The Role And Mechanism Of NDRG2 Promotes The Uptake Of Glutamate In Astrocytes

Glutamate, which is the major excitatory neurotransmitter in the central nervous system, plays an important role in the development and maintenance of synaptic plasticity,. In the physiological condition, the glutamate plays the role of signal transduction. In pathological conditions, glutamate accumulates in the synaptic cleft[1]. High concentration of glutamate overstimulating glutamate receptors causes intracellular Ca²⁺ overload, which eventually lead to the death of neurons[1], that is, the excitatory toxicity of glutamate[2]. Therefore, rapid and effective removal of glutamate from the synaptic cleft is beneficial to maintain normal synaptic transmission.Since there is no enzyme inactivating glutamate in the synaptic cleft, the main way to terminate the role of glutamate is the uptake of glutamate. The removal of glutamate is mainly dependent on the excitatory amino acid transporter EAATs, which works against the concentration gradient to reuptake glutamate. In the central nervous system, astrocytes are the main responsible cells for glutamate uptake[3,4]. Na⁺-dependent glutamate transporters in astrocytes are EAAT1 and EAAT2. EAATs transporter 1 glutamate molecule, accompanied by 3 Na⁺ and 1 H⁺ into the cell and 1 K⁺ out of the cell[5]. The energy source of the transmembrane transport is the Na⁺ and K⁺ electrochemical concentration gradient induced by Na⁺/K⁺-ATPase. However, the key molecules and precise mechanisms involved in modulating Glu uptake in astrocytes remain poorly understood.In the central nervous system, NDRG2 is specifically expressed in glial cells, especially astrocytes[6,7]. NDRG2 expression is upregulated under excitotoxic conditions, including ischemia[8], trauma[9], and Alzheimer’s disease[10]. Previous studies demonstrated that Na⁺/K⁺-ATPase β1 subunit was one of the moleculars interaction with NDRG2, and NDRG2 could stabilize the Na⁺/K⁺-ATPase β1 through inhibition ubiquitination of the protein[11]. To evaluate the potential physiological or pathological roles of NDRG2 in the brain, we generated NDRG2 knockout（Ndrg2-/-） mice. In a pilot study, these mice exhibited locomotor hyperactivity in an open-field test and a parallel increase in brain extracellular glutamate, suggesting that NDRG2 might be the key molecule controlling the glutamate clearance. The purpose of this study was to investigate whether NDRG2, which was expressed in astrocytes, affected the uptake of glutamate by EAAT1 and EAAT2 by influencing the activity of Na⁺/K⁺-ATPase.The first part, NDRG2 regulated the concentration of extracellular glutamate inbrain tissue, and maintained the dynamic balance of glutamateObjective: To investigate the effects of NDRG2 on locomotor activity and on concentration of extracellular glutamate in the brain of the mice.Methods: Six male C57BL/6J and Ndrg2-/- mice ageing 6 to 8 week were selected for experiment, and LV-NDRG2 or LV-Ctrl were injected into the lateral ventricles of the Ndrg2-/- mice. 5 days later, the open-field test was carried out to record total distance travelled and velocity in five minutes and HPLC was carried out to determine the concentration of glutamate and GABA gathered by microdialysis from striatum.Results: Compared with the WT mice, total distance travelled and velocity in 5 min were significantly higher in Ndrg2-/- mice, which indicated the mouse was hyperactive（p < 0.01）. Further investigation showed that glutamate gathered by microdialysis was higher than that of WT mice（p < 0.01）, but no obvious change was observed in the content of GABA. Rescue of NDRG2 expression with the LV-NDRG2 partly abolished the phenomenonConclusion: NDRG2 in the central nervous system affected the excitability of mice. NDRG2 deficiency, the mice showed hyperactivity. Our results indicated that the increased excitement was related to the increase of excited amino acid-glutamate in the extracellular fluid of brain.The second part, NDRG2 promoted the uptake of glutamate by astrocytesObjective: to observe the effect of NDRG2 on the ability of glutamate uptake by astrocytes.Methods: Primary astrocytes were obtained from WT and Ndrg2-/-mice. Ndrg2-/-astrocytes were tranfected with LV-Ctrl or LV-NDRG2 respectively. The cells were treated with 200 μM FITC-glutamate for different time（0, 5 min, 30 min, 60 min, 120 min）, then fluorescence intensity of cells was observed and recorded with confocal microscope.Results: The fluorescence intensity in Ndrg2-/- astrocytes was significantly lower than that of the wild astrocytes at different time points（0, 5 min, 30 min, 60 min, 120 min）, which indicated the amount and speed of glutamate uptake in Ndrg2-/- astrocytes were inhibited（p < 0.01）. NDRG2 expression was rescued by LV-NDRG2 in Ndrg2-/- astrocytes, and the FITC- glutamate uptake ability of astrocytes was restored.Conclusion: NDRG2 promoteed the uptake of glutamate by astrocytes.The third part, NDRG2 promoted Na⁺-dependent glutamate uptake in astrocytesObjective: To investigate the possible mechanism of NDRG2 affecting the uptake of glutamate by astrocytes.Methods: The purified astrocytes were isolated from WT and Ndrg2-/- mice, and Ndrg2-/- astrocytes were tranfected with LV-Ctrl or LV-NDRG2 respectively. Na⁺/K⁺-ATPase kit was used to detect Na⁺/K⁺-ATPase activity and CoroNa Green was used to detect intracellular Na⁺ concentration. Western blot was used to measure the expression level of NDRG2、EAAT1、EAAT2、Na⁺/K⁺-ATPase α1 and Na⁺/K⁺-ATPase β1.Results: Compared with the WT astrocytes, the activity of Na⁺/K⁺-ATPase was reduced（p < 0.05）, and fluorescence intensity of Na⁺ increased significantly in Ndrg2-/-astrocytes（p < 0.01）. The expression level of EAAT1, EAAT2 and Na⁺/K⁺-ATPase β1 was significantly lower in Ndrg2-/- astrocytes than that of WT astrocytes（p < 0.01）, whereas the expression of Na⁺/K⁺-ATPase α1 was not changed. NDRG2 expression was rescued by LV-NDRG2 in Ndrg2-/- astrocytes, and the changes were reversed.Conclusion: NDRG2 enhanced the activity of Na⁺/K⁺-ATPase, reduced intracellular Na⁺ concentration, and promoted the expression of EAAT1, EAAT2 and Na⁺/K⁺-ATPase β1. Taken together, these results indicated NDRG2 promoted Na⁺-dependent glutamate uptake and exerted a protective effect.The fourth part, NDRG2 interacted with Na⁺/K⁺-ATPase to regulate the uptake ofglutamate by astrocytesObjective: To investigate the molecular target of NDRG2 on glutamate uptake by astrocytes.Methods: The astrocytes were isolated and purified from WT and Ndrg2-/- mice. Ndrg2-/- astrocytes were transfected with Na⁺/K⁺-ATPase LV-β1 or LV-Ctrl, then the protein level of EAAT1, EAAT2, Na⁺/K⁺-ATPase α1 and Na⁺/K⁺-ATPase β1 was examined by western blot. Co-immunoprecipitation test was used to test if NDRG2 interacted with Na⁺/K⁺-ATPase β1 subunit in WT astrocytes. The interaction of NDRG2 and Na⁺/K⁺-ATPase β1 was disrupted, then the glutamate uptake ability, Na⁺/K⁺-ATPase activity and Na⁺ in the astrocytes was measured.Results: Ndrg2-/- astrocytes upregulated the expression of Na⁺/K⁺-ATPase β1 with transfection of LV-β1, at the same time, EAAT1, EAAT2 expression were increased significantly（p < 0.01）. We determined that NDRG2 interacted with Na⁺/K⁺-ATPase β1 subunit by co-immunoprecipitation. The interaction of NDRG2 and Na⁺/K⁺-ATPase β1 was disrupted, then the glutamate uptake ability（p < 0.01） and Na⁺/K⁺-ATPase activity（p < 0.05） significantly decreased and intracellular Na⁺ increased（p < 0.01）.Conclusion: NDRG2 interacted with Na⁺/K⁺-ATPase β1. Upregulation the expression of Na⁺/K⁺-ATPase β1 subunit or enhancing the interaction between NDRG2 and Na⁺/K⁺-ATPase β1 could promote Na⁺ dependent glutamate uptake in astrocytes.The fifth part, NDRG2 reduced excitotoxicity induced by glutamate bypromoting glutamate uptake by astrocytesObjective: To observe the effect of NDRG2 on excitotoxicity to neurons by promoting glutamate uptake by astrocytes.Methods: The direct and indirect coculture system were established with astrocytes and neurons. In the direct coculture system, the effect of glutamate on NMDAR1 translocation in neurons was measured. In the indirect coculture system, neurons and astrocytes were treated with 200 μM glutamate for 120 min, then the supernatant was collected for measurement of glutamate.The neurons were incubated in Neurobasal medium without glutamate for 24 hours, then LDH in the medium was measured and neuronal death was determined. The interaction of NDRG2 and Na⁺/K⁺-ATPase β1 was disrupted, then the glutamate uptake ability by astrocytes was measured and the excitotoxicity of glutamate to neurons were calculated.Results: In the direct coculture system, neurons in Ndrg2-/- groups, NMDAR1 membrane translocation increased significantly, and NMDAR1 on the neuronal cell bodies and neurites increased significantly, compared with the WT astrocytes group. In the indirect coculture system, the glutamate and LDH were significantly increased in the supernatant of Ndrg2-/- astrocytes culture compared with the WT astrocytes（p < 0.01）, and the mortality was significantly increased（p < 0.01）. NDRG2 expression was rescued by LV-NDRG2 in Ndrg2-/- astrocytes, these phenomenon was reversed. The interaction of NDRG2 and Na⁺/K⁺-ATPase β1 was disrupted, the glutamate uptake ability by astrocytes decreased and the excitotoxicity of glutamate to neurons was increased. significantly.Conclusion: NDRG2 or enhancement the interaction of NDRG2 with Na⁺/K⁺-ATPase β1 reduced the toxicity of glutamate to neurons by promoting glutamate uptake by astrocytes, which exerted a protective effect.