NDRG2 Is Critical For Neuroprotection In Ischemic Brain Injury Through Facilitating The Glutamate Transport

【Background】Stroke, with high mortality and disability rate, badly threatens human health and brings heavy economic burden to society and family. In decades, many promising therapies and drugs of stroke failed in the process of clinical transformation because of the limited efficacy and side effects. It is an urgent task to comb a new treatment targets for stroke. Reducing the excitatory neurotoxicity damage which is the basic pathological of stroke is an important way to save the neurons. So far, the treatments targeted neurons are less than satisfactory. Astrocytes which are 5 to 10 times the size of the neurons affect the neurons outcome with their high sensitivity, tolerance and adjustment to injury. Think out of the box, focusing the “glia-neuron unit” will open a new pathway for the treatment of stroke. NDRG2 firstly identified by our school professor Yao is a new gene associated with differentiation, proliferation and stress. NDRG2 is mainly expressed in astrocytes in CNS, suggesting NDRG2 may involve in the pathological and physiological process modulated by astrocytes. Our previous studies have showed, the protein levels of NDRG2 showed a time dependent increase in the ischemic reperfusion brain injury. NDRG2 also involved in the neuroprotection induced by EA or sevoflurane pretreatment. These researches indicated that NDRG2 involved in the pathological and physiological process of ischemic brain injury. While in these studies, NDRG2 acted as a stress responsor was always used as an observation index of pretreatment-induced cerebral ischemic tolerance, its temporal and spatial expression was not only regulated by ischemic brain injury but also by the pretreatment initially. Up to day, the role of NDRG2 itself in ischemic brain injury is unclear, our study will explore it for the first time.On the basis of these data, this study will investigate the role of NDRG2 in the ischemic brain injury, and further explore its mechanisms. 【Objectives】1. To clear the effect of NDRG2 in cerebral ischemia-reperfusion injury. 2. To investigate the influence of NDRG2 on which pattern and way of excitatory amino acid——glutamate level were regulated by, and elucidate the mechanism of the neuroprotective role of NDRG2 in cerebral ischemia-reperfusion injury. 3. To screen the efficient peptide which NDRG2 and Na⁺-K⁺-ATPase β1 interacted with, and confirm its essential role in neuroprotection of NDRG2 in vitro and in vivo. 【Methods】1. Mice carrying NDRG2 deletion that targeted exons 2-6,and their wild-type（WT） littermates were constructed. NDRG2 lentivirus or control virus was injected into the right lateral ventricle of Ndrg2-/- mice to regulate the expression of NDRG2 in time and space. Mice were subjected 60-min middle cerebral artery occlusion（MCAO） mimic transient focal cerebral ischemia, neurobehavioral scores, infact volumes and quantification of neuron-specific nuclear protein（NeuN） positive neurons in ischemic penumbra were determined at 24 hr reperfusion to clear the effect of NDRG2 in cerebral ischemia-reperfusion injury. 2. Microdialysis of extracellular neurotransmitter in mouse brain, electrophysiology analysis of amplitude and frequency of sEPSC of glutamatergic neuron, Western blot analysis of NMDAR1 were used to observe the influncence of NDRG2 in synaptic excitatory electrical activity; Glutamate uptake rate and Western blot were applied to detect the effects of NDRG2 on the the pattern of glutamate transpor and the expression of EAATs. Plasmid transfection、co-immunoprecipitation were used to observe the possible co-localization between NDRG2 and Na⁺-K⁺-ATPase β1 in HEK293 cells. Lentivirus transfection and glutamate uptake rate were practice to evaluate the effect of Na⁺-K⁺-ATPase β1 on NDRG2 regulated glutamate transportation. 3. Co-immunoprecipitation was devoted to screen the efficient peptide which NDRG2 and Na⁺-K⁺-ATPase β1 interacted with. Co-immunoprecipitation, Western blot and glutamate uptake rate were applied to confirm the effectiveness of the key peptide on the interaction and the collaboration of NDRG2 and Na⁺-K⁺-ATPase β1 in vivo. Neurobehavioral scores, infact volumes and quantification of neuron-specific nuclear protein（NeuN） positive neurons in ischemic penumbra were determined at 24 hr reperfusion followed with 60-min MCAO to define the role of the key peptide in transient focal cerebral ischemia in vitro. 【Results】 1. NDRG2 played a neuroprotection role in cerebral ischemia-reperfusion injuryNDRG2 deficiency markedly enlarged the infarct volume and neurological deficits（p < 0.01）. The number of positive NeuN which stained to mark healthy neurons in the ischemic penumbra region was significantly decreased（p < 0.01）. NDRG2 lentivirus rescued NDRG2 expression in Ndrg2-/- astrocytes, and attenuated the infarct volume and neurological deficit in Ndrg2-/- mice after MCAO（p < 0.05）. The number of NeuN-positive neurons in the ischemic penumbra region was also notably recovered after NDRG2 lentivirus injection（p < 0.05）. 2. NDRG2 interacted with Na⁺-K⁺-ATPase β1 to regulate the glutamate uptakeNDRG2 deficiency markedly increased the level of extracellular L-Glu in mice brain（p < 0.01）. Amplitude and frequency of sEPSC of glutamatergic neuron were markedly increased in Ndrg2-/- mice（p < 0.01）. NMDAR1 membrane translocation in Ndrg2-/- mice was observably increased（p < 0.01）. The glutamate clearance ability of Ndrg2-/- astrocytes and the expression of the Na⁺-dependent glutamate transporters including Na⁺-K⁺-ATPase β1、EAAT1 and EAAT2 in Ndrg2-/- mice were all decreased significantly（p < 0.01）. Strikingly,NDRG2 lentivirus treatment reversed the above indicators（p < 0.05）. The interaction between NDRG2 and Na⁺-K⁺-ATPase β1 was identified in HEK293 cells. Glutamate clearance was markedly rescued after the overexpression of Na⁺-K⁺-ATPase β1 in Ndrg2-/- astrocytes（p < 0.05）. 3. Peptide Na⁺-K⁺-ATPase244-263 was critical for the interaction btween NDRG2 and Na⁺-K⁺-ATPase β1 to facilitate the glutamate transport further influence the NDRG2 mediated neuroprotection in ischemic brain injuryAmino acids 244-263 of Na⁺-K⁺-ATPase β1 were critical for the NDRG2-Na⁺-K⁺-ATPase β1 interaction. Na⁺-K⁺-ATPase β1_244-263 disturbs the interaction between endogenous NDRG2 and Na⁺-K⁺-ATPase β1 in vivo and in vitro. The halflife of endogenous Na⁺-K⁺-ATPase β1 was shorten. The glutamate clearance ability of astrocytes was remarkly reduce after the Na⁺-K⁺-ATPase β1_244-263 exposure（p < 0.05）. The mice pretreated with Na⁺-K⁺-ATPase β1_244-263 subjected MCAO 60 min followed by 24 hr reperfusion, the level of extracellular glutamate in mice brain were increased（p < 0.01）, the infarct volume and neurological deficits were obviously increased（p < 0.01）, NeuN-positive cells in the ischemic penumbra was distinctly decreased（p < 0.01）. 【Conclusion】We evidenced that NDRG2 interacts with Na⁺-K⁺-ATPase β1 through the site of 244-263 amino acids sequence of Na⁺-K⁺-ATPase to protect Na⁺-K⁺-ATPase β1 from degrading, and collabrates with Na⁺-K⁺-ATPase β1 to regulate the expression and function of Na⁺-dependent glutamate transporters EAAT1 and EAAT2, further facilitates the glutamate transport mediated by astrocyte to maintain the proper excitatory synaptic transmission and play a critical role of neuroprotection finally. Thie model illuminate the NDRG2 mediated neuroprotection in ischemic brain injury. This study enriches the biological function of NDRG2 and provides a novel target for the prevention and treatment of the stroke.