyramidal cells of σ1R+/-mice showed no difference. However, the number of surviving pyramidal cells reduced approximately25%in Aβ25-35-WT mice, but was not changed in Aβ25-35-σ1R+/-mice, compared to that in WT mice.3. The apoptotic cells in CA1:The TUNEL-positive cells and hoechst-positive cells were hardly observed in WT or σ1R+/-mice. The number of TUNEL-positive cells and Hoechst-positive cells were significantly increased in Aβ25-35-WT mice, but not in σ1R+/-or Aβ25-35-σ1R+/-mice.4. Effect of σ1R antagonist on AP25-35-WT mice:NE100was able to improve the Aβ25-35-induced pyramidal cell loss and spatial cognitive impairment in Aβ25-35-WT mice.5. Effect of σ1R agonist on Aβ25-35-σ1R+/-mice:PRE084can aggravate the Aβ25-35-induced pyramidal cell loss and spatial cognitive impairment in Aβ25-35-σ1R+/-mice. Conclusions1. The injection (i.c.v.) of Aβ25-35in WT mice impaires the cognitive performance and induces the death of hippocampal pyramidal cells.2. The σ1R deficiency can suppress the cognitive deficits and the apoptosis of neuronal cells induced by Aβ.—The Second PartMethods1. Electrophysiological analysis:At48h and72h after Aβ25-35-injection, the brains were rapidly removed and the coronal brain slices were cut in order to examine NMDA activated currents (INMDA) in hippocampal CA1pyramidal cells.2. Western blot analysis:the levels of phospho-NR2B in hippocampus at48h and72h after Aβ25-35-injection were examined.3. Drugs administration:NE100(1mg/kg), Ro25-6981(NR2B inhibitor,6mg/kg), PRE084(lmg/kg) or NMDA (NMDAr agonist,30mg/kg) were injected (i.p.) once daily on days1-4after Aβ25-35-injection.Results1. NMDA-activated currents (INMDA) in hippocampal CA1pyramidal cells:In comparison with WT mice,INMDA of σ1R+/-mice in hippocampal CA1pyramidal cells was reduced, consisting with our previous studies of σ1R-/-mice.INMDA in Aβ25-35-WT mice were increased nearly2-folds at48h after Aβ25-35-injection (post-AP25-35) followed by approximately40%decline at72h post-Aβ25-35. The INMDA density in Aβ25-35-σ1R+/-mice almost shows no difference at48h or72h post-Aβ25-35compared to σ1R+/-mice.2. Level of phospho-NR2B in hippocampus:The level of phospho-NR2B in σ1R+/- mice was obviously lower than WT mice, consisting with our previous studies of σ1R-7-mice as well. The phospho-NR2B levels of hippocampus in Aβ25-35-WT mice were increased nearly2-folds at48h post-Aβ25-35followed by obiviously decline at72h post-Aβ25-35. The levels of phospho-NR2B in AP25-35-σ1R+/-mice almost had no difference at48h or72h post-Aβ25-35compared to σ1R+/-mice.3. Effect of σ1R antagonist on AP25-35-WT mice:The changes in INMDA density and phospho-NR2B in AP25-35-WT mice could be inhibited by the σ1R antagonist NE100.4. Effect of σ1R agonist on Aβ25-35-σ1R+/-mice:INMDA density and phospho-NR2B in PRE084-treated Aβ25-35-σ1R+/-mice increaed at48h but decreased at72h post-Aβ25-35, which is comparable to those in Aβ25-35-WT mice.5. Effect of NR2B inhibitor on Aβ25-35-WT mice:Ro25-6981can improve Aβ25-35-induced neuron loss and spatial cognitive impairment in Aβ25-35-WT mice.6. Effect of NMDAr agonist on Aβ25-35-σ1R+/-mice:NMDA can aggravate the Aβ25-35-induced neuron loss and spatial cognitive impairment in mice.ConclusionsThe σ1R deficiency, through depressing Aβ25-35-induced phosphorylation of NR2B and over-activation of NMDAr, inhibits intracellular calcium overload and prevents early neuronal apoptosis. |
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