Function And Mechanism Of NDRG2 In Attention-deficit/hyperactivity Disorder

The worldwide prevalence of attention deficit/hyperactivity disorder?attention-deficit/hyperactivity disorder,ADHD?in school-age children is about 5%.Its core characteristics include hyperactivity,impulsiveness and/or attention deficit.However,the etiology of ADHD has not been determined.The effect of methylphenidate,the most commonly used in the treatment of ADHD,is not so satisfactory.About 1/3 patients have no response to methylphenidate.Currently the dominant perception is that the occurrence of ADHD is related to the decrease of dopamine concentration in synaptic cleft.The mechanism of methylphenidate is to increase the concentration of dopamine and norepinephrine in synaptic cleft.In patients with poor response to methylphenidate treatment,it is not clear whether there are changes in neurotransmitters other than dopamine.NDRG2 is highly expressed in mammalian central nervous system and mainly expressed in astrocytes.Its biological function is not completely clear.One of the main roles of astrocytes in the central nervous system is the reuptake of glutamate in the synaptic cleft.Some studies on ischemic stroke have shown that the increased expression of NDRG2 after stroke leads to the activation of astrocytes,suggesting that NDRG2 may be related to the excitotoxic injury of glutamate.Hyperactivity and other behaviors in patients with ADHD indicate that the excitability of the central nervous system is increased.Is this increase related to the excitatory neurotransmitter glutamate of the central nervous system?Therefore,the purpose of this study is to reveal the role and mechanism of NDRG2in the occurrence and development of ADHD,and to reveal a new pathogenic mechanism of ADHD in addition to dopamine hypothesis.It will help us to better understand the etiology of ADHD and lay a theoretical foundation for the search for NDRG2-based ADHD drugs or interventions.Part One:Ndrg2^-/-mice showed ADHD-like symptoms:increased locomotor activity,attention deficit and increased impulsive behaviorObjective:To explore whether Ndrg2^-/-mice have all the behavioral characteristics of ADHD,so as to become a new ADHD experimental animal model.Methods:The open field test was used to detect the locomotor activity of Ndrg2^-/-?KO?mice and their wild-type?WT?littermates in different months of age.Gait and muscle strength test were used to detect the muscle strength and coordination between KO mice and WT mice.The 5-CSRTT was used to detect the attention and impulse behavior between KO mice and WT mice.The novel object recognition test was used to test the short-term and long-term memory between KO mice and WT mice.All these behavioral tests were designed to confirm whether Ndrg2^-/-mice have all the behavioral characteristics of ADHD,and whether it can be used as a new experimental animal model of ADHD.Results:Compared with WT mice,2-month-old KO mice showed increased locomotor activity?total distance,number of lines crossed and velocity,P<0.01?.There was no significant difference in locomotor activity between 8-month-old and 12-month-old KO mice and WT mice,which accorded with the self-healing characteristic of clinical ADHD patients.5-CSRTT test comfirmed that 2-month-old KO mice showed attention deficit?P<0.01?,more impulsive behavior?P<0.05?.Novel object recognition test showed that KO mice had impairment of both short-term and long-term memory?short-term memory,P<0.01,long-term memory,P<0.05?.Conclusion:Ndrg2^-/-mice shows all the behavioral characteristics of ADHD and can be used as a new experimental animal model of ADHD.Part Two:NDRG2 knockout leads to the accumulation of glutamate in intercellular space and the increase of nervous excitatory conductionObjective:To explore the effect of NDRG2 knockout on the content of neurotransmitters in the central nervous system?CNS?and the discharge of the glutamatergic pyramidal neurons in the hippocampus.Methods:We recorded and analysed the electroencephalogram?EEG?of KO mice and WT mice.Using microdialysis we tested the contents of different kinds of neurotransmitters in central nervous system of KO mice and WT mice.And we used electrophysiological tools to investigate the discharge of the glutamatergic pyramidal neurons in hippocampus of KO mice and WT mice.Results:Ndrg2^-/-mice showed more Theta waves than WT mice?P<0.05?.The concentration of glutamate,an excitatory neurotransmitter,was significantly increased in KO mice?P<0.01?.And the glutamatergic pyramidal neurons in the hippocampus of Ndrg2^-/-mice showed increased amplitude of spontaneous excitatory postsynaptic currents?P<0.01?.Conclusion:NDRG2 knockout leads to the accumulation of glutamate in synaptic cleft and the increase of excitatory conduction.Part Three:The molecular mechanism of glutamate reuptake disorder induced by NDRG2 deletion in astrocytesObjective:To explore the role of NDRG2 in glutamate reuptake.Methods:To clarify the molecular mechanism of NDRG2 in glutamate reuptake,we used immunoblotting and immunoprecipitation to detect the level of glutamate transportation related proteins?EAAT1,EAAT2 and Na⁺/K⁺-ATPase?1?and glutamate receptors?NR1,NR2A,NR2B and GluR1,GluR2,GluR3?in the brains of KO mice and WT mice.Results:Immunoblotting showed that the expression of glutamate reuptake related proteins?EAAT1,EAAT2 and Na⁺/K⁺-ATPase?1?on astrocyte membrane dramatically decreased after NDRG2 knockout?P<0.01?.While there was no difference of the glutamate receptor expression?NR1,NR2A,NR2B and GluR1,GluR2,GluR3?in hippocampus of KO mice and WT mice.The results of immunoprecipitation experiment suggested that NDRG2-Na⁺/K⁺-ATPase?1-EAAT1/2 could form a potential molecular complex to facilitate the reuptake of glutamate.Conclusion:NDRG2 knockout leads to the dysfunction of NDRG2-Na⁺/K⁺-ATPase?1-EAAT1/2 molecular complex,which hinders the reuptake of glutamate by EAAT1/2 on the surface of astrocyte membrane and results in the accumulation of glutamate in synaptic cleft.Part Four:NDRG2 peptide can restore glutamate reuptake by astrocytes and reverse the ADHD-like symptoms of Ndrg2^-/-miceObjective:To screen the key segment of NDRG2 that can interact with Na⁺/K⁺-ATPase?1 subunit,and explore the function of TAT-NDRG2.Methods:We truncated NDRG2 into different length of peptides.Then we conducted immunoprecipitation and immunoblotting to find the key segment of NDRG2 that can interact with Na⁺/K⁺-ATPase?1 subunit.Then we combined the NDRG2?aa 141-160?with TAT?human immunodeficiency virus-type 1 transactivator of transcription?.The ability of TAT-NDRG2?aa 141-160?to enter cells was verified at both the cellular and animal levels.The effects of TAT-NDRG2?aa 141-160?on glutamate reuptake in astrocytes were detected at cellular and animal levels,respectively.The therapeutic effect of TAT-NDRG2?aa 141-160?on ADHD-like symptoms in Ndrg2^-/-mice was tested by behavioral experiments.Results:We found that NDRG2?aa 141-160?is the key segment of NDRG2 that can interact with Na⁺/K⁺-ATPase?1 subunit.TAT-NDRG2?aa 141-160?had the ability to penetrate the blood-brain barrier into cells.TAT-NDRG2?aa 141-160?could restore glutamate reuptake in both cultured astrocytes and different brain regions.TAT-NDRG2?aa 141-160?could correct most of the ADHD-like symptoms in Ndrg2^-/-mice.Conclusion:TAT-NDRG2?aa 141-160?can restore glutamate reuptake by astrocytes and improve the ADHD-like phenotype of Ndrg2^-/-mice.Part Five:A SNP of NDRG2 gene is strongly associated with the risk of ADHDObjective:To explore the SNPs of NDRG2 gene may be associated with the risk of clinical ADHD patients.Methods:The peripheral blood genomes from two independent cohorts were extracted and analyzed for SNP.The first cohort included 70 ADHD patients and 82 healthy controls,and the second cohort included 81 ADHD patients and 80 healthy controls.The luciferase activity of SNP site was detected by reporter gene activity.The expression of NDRG2 mRNA in peripheral blood of homozygous?CC?patients and homozygous?CC?healthy controls and heterozygous?CT?patients was detected by real-time quantitative PCR,respectively.Results:SNP rs1998848 was associated with susceptibility to ADHD.Compared with rs1998848 homozygous?CC?population,heterozygous?CT?population was significantly associated with the risk of ADHD.The results of two independent cohort studies showed that the risk of heterozygous was 6.3 times and 8.1 times higher than that of homozygous,respectively.The results of reporter gene experiment showed that the luciferase activity of homozygous?CC?rs1998848 was 2³ times higher than that of heterozygous?CT? rs1998848.The results of real-time quantitative PCR showed that the expression of NDRG2 mRNA in patients with heterozygous?CT?was significantly lower than that of patients with homozygous?CC?and healthy controls of homozygous?CC?.Conclusion:SNP rs1998848 of NDRG2 gene was strongly associated with susceptibility to ADHD.