Abstract
The over-activation of NMDA receptor via the excessive glutamate is believed to one of the most causal factors associated with Alzheimer’s disease (AD), a progressive neurodegenerative brain disorder. Molecules that could protect against glutamate-induced neurotoxicity may hold therapeutic values for treating AD. Herein, the neuroprotective mechanisms of dimeric DT-010, a novel derivative of naturally occurring danshensu and tetramethylpyrazine, were investigated using primary rat cerebellar granule neurons (CGNs) and hippocampal neurons. It was found that DT-010 (3–30 μM) markedly prevented excitotoxicity of CGNs caused by glutamate, as evidenced by the promotion of neuronal viability as well as the reversal of abnormal morphological changes. While its parent molecules did not show any protective effects even when their concentration reached 50 μM. Additionally, DT-010 almost fully blocked intracellular accumulation of reactive oxygen species caused by glutamate and exogenous oxidative stimulus. Moreover, Western blot results demonstrated that DT-010 remarkably attenuated the inhibition of pro-survival PI3K/Akt/GSK3β pathway caused by glutamate. Ca2+ imaging with Fluo-4 fluorescence analysis further revealed that DT-010 greatly declined glutamate-induced increase in intracellular Ca2+. Most importantly, with the use of whole-cell patch clamp electrophysiology, DT-010 directly inhibited NMDA-activated whole-cell currents in primary hippocampal neurons. Molecular docking simulation analysis further revealed a possible binding mode that inhibited NMDA receptor at the ion channel, showing that DT-010 favorably binds to Asn602 of NMDA receptor via arene hydrogen bond. These results suggest that DT-010 could be served as a novel NMDA receptor antagonist and protect against glutamate-induced excitotoxicity from blocking the upstream NMDA receptors to the subsequent Ca2+ influx and to the downstream GSK3β cascade.
Highlights
Alzheimer’s disease is a devastating progressive brain disorder that severely destroys the learning and memory function of patients
We had successfully established the cellular model in which the neurotoxicity is triggered by glutamate in primary cerebellar granule neurons (CGNs) (Hu et al, 2013)
We identified DT-010, a dimeric derivative of DSS and TMP, as a novel NMDA receptor antagonist by whole-cell patching of hippocampal neurons, and systematically examined its protective effects against glutamate-mediated neurotoxicity in CGNs
Summary
Alzheimer’s disease is a devastating progressive brain disorder that severely destroys the learning and memory function of patients. The NMDA receptor is a glutamate receptor and ion channel protein mainly existing in neurons. These receptors gate the cytoplasmic influx of Ca2+, depending on the intensity of the stimulus, and subserve either physiological functions or neuronal death (Contestabile, 2012; Korde and Maragos, 2012). Overactivation of NMDA receptors by abnormal release of glutamate has been demonstrated to mediate excitotoxic neuronal damage observed in AD and other neuropathological conditions (Luo et al, 2010). Excessive glutamate pathologically over-activates NMDA receptors in the post-synaptic neurons, causes a massive abnormal influx of extracellular Ca2+ into cells and leads to neuronal death. A shift in the balance of these proteins in turn activates caspase-3 and disrupts mitochondrial membrane potential, and causes extensive neuronal death
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