Studies of pathology and pharmacology of diabetic encephalopathy with KK-Ay mouse model.

AIMS: Pathogenesis of diabetic encephalopathy (DE) is not completely understood until now. The purposes of this study were to illustrate the changes in morphology, function, and important transporters in neurons and glia during DE, as well as to reveal the potential therapeutic effects of medicines and the diet control on DE. METHODS: Spontaneous obese KK-Ay mice were used to investigate diabetes-induced cognitive disorder, the morphology, function, and protein expression changes in impact animal and the cell level studies. The new drug candidate PHPB, donepezil, and low-fat food were used to observe the therapeutic effects. RESULTS: KK-Ay mice at 5 months of age showed typical characteristics of type 2 diabetes mellitus (T2DM） and appeared significant cognitive deficits. Morphological study showed microtubule-associated protein 2 (MAP2) expression was increased in hippocampal neurons and glial fibrillary acidic protein (GFAP) expression decreased in astrocytes. Meanwhile, the vesicular glutamate transporter 1 (vGLUT1) expression was increased and glucose transporter 1 (GLUT1) decreased, and the expression of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was also reduced in KK-Ay mice. Microglia were activated, and IL-1ß and TNF-α were increased obviously in the brains of the KK-Ay mice. Most of the above changes in the KK-Ay mice at 5 months of age could be relieved by diet intervention (DR) or by treatment of donepezil or new drug candidate PHPB. CONCLUSION: KK-Ay mouse is a useful animal model for studying DE. The alterations of morphology, structure, and function of astrocyte and microglia in KK-Ay mice might be rescued by DR and by treatment of medicine. The proteins we reported in this study could be used as biomarkers and the potential drug targets for DE study and treatment.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate-potentiated GABAA receptor response in hippocampal neurons.

AIMS: 2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate (NMZM), a derivative of clomethiazole (CMZ), had been investigated for the treatment of Alzheimer's disease (AD). The beneficial effects of NMZM in AD included reversing cognitive deficit, improving learning and memory as well as neuroprotection. The pharmacological effects of NMZM on GABAA receptors were reported previously; however, the mechanisms were unclear and were explored therefore. RESULTS: In this study, we demonstrated that NMZM improved learning and memory by alleviating scopolamine-induced long-term potentiation (LTP) suppression in the dentate gyrus of rats, indicating that NMZM had protective effects against scopolamine-induced depression of LTP. Next, we investigated the action of NMZM on GABAA receptors in hippocampal neurons and the binding site of NMZM on GABAA receptors. NMZM directly activated GABAA receptors in hippocampal neurons in a weak manner. However, NMZM could potentiate the response of GABAA receptors to GABA and NMZM positively modulated GABAA receptors with an EC50 value of 465 µmol/L at 3 µmol/L GABA while this potentiation at low concentration of GABA (1, 3 µmol/L) was more significant than that at high concentration (10, 30 µmol/L). In addition, NMZM could enhance GABA currents after using diazepam and pentobarbital, the positive modulators of GABAA receptors. NMZM could not affect the etomidate-potentiated GABAA current. It suggested that the binding site of NMZM on GABAA receptors is the same as etomidate. CONCLUSIONS: These results provided support for the neuroprotective effect of NMZM, which was partly dependent on the potentiation of GABAA receptors. The etomidate binding site might be a new target for neuronal protection and for drug development.

The pathological roles of NDRG2 in Alzheimer's disease, a study using animal models and APPwt-overexpressed cells.

AIMS: To investigate the roles of N-myc downstream-regulated gene 2 (NDRG2) in the pathology of aging and neurodegenerative disease such as Alzheimer's disease (AD). RESULTS: In this study, we confirmed the upregulation of NDRG2 in the brains of aging and AD animal models. To explore the role of NDRG2 in the pathology of AD at molecular level, we conducted a cell-based assay of highly expressed wild-type human APP695 SK-N-SH cells (SK-N-SH APPwt). By silencing and overexpressing gene of NDRG2, we demonstrated that NDRG2-mediated increase in Aß1-42 was through the pathways of BACE1 and GGA3. NGRG2 improved tau phosphorylation via enhanced activity of CDK5 and decreased Pin1, but it was not affected by GSK3ß pathway. NDRG2 might also induce cell apoptosis through the extrinsic (caspase 8) apoptotic pathway by interaction with STAT3. CONCLUSION: Our study confirmed the upregulation of NDRG2 in AD animal models and demonstrated its important roles in AD pathology. NDRG2 might be a potential target for studying and treatment of AD.

Over-expressed human TREK-1 inhibits CHO cell proliferation via inhibiting PKA and p38 MAPK pathways and subsequently inducing G1 arrest.

AIM: Recent studies have shown that the two-pore-domain potassium channel TREK-1 is involved in the proliferation of neural stem cells, astrocytes and human osteoblasts. In this study, we investigated how TREK-1 affected the proliferation of Chinese hamster ovary (CHO) cells in vitro. METHODS: A CHO cell line stably expressing hTREK-1 (CHO/hTREK-1 cells) was generated. TREK-1 channel currents in the cells were recorded using whole-cell voltage-clamp recording. The cell cycle distribution was assessed using flow cytometry analysis. The expression of major signaling proteins involved was detected with Western blotting. RESULTS: CHO/hTREK-1 cells had a high level of TREK-1 expression, reached up to 320%±16% compared to the control cells. Application of arachidonic acid (10 µmol/L), chloroform (1 mmol/L) or etomidate (10 µmol/L) substantially increased TREK-1 channel currents in CHO/hTREK-1 cells. Overexpression of TREK-1 caused CHO cells arresting at the G1 phase, and significantly decreased the expression of cyclin D1. The TREK-1 inhibitor l-butylphthalide (1-100 µmol/L) dose-dependently attenuated TREK-1-induced G1 phase cell arrest. Moreover, overexpression of TREK-1 significantly decreased the phosphorylation of Akt (S473), glycogen synthase kinase-3ß (S9) and cAMP response element-binding protein (CREB, S133), enhanced the phosphorylation of p38 (T180/Y182), but did not alter the phosphorylation and expression of signal transducer and activator of transcription 3 (STAT3). CONCLUSION: TREK-1 overexpression suppresses CHO cell proliferation by inhibiting the activity of PKA and p38/MAPK signaling pathways and subsequently inducing G1 phase cell arrest.