To screen Chinese herbal medicines for ameliorating Aβ-induced pathophysiological neurotransmission

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Alzheimer’s disease (AD), the most prevalent form of dementia worldwide, is a progressive neurodegenerative disorder characterized by the presence of two hallmark lesions: extracellular amyloid β (Aβ)-containing neuritic plaques and intracellular phospho-tau-positive neurofibrillary tangle. Recent studies have revealed that Aβ reduces levels of vGLUT1/2 in presynaptic glutamatergic terminals and inhibits clearance of glutamate by astrocytes. This leads to increased concentration of glutamate at the synaptic cleft and subsequently trigger excitotoxicity. However, the Aβ-induced activation of NMDA receptor can be ameliorated by the synthetic NMDA antagonist, memantine. Hence, glutamate receptor antagonists have been regarded as a therapeutic strategy in Aβ-induced abnormality. In this study, we intend to find natural ingredients instead of synthetic drugs by screening Chinese herbal medicines (CHMs) for attenuating Aβ-induced abnormal neurotransmission in mouse primary cortical neurons. Judging from vGLUT1/2 staining in immunocytochemistry, we obtained an almost uniform population of glutamatergic neurons. Synaptotagmin staining showed the formation of neuronal network within these neurons. The property of these primary cortical neurons was characterized by Western blot analysis by using antibodies such as type III β-tubulin, PSD95, and AMPAR. In order to observe the changes of membrane potential after Aβ stimulation, bis-(1,3-dibutylbarbituric acid)-trimethineoxonol (DiBAC4(3)), a slow-response voltage-sensitive fluorescent dye, was employed to perform the drugs screening. DiBAC4(3) can enter depolarized cells where it binds to intracellular proteins or membrane proteins as to exhibit enhanced fluorescence and a red spectral shift. Increased depolarization results in additional influx of DiBAC4(3) anionic dye and an increase in fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. After treating mouse primary cortical neurons with KCl, glutamate, or Aβ, the fluorescence of DiBAC4(3) was increased as expected. Moreover, we found that pretreatment of memantine can ameliorate the Aβ-induced depolarization by assessing changes of DiBAC4(3) fluorescence intensity. These results indicated that the platform of drug screening has been established. Meanwhile, we measured the cell viabilities of primary neurons treated with CHMs to assess their IC50. We found some effective CHMs against the Aβ-induced excessive depolarization in neurons. The molecular mechanisms of effective compounds from CHMs involved in signaling in AD pathology remain to be further investigated.