Mechanisms Underlying ?-amyloid And Oxidative Stress-induced Neuronal Injury

Background: Alzheimer's disease is the most common neurodegenerative disease characterized by two hallmark pathologies,i.e.neurofibrillary tangles(NFTs)and senile plaques(SP).The senile plaques are formed by the extracellular aggregation of an amyloid peptide containing the structure of ?-sheet,which are mainly distributed in the hippocampus and cortex.It is generally believed that A? is able to cause neurotoxic effects by inflammatory reaction and oxidative stress.Microglia are the main immune cells of the central nervous system,participate in the immune defense in the brain,and keep the internal environment stable.Studies have shown that A? can damage neurons;however,the mechanisms underlying the toxical effects of A? on neurons are not fully understood.Objective: This paper was aimed to investigate the mechanisms underlying the A?-induced acute neuronal damages.Methods: Western blot was used to detect the alterations of total protein levels both in vivo and in vitro.Immunohistochemitry was used to detect the expression level and distribution of the target proteins in brain slices.Immunofluorescence double/triple co-labeling using two-photon confocal microscopy was used to detect the co-location of the related proteins.Eenzyme-linked immuno sorbent assay(ELISA)was used to detect the level of inflammatory cytokine in the cell and tissue extracts.Chromatin immunoprecipitation assay(CHIP)was used to detect the association of the protein and DNA.Golgi staining was used to measure the morphology and number of neuronal dendritic spines.The effect of microglia on the neuron was confirmed by co-calture of the conditioned microglial medium with primary hippocampal neurons.Novel object recognition test was used to detect the learning and memory abibilities of the mice.Results: 1.Lateral ventricle infusion of A?42 only induces cognitive deficit in acute phase.The aged A?42 or same volume of DMSO(the viechle control)was infused to the brain lateral ventricle of mice.The cognitive function was measured by using novel object recognition test at 24 h and 7 days respectively after the infusion.Compared with the control group,the mice at 24 h after A?42 infusion showed learning and memory deficits.No difference was detected between control and 7 days groups.2.Effects of A?42 on expression of synapse-associated proteins.The level of synaptic-associated protein was detected 24 h,7 days and 14 days respectively after intracerebroventricular infusion of A?42.At 24 h after A?42 infusion,the protein level of Glu A2 was significantly decreased accompanied with a marked increase in p S133-CREB compared with the control group.Golgi staining showed that the number of dendritic spines was significantly reduced in the A?42 group compared to the control group.At 7 days,no difference of the protein levels was detected.At 14 days,significant reduction of Glu N2 B was shown.3.A?42 activates microglia in the acute phase.At 24 h after intracerebroventricular injection of A?42,the microglial activation was detected,shown by the significantly increased number and the enlarged diameter(>1.5 times that of the control group)of the microglial cells.However,no differences of the microglial number and the morphology were detcetd between control and the A?42 infused groups at 7 days.4.Infusion of A?42 does not change numbers of astrocytes and neurons.By using GFAP(astrocyte marker)and Neu N(neuron marker)staining,no differences in astrocyte and neuron numbers were detected between A?42(24 h)and the veichle control groups.5.A?42 increases microglial level of p S133-CREB in the acute phase.At 24 h after infusion of A?42,the expression level of p S133-CREB was significantly increased in the hippocampal microglial cells,whereas the immunoreactivity of p S133-CREB in the granular cell layer and the pyramidal layer was significantly decreased.These changes were not detected at 7 days after A?42 exposure.6.A?42 decreases neuronal level of p S133-CREB in the acute phase.By immunofluorescence co-staining Neu N and p S133-CREB,we observed that intracerebroventricular infusion of aged A?42 remarkably decreased the level of p S133-CREB compared with the control group.7.Inhibition of microglial activity attenuates A?-induced microglial elevation of p S133-CREB with improved memory.Simultaneous inhibition of microglial activity by using minocycline attenuates A?42-induced CREB phosphorylation with reduction of inflammatory factors and improvement of synaptic and cognitive impairments.8.A?42 activates microglia by PKA-dependent CREB phosphorylation.The activity of PKA in Bv2 cells after A? treatment was increased,the phosphorylation level of Ser133 in CREB was also increased,and the secretion of inflammatory cytokine IL-6 was increased and the transcription of metalloproteinase-9 was increased.When phosphorylation of CREB was inhibited by PKA inhibitor H89 2HCl,IL-6 secretion was reduced and MMP-9 transcription was also reduced.9.A?42-conditioned bv2 culture medium inhibits neuronal expression of synapse-associated proteins in CREB phosphorylation-dependent manner.Treatment of primary neurons with A?42-conditioned bv2 culture medium results in decreased expression of synaptic-associated proteins in neurons,whereas inhibition of CREB phosphorylation in bv2 cells abolishes the effect.HVCN1(voltage-gated proton channels,VSOP/Hv1)is a voltage-gated proton channel expressed in immune cells.In the central nervous system,it is mainly expressed in microglia and participates in oxidative stress and immune phagocytosis.Some studies have found that HVCN1 may be a therapeutic target for cerebral ischemic injury because HVCN1 knockout can reduce the generation of reactive oxygen species(ROS)in microglia and prevent neuronal damage.Alzheimer's disease(AD)is a multi-factor chronic neurodegenerative disease.Oxidative stress and inflammation are also considered to be some of its pathogenesis.Our study found that in animal models of oxidative stress,the expression of HVCN1 was elevated with the increase of two major pathological proteins of AD,one is the phosphorylation of tau and the other is the ratio of A?42/A?40.In the A?-associated AD model,there is also an increase in HVCN1 and clustering around phosphorylated tau,suggesting that HVCN1 may participate in the pathogenesis of AD.