The Role Of Homocysteine-mediated Methylation In Stress-induced Cognitive Impairment In Rats

Stress is defined as the general non-specific response to adapt the internal or external challenges followed by alterations in environment,society and psychology.Appropriate stresses can enhance the ability of our body in coping with the adverse stimulates,while the prolonged or overwhelming stress would exhaust the potential energies and induce stress injury in our body.Brain is the most critical organ in stress coping,which makes it one of the most susceptible target of stress.However the mechanism underling remains unclear yet.Previous studies have demonstrated that the Hcy with higher concentration in plasma was probably be one of the pathogenic factors involved in stress-induced cognitive impairment.As an important intermediate product of methionine cycle,Hcy is closely linked to the process of methylation and plays an important role in DNA methylation.In recent years,more and more studies have found that BDNF,a protein closely related to nervous system function,can be regulated by DNA methylation.which affects the development of some mental disorders.We hypothesized that the stress-induced Hcy metabolic dysfunction could down-regulate the expression of BDNF,and thus as an important factors involved in the development of cognitive decline.Therefore,the present study choose chronic unpredictable mild stress(CUMS)for stressful model establishment.Neural pharmacological,behavioral and molecular methods would be used to reveal the effect of Hcy metabolism on BDNF expression and the mechanism underlying it.This research would be helpful for the prevention and treatment of stress-associate cognitive impairment.Chapter 1.Role of plasma Hcy level in stress cognitive impairment in ratsObjective: To observe the effect of CUMS on cognitive function of rats and explore the effect of plasma Hcy level on stress cognitive impairment in rats.Methods:(1)Animal models: stressful rat models were established by CUMS method,and the high methionine diet and compound B vitamins interference were used to up-or down-regulate the level of plasma Hcy level.(2)Behavioral evaluation:Open-field test was used to detect the basic locomotion in rats.Morris water maze and step-down test were chosen in evaluating the spatial memory and avoidance memory respectively.Cognitive performance was estimated by object recognition experiments.(3)Plasma Hcy measurement: the level of plasma Hcy was detected by highperformance liquid chromatography(HPLC).Results: CUMS induced the worse cognitive performance in rats,and all cognitive functions showed significant decline(p < 0.05)since the sixth week after CUMS.At the same time,more than four weeks CUMS also lead to statistical elevation in plasma Hcy level(p < 0.05).The correlation analysis showed that the level of Hcy in rat plasma was negatively correlated with the cognitive index,the memory for bad stimulation and spatial learning.In addition,diet-induced higher Hcy could mimic the cognitive impairment in the same manner as CUMS,while Hcy reduction by means of vitamin B complex supplements and betaine could alleviate the cognitive deficits in CUMS rats.Conclusion:(1)Chronic stress can induce cognitive dysfunction in rats.(2)Chronic stress can lead to the increase of plasma Hcy level in rats.(3)The increase of plasma Hcy level is one reason for stress-induced cognitive impairment in rats.Chapter 2.Mechanisms of Hcy in cognitive regulation in stressed ratsObjective: To detect the effects of CUMS on rat plasma methylation potential(SAM/SAH),expression of methyltransferase,and bdnf promoter methylation.The effects of TET1 overexpression on BDNF expression and behavioral performance were also investigated to explore the possible methylational mechanism of plasma Hcy in cognitive regulation.Methods:(1)Animal models: besides the animal models in Chapter 1,TET1-overexpression AAV injection was used to block DNA methylations in rats’ hippocampus.(2)Behavioral evaluation: the same to Chapter 1.(3)Plasma Hcy measurement: the same to Chapter 1.(4)Methylational potential detection: was administrated by enzyme labeled immune sorbent assay(ELISA)test.(5)Hippocampal methyltransferase DNMT1 and DNMT3b: was administrated by protein western blotting assay(WB).(6)The expression of BDNF protein in the hippocampus was detected by WB and immunofluorescence staining(IF).(7)The methylation level of the hippocampal bdnf promoter was detected by methyl specific polymerase chain reaction(MSP).Results: CUMS induced a decreasing trend of methylation potential in rats.The methylation potential(p < 0.01)of rats was also significantly decreased by high methionine diet feeding,while the methylation potential remained unchanged in those CUMS rats with Hcy controlling.At the same time,both CUMS and high methionine diet feeding lead to the up-regulated methyltransferase DNMT1,DNMT3 b expression(p < 0.05),and the stress-induced elevation in hippocampus DNMT1,DNMT3 b could be blocked by Hcy controlling(p < 0.05).In addition,8 weeks CUMS increased the DNA methylation of bdnf promoter significantly(p < 0.05)in hippocampus,and decreased the expression of BDNF(p < 0.01)there.Diet-induced higher Hcy could result in the higher DNA methylation of bdnf promoter and lower BDNF expression similar to CUMS,while Hcy reduction by means of vitamin B complex supplements and betaine could reverse the changes caused by CUMS.DNA demethylation induced by AAV-TET1 injection could also increase the expression of BDNF protein in hippocampus(p < 0.01),and improve the cognitive function significantly in CUMS rats.Conclusion:(1)Chronic stress increased the level of plasma Hcy and lead to the decrease of methylation potential and the increase the expression of DNMT1 and DNMT3 b in hippocampus.(2)Chronic stress increased the level of plasma Hcy,and then induced the hypermethylation of bdnf promoter in the hippocampus,and ultimately reduce the expression of BDNF protein.(3)TET1-induced DNA demethylation could increase the expression of BDNF in hippocampus and alleviate the cognitive impairment caused by stress.