Cannabinoid Type 2 Receptor Protects Hippocampal Neurons Against A?_1-42-induced Apoptosis

Alzheimer's disease?AD?is a common neurodegenerative disease,which is characterized by deposition and aggregation of amyloid?-protein,which may lead to a progressive degeneration of hippocampal neurons.The main feature of AD is that a large number of senile plaques?SP?appear in the brain,and neuronal fiber tangles?NFT?appear in the neurons.However,the etiology of AD has not yet been fully clarified until now.Studies have shown that in the patient's brain,especially the hippocampus,the accumulation of amyloid?-protein_1-42(A?_1-42)is significantly increased,which exhibits significant relationship with the progress of AD pathogenesis.Inhibiting the toxic effects of A?_1-42 may be an important pathological strategy to protect hippocampal neurons.A?_1-42 is a kind of peptide that is very easy to aggregate.A?_1-42 in the brain will form oligomers and fibrils.Many studies have shown that A?_1-42 oligomers have stronger toxicity.It has been reported that A?_1-42 oligomers can either activate microglia to cause inflammation,or directly damage hippocampal neurons,which eventually leads to a decline in learning and memory.Brain contains endogenous cannabinoid system including endogenous materials and cannabinoid receptors.In many neurodegenerative diseases,the expression of cannabinoid receptor 2?CB₂R?is significantly increased,suggesting that CB₂R may be involved in the pathogenesis and process of these diseases,and pharmacological manipulations of CB₂R exhibit neuron protective effects.CB₂R is a G protein-coupled receptor.Studies have shown that the activation of CB₂R can inhibit microglia activation,and reduce the damage caused by traumatic brain injury and cerebral ischemia.However,whether CB₂R activation plays a role in hippocampal neuron apoptosis induced by chronic A?_1-42 and its possible signal pathway is unclear.In this study,we address these questions.Using primary cultured rat hippocampal neurons,we chronically applied A?_1-42 to induce cell apoptosis,and examined the effects of CB₂R agonist JWH133 on the A?_1-42-induced neuronal toxicity.We used flow cytometry,LDH kit,Quantitative Real-time PCR,immunoblotting and other experimental methods to evaluate the roles and possible signal pathway of CB₂R activation in protection of hippocampal neurons against the A?_1-42-induced toxicity.The results were shown as followings:1.In primary cultured hippocampal neurons,compared to control group(without A?_1-42treatment),7 days treatment with 100 nmol/L A?_1-42 oligomer decreased the cell survival rate?P<0.05?,increased LDH release level?P<0.01?.2.Compared to Control group,7 days treatment with 100 nmol/L A?_1-42 oligomer increased CB₂R mRNA expression in hippocampal cultures detected by qRT-PCR?P<0.001?.3.Pre-incubate hippocampal neurons with 10?mol/L CB₂R agonist,JWH133 for 50 minutes in advance,and then 100 nmol/L A?_1-42 oligomer treatment,the A?_1-42-induced increase of LDH release level was prevented?P<0.01?,and this JWH133's protective effect was abolished by the addition of CB₂R antagonist,AM630?P<0.01?.4.We used Flow Cytometry to detect the change of mitochondrial membrane potential of hippocampal neurons,and the ratio of subdivision,??m in A?group decreased significantly?P<0.01?;Compared with A?group,the??m in A?+JWH133 group increased?P<0.01?;but the??m in A?+AM630+JWH133 group was significantly decrease compared with A?+JWH133 group?P<0.01?.5.We used Flow Cytometry to detect changes in ROS levels in hippocampal neurons.Compared with the control group,the expression of ROS in the A?group was significantly increased?P<0.01?;Compared with A?group,the production of ROS in A?+JWH133 group decreased?P<0.01?;the expression of ROS in A?+AM630+JWH133 group increased compared with A?+JWH133 group?P<0.05?.6.We used qRT-PCR to detect the expression of Caspase-3 mRNA.Compared to control group,the expression of Caspase-3 mRNA in the 100 nmol/L A?oligomer treated group was increased significantly?P<0.01?;Pre-incubation of JWH133 prevented the A?-induced increase of Caspase-3 mRNA?P<0.01?,and this neuron protective effect was abolished by CB₂R antagonist,AM630?P<0.001?.7.We used qRT-PCR to detect the mRNA expression of Bcl-2 and Bax.Bax expression increased in A?group?P<0.001?,Bcl-2 expression decreased?P<0.001?;while Bax in A?+JWH133 group decreased compared with A?group?P<0.001?,Bcl-2 expression increased?P<0.001?;compared with A?+JWH133 group,Bax mRNA expression of A?+AM630+JWH133 group increased?P<0.01?,Bcl-2 mRNA expression decreased?P<0.01?.8.We used Western Blot to detect changes in protein levels of Bcl-2 and Bax.Compared with the control group,the expression of Bax protein in the A?group was significantly increased?P<0.05?,and the Bcl-2 expression was decreased?P<0.05?;The expression of Bax in the A?+JWH133 group was significantly decreased compared with A?group?P<0.01?,and the expression of Bcl-2 increased?P<0.01?;The expression of Bax protein in A?+AM630+JWH133 group increased?P<0.05?and expression of Bcl-2 decreased compared with A?+JWH133 group?P<0.01?9.We used Western Blot to detect the phosphorylation of protein kinase B?Akt?and phosphoinositide 3 kinase?PI3K?.Compared with the control group,the phosphorylation of Akt and PI3K in the A?_1-42 group was significantly decreased?P<0.05?;The phosphorylation of Akt/PI3K in the A?+JWH133 group was significantly increased compared to the A?group?P<0.05?;The phosphorylation of Akt and PI3K in the A?+AM630+JWH133 group was significantly decreased compared to the A?+JWH133 group?P<0.05?.In summary,after chronic treatment with A?_1-42 oligomers,hippocampal neurons show a reduced cell survival rate,neurons undergo apoptosis,and at the same time,lead to increased mRNA expression of CB₂R.This may be an endogenous protective mechanism produced by neurons when neurons are damaged.After pre-activating CB₂R with JWH133,the cell survival rate??m and Bcl-2 increased,and it could inhibit the increase of Caspase-3 mRNA and Bax expression,to a certain extent,it could inhibit neurons from being damaged by A?_1-42.Further experiments showed that the activation of CB₂R can increase the phosphorylation of Akt and PI3K,which indicates that the activation of CB₂R may play a protective role through the Akt/PI3K signaling pathway.The results suggest that hippocampal CB₂Rs exhibit neuronal protection against A?_1-42 toxicity.Therefore,CB₂R may be a new target for the treatment of the AD.