The Protective Role And Mechanism Of Stress Response Gene NDRG2 In AD-induced Nerve Pathological Damage

Alzheimer’s disease(AD)is a advanced degenerative disease of central nervous system.It is the most common type of dementia in the elderly population,with continuous progress of memory disorders,general intelligence decline,personality changes and abnormal mental behaviorsas main clinical manifestations.AD has become one of the major disabling and lethal diseases of the elderly in the world.With the intensified global aging population,the incidence of AD in the world has increased year by year.According to dataes released by the Alzheimer’s Disease International(ADI)in 2016,there are about 46.8 million AD patients worldwide,which means a new case appears every 3 seconds.It is predicted that the number of AD patients around the world will reach 74.7 million by 2030 and 131.5 million by 2050.In 2010,ADI reported that the annual costs of treating people who suffered AD were more than 604 billion dollars.With the largest population of the elderly in the world,AD patients in China has been estimated from 8 million to 10 million.China and the world will face huge burdens for the treatments and cares of AD patients in the future.The typical pathological features of AD are the senile plaques formed by the accumulation of β-amyloid proteins(Aβ)between the brain nerve cells.Tau proteins accumulated abnormally in the brain nerve cells form neurofibrillary tangles and lead to neuron degenerations and necrosis.As the current etiology and pathogenesis of AD are not yet clear,the treatments of AD mainly rely on oral drug therapies,including improved cholinergic neurotransmission drugs and antioxidant drugs for symptomatic treatments.Due to concentrations of drugs in the lesion are limited,so there is no ideal radical effect.As a result,it is of great significance to further study the key molecular mechanisms of AD pathogenesis for the basic researches and clinical diagnosis and treatment.Human-derived NDRG2 gene was firstly found and cloned by the Biochemistry Department of the Fourth Military Medical University in 1999,which was located on chromosome 14q11.2,containing 16 exons and 15 introns,encoding a protein with 371 amino acid residues and the molecular weight of 41 kDa.Mitchelmore et al.found that NDRG2 expression in brain tissues of AD patients was significantly higher than that of control subjects.However,whether NDRG2 expression is involved in promoting the development of AD,or an endogenous protection response of cellular activation to Aβ and other stresses,the mechanisms are not clear.As a new cellular stressor gene,NDRG2 is involved in a variety of cellular stress responses and that is mainly expressed in astrocytes of mouse brain tissue.Astrocytes are mainly involved in a variety of cell stress responses.It suggests that NDRG2 may play an important role in the crucial physiological and pathological responses of the central nervous system.In summary,this study aims to explore the role of NDRG2 in the pathogenesis of AD,providing more scientific basis for AD prevention and treatment,furthermore laying the foundation for future clinical transformation.Experiment 1: The expression level of NDRG2 changes in the pathogenesis of AD.Objective: To observe the changes of NDRG2 expression in hippocampus with different ages of mice in the pathogenesis of AD.Methods: APP/PS1 male mice were randomly divided into four groups: 2 months group,5 months group,8 months group and 11 months group.Each group had 3 mice.The right hemisphere of brain was harvested as frozen slices,where NDRG2 and GFAP were detected by immunofluorescence staining.Western blot was used to detect the expression of NDRG2,GFAP and β-amyloid proteins in the right hemisphere hippocampus.Results:Immunofluorescence staining results showed that the expression of NDRG2 and GFAP in hippocampus increased in time manner.Western blot showed that the expression of GFAP in 11 months old group was significantly higher than that in the other three groups(P <0.05).The levels of Aβ1-42 and NDRG2 in hippocampus significantly increased in time manner(P <0.05).Experiment 2: Detecting the learning and memory ability in NDRG2-/-and NDRG2+/+ mice with injection of Aβ Oligomers.Objective: To observe the role of NDRG2 in the pathogenesis of AD,with detecting the learning and memory ability in NDRG2-/-and NDRG2+/+ mice with injection of Aβ oligomers in the lateral ventricle.Methods: Aβ monomer was first subjected to aging treatment for obtaining Aβ oligomers.2-month-old APP/PS1 male mice were divided into 4 groups with bilateral lateral ventricles injection.Each group had 8 mice respectively.The first group was wild type mice injected with 5ul Aβ oligomers in the bilateral ventricles.The second group was NDRG2 knockout mice with 5μl Aβ oligomer injection.The third group was wild type mice injected with 5μl saline in the bilateral ventricles.The fourth group was NDRG2 knockout mice with 5μl saline.After the injection,the mice were housed in the animal room for 2 weeks,and sacrificed after behavioral experiment over a period of 3 days.The expressions of NDRG2 and β-amyloid proteins were detected by Western Blot to verify AD modeling.Results:1.The indexes,containing total distances,average speed of movement and number of crossing,in NDRG2 knockout mice group with injection of Aβ oligomers in the lateral ventricle were significantly higher than the control group(P < 0.01).2.Four groups of mice had similar ratio of exploration time on the same bricks;After one hour,the mice injected with saline showed significantly higher ratios of exploration time on the different bricks than other groups(P< 0.05);after 24 hours,the NDRG2 knockout mice with AβO injection showed significantly lower ratio of exploration time on the different bricks than other groups(P < 0.01);there were no significant differences in the ratios of exploration time on the different bricks in other three groups.Experiment3: Detecting the learning and memory abilities in NDRG2-/-APP/PS1 genetically modified mice.Objective:To obtained NDRG2-/-APP/PS1 mice by breeding and to verify the role of NDRG2 in pathological process of AD.Methods: Two mouse models,namely NDRG2-/-and APP/PS1 were interbred,and the first-generation offsprings that through the use of PCR with the NDRG2+/-APP/PS1 genotype were backcrossed with NDRG2-/-to obtain the NDRG2-/-APP/PS1 mice.To set mice into two groups,the first group was 8-month-old NDRG2 knockout APP/PS1 male mice(n=4),the second group is 8-month-old common APP/PS1 male mice(n=4).Two groups of mice are brought into the novel object recognition test for three days and then compared the results.Results:1.The total distances,average speed of movement and number of crossing in NDRG2 knockout APP/PS1 mice groupare significantly higher than the control groups(P < 0.01).2.Two groups of mice have similar ratio of exploration time on the same bricks;1 hour later,there is no significant difference between the ratios of exploration time on the different bricks;after 24 hours,the NDRG2 knockout APP/PS1 mice group showed significantly lower ratio of exploration time on the different bricks than the control group(P < 0.05).Experiment 4: Effects of NDRG2 gene deletion on the expression levels of proteasome functional subunits in AD mice.Objective: To observe the effect of NDRG2 on the expression levels of proteasome active subunits in AD mice.Methods:The hippocampus of 8-month-old APP/PS1 male mice and NDRG2 knockout APP/PS1 male mice were harvested respectively.After the extraction of RNA,real-time PCR was using to detect the expression of PSMB5 PSMB6 and PSMB7,which were regarded as the core active subunits of proteasome.Results:Compared with the APP/PS1 mice,the PSMB6 in the hippocampal significantly decreased in the NDRG2 knockout APP/PS1 mice(P< 0.05),while the PSMB5 and PSMB7 were not statistically different.Conclusions:1.In the pathogenesis of AD,the expression of NDRG2 and Aβ proteins in hippocampus of mice increased in time manner.2.The long-term memory of the NDRG2 knockout micewere damaged after injection with the Aβ oligomer in the lateral ventricles,and the short-term memory did not change significantly.3.Compared with 8-month-old APP/PS1 mice,the long-term cognitive memory of NDRG2 knockout APP/PS1 mice was damaged,while the short-term cognitive memory was not significantly different.4.The PSMB6 of the hippocampal in the NDRG2 knockout APP/PS1 mice was significantly decreased.Summary:In this study,we used Western Blot and Immunofluorescence staining in orderto verify the expression changes of NDRG2 and β-amyloid protein in the hippocampus of AD pathological progress,and then constructed AD mice models by lateral ventricle injection of β-amyloid protein oligomers.Finally we got the NDRG2 knockout AD mice.The long-term cognitive memory of NDRG2 knockout mice was damaged,but the short-term cognitive memory was not significantly different in AD model.Meanwhile,the PSMB6 m RNA of the hippocampals in NDRG2 knockout AD mice was significantly decreased to APP/PS1 mice without processing.The results showed that NDRG2 could play a protective role in the pathogenesis of AD neuropathic injury.This study reveals a new molecular mechanism of Alzheimer’s disease,and provides an important support for the new strategy of AD treatment and prevention.