阿茲海默症是世界上最普及的一種失智症，其中有兩個主要病徵與該疾病漸進的神經退化失調有關：細胞外由類澱粉蛋白貝塔(Aβ)組成的斑塊，以及細胞內由過度磷酸化的tau所導致的神經原纖維纏結。近期研究指出Aβ蛋白會減低第一型與第二型囊泡麩氨酸轉運體(vGLUT1/2)在前突觸麩氨酸性區的表現，以及抑制麩氨酸被星狀細胞回收清除。這使得過多的麩氨酸累積在突觸間隙而後引發神經興奮毒性。然而Aβ蛋白過度活化N-甲基-D-天門冬胺酸受體(NMDAR)的現象可被人工合成的NMDAR拮抗藥物美金剛(memantine)所改善。因此找出麩氨酸受體的拮抗劑被視為Aβ蛋白所引發之異常現象的治療策略。於本研究中，我們試圖以小鼠初級大腦皮質神經細胞篩選中草藥以找出能改善Aβ蛋白導致的過量麩氨酸神經傳遞的非人工合成之天然成分。由免疫細胞化學法得知，vGLUT1/2染色呈現幾乎一致的麩氨酸性神經細胞群，突觸結合蛋白(synaptotagmin)染色亦在這些細胞之間呈現神經網絡的形式。而該初級神經細胞的特徵也透過西方墨點法中的抗體訊號如第三型貝塔微管蛋白、PSD-95蛋白以及α-氨基烴甲基嘿挫丙酸型受體(AMPAR)得以確認。為了在篩選中草藥的過程中能偵測並量化受Aβ蛋白刺激所造成的膜電位變化，DiBAC4(3)，一個慢反應電位敏感染劑，被用來做為藥物篩選的工具。DiBAC4(3)可進入去極化細胞並與胞內或膜蛋白結合隨後引發增加的紅螢光強度。去極化程度越高紅螢光越強，反之過極化則導致紅螢光減弱。為了建立DiBAC4(3)的中草藥篩選平台，我們對初級神經細胞刺激氯化鉀、麩氨酸以及Aβ蛋白後發現DiBAC4(3)螢光強度如預期般地增強。此外我們藉由DiBAC4(3)方法發現於Aβ蛋白刺激前加入memantine可減弱Aβ蛋白所導致的去極化現象。由上述結果可知DiBAC4(3)的中草藥篩選平台已被成功建立。在篩藥之前，我們亦先量測了處理不同濃度的中草藥對細胞存活的半抑制濃度(IC50)以便日後篩藥。在我們近期研究中，我們找到一些能抑制Aβ蛋白所引發的過度去極化現象的中草藥，也希望之後能找出該中草藥之有效成分如何參與阿茲海默病理現象中的麩氨酸受體訊息傳遞機轉。

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.

Alvarez G, Munoz-Montano JR, Satrustegui J, Avila J, Bogonez E, Diaz-Nido J (2002) Regulation of tau phosphorylation and protection against beta-amyloid-induced neurodegeneration by lithium. Possible implications for Alzheimer's disease. Bipolar Disord 4:153-165.
Anderson CM, Swanson RA (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32:1-14.
Bettini E, Sava A, Griffante C, Carignani C, Buson A, Capelli AM, Negri M, Andreetta F, Senar-Sancho SA, Guiral L, Cardullo F (2010) Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors. J Pharmacol Exp Ther 335:636-644.
Blurton-Jones M, Laferla FM (2006) Pathways by which Abeta facilitates tau pathology. Curr Alzheimer Res 3:437-448.
Brewer GJ, Torricelli JR (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc 2:1490-1498.
Brion JP, Anderton BH, Authelet M, Dayanandan R, Leroy K, Lovestone S, Octave JN, Pradier L, Touchet N, Tremp G (2001) Neurofibrillary tangles and tau phosphorylation. Biochem Soc Symp 67:81-88.
Bussiere T, Giannakopoulos P, Bouras C, Perl DP, Morrison JH, Hof PR (2003) Progressive degeneration of nonphosphorylated neurofilament protein-enriched pyramidal neurons predicts cognitive impairment in Alzheimer's disease: stereologic analysis of prefrontal cortex area 9. J Comp Neurol 463:281-302.
Butterfield DA, Pocernich CB (2003) The glutamatergic system and Alzheimer's disease: therapeutic implications. CNS Drugs 17:641-652.
Chen JH, Lin KP, Chen YC (2009) Risk factors for dementia. J Formos Med Assoc 108:754-764.
Cummings JL (2004) Alzheimer's disease. N Engl J Med 351:56-67.
Danysz W, Parsons CG (2012) Alzheimer's disease, beta-amyloid, glutamate, NMDA receptors and memantine--searching for the connections. Br J Pharmacol 167:324-352.
de la Torre JC (2006) How do heart disease and stroke become risk factors for Alzheimer's disease? Neurol Res 28:637-644.
Domingues A, Almeida S, da Cruz e Silva EF, Oliveira CR, Rego AC (2007) Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits. Neurochem Int 50:872-880.
Esposito Z, Belli L, Toniolo S, Sancesario G, Bianconi C, Martorana A (2013) Amyloid beta, glutamate, excitotoxicity in Alzheimer's disease: are we on the right track? CNS Neurosci Ther 19:549-555.
Fa M, Orozco IJ, Francis YI, Saeed F, Gong Y, Arancio O (2010) Preparation of oligomeric beta-amyloid 1-42 and induction of synaptic plasticity impairment on hippocampal slices. J Vis Exp 14.
Francis PT (2003) Glutamatergic systems in Alzheimer's disease. Int J Geriatr Psychiatry 18:S15-21.
Gao J, Inagaki Y, Li X, Kokudo N, Tang W (2013) Research progress on natural products from traditional Chinese medicine in treatment of Alzheimer's disease. Drug Discov Ther 7:46-57.
Goedert M (1993) Tau protein and the neurofibrillary pathology of Alzheimer's disease. Trends Neurosci 16:460-465.
Goedert M (1998) Neurofibrillary pathology of Alzheimer's disease and other tauopathies. Prog Brain Res 117:287-306.
Guerreiro RJ, Gustafson DR, Hardy J (2012) The genetic architecture of Alzheimer's disease: beyond APP, PSENs and APOE. Neurobiol Aging 33:437-456.
Guskiewicz KM, Marshall SW, Bailes J, McCrea M, Cantu RC, Randolph C, Jordan BD (2005) Association between recurrent concussion and late-life cognitive impairment in retired professional football players. Neurosurgery 57:719-726.
Herrmann N, Li A, Lanctot K (2011) Memantine in dementia: a review of the current evidence. Expert Opin Pharmacother 12:787-800.
Hillen H, Barghorn S, Striebinger A, Labkovsky B, Muller R, Nimmrich V, Nolte MW, Perez-Cruz C, van der Auwera I, van Leuven F, van Gaalen M, Bespalov AY, Schoemaker H, Sullivan JP, Ebert U (2010) Generation and therapeutic efficacy of highly oligomer-specific beta-amyloid antibodies. J Neurosci 30:10369-10379.
Holscher C (2011) Diabetes as a risk factor for Alzheimer's disease: insulin signalling impairment in the brain as an alternative model of Alzheimer's disease. Biochem Soc Trans 39:891-897.
Huang XT, Qian ZM, He X, Gong Q, Wu KC, Jiang LR, Lu LN, Zhu ZJ, Zhang HY, Yung WH, Ke Y (2014) Reducing iron in the brain: a novel pharmacologic mechanism of huperzine A in the treatment of Alzheimer's disease. Neurobiol Aging 35:1045-1054.
Huang Y, Mucke L (2012) Alzheimer mechanisms and therapeutic strategies. Cell 148:1204-1222.
Hynd MR, Scott HL, Dodd PR (2004) Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease. Neurochem Int 45:583-595.
Jurgensen S, Ferreira ST (2010) Nicotinic receptors, amyloid-beta, and synaptic failure in Alzheimer's disease. J Mol Neurosci 40:221-229.
Kim S, Ahn K, Oh TH, Nah SY, Rhim H (2002) Inhibitory effect of ginsenosides on NMDA receptor-mediated signals in rat hippocampal neurons. Biochem Biophys Res Commun 296:247-254.
Klyubin I, Betts V, Welzel AT, Blennow K, Zetterberg H, Wallin A, Lemere CA, Cullen WK, Peng Y, Wisniewski T, Selkoe DJ, Anwyl R, Walsh DM, Rowan MJ (2008) Amyloid beta protein dimer-containing human CSF disrupts synaptic plasticity: prevention by systemic passive immunization. J Neurosci 28:4231-4237.
Koch G, Di Lorenzo F, Bonni S, Ponzo V, Caltagirone C, Martorana A (2012) Impaired LTP- but not LTD-like cortical plasticity in Alzheimer's disease patients. J Alzheimers Dis 31:593-599.
Kowall NW, Beal MF (1991) Glutamate-, glutaminase-, and taurine-immunoreactive neurons develop neurofibrillary tangles in Alzheimer's disease. Ann Neurol 29:162-167.
Liang W, Lam WP, Tang HC, Leung PC, Yew DT (2013) Current Evidence of Chinese Herbal Constituents with Effects on NMDA Receptor Blockade. Pharmaceuticals 6:1039-1054.
Liu Q, Huang Y, Xue F, Simard A, DeChon J, Li G, Zhang J, Lucero L, Wang M, Sierks M, Hu G, Chang Y, Lukas RJ, Wu J (2009) A novel nicotinic acetylcholine receptor subtype in basal forebrain cholinergic neurons with high sensitivity to amyloid peptides. J Neurosci 29:918-929.
Mattson MP (2004) Pathways towards and away from Alzheimer's disease. Nature 430:631-639.
Morgan D (2011) Immunotherapy for Alzheimer's disease. J Intern Med 269:54-63.
Ni R, Marutle A, Nordberg A (2013) Modulation of alpha7 nicotinic acetylcholine receptor and fibrillar amyloid-beta interactions in Alzheimer's disease brain. J Alzheimers Dis 33:841-851.
Nimmrich V, Reymann KG, Strassburger M, Schoder UH, Gross G, Hahn A, Schoemaker H, Wicke K, Moller A (2010) Inhibition of calpain prevents NMDA-induced cell death and beta-amyloid-induced synaptic dysfunction in hippocampal slice cultures. Br J Pharmacol 159:1523-1531.
Okamoto S, Pouladi MA, Talantova M, Yao D, Xia P, Ehrnhoefer DE, Zaidi R, Clemente A, Kaul M, Graham RK, Zhang D, Vincent Chen HS, Tong G, Hayden MR, Lipton SA (2009) Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin. Nat Med 15:1407-1413.
Pahnke J, Walker LC, Scheffler K, Krohn M (2009) Alzheimer's disease and blood-brain barrier function-Why have anti-beta-amyloid therapies failed to prevent dementia progression? Neurosci Biobehav Rev 33:1099-1108.
Palop JJ, Mucke L (2010) Amyloid-beta-induced neuronal dysfunction in Alzheimer's disease: from synapses toward neural networks. Nat Neurosci 13:812-818.
Querfurth HW, LaFerla FM (2010) Alzheimer's disease. N Engl J Med 362:329-344.
Rammes G, Hasenjager A, Sroka-Saidi K, Deussing JM, Parsons CG (2011) Therapeutic significance of NR2B-containing NMDA receptors and mGluR5 metabotropic glutamate receptors in mediating the synaptotoxic effects of beta-amyloid oligomers on long-term potentiation (LTP) in murine hippocampal slices. Neuropharmacology 60:982-990.
Revett TJ, Baker GB, Jhamandas J, Kar S (2013) Glutamate system, amyloid ss peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology. J Psychiatry Neurosci 38:6-23.
Santangelo RM, Acker TM, Zimmerman SS, Katzman BM, Strong KL, Traynelis SF, Liotta DC (2012) Novel NMDA receptor modulators: an update. Expert Opin Ther Pat 22:1337-1352.
Schmitt F, Ryan M, Cooper G (2007) A brief review of the pharmacologic and therapeutic aspects of memantine in Alzheimer's disease. Expert Opin Drug Metab Toxicol 3:135-141.
Selkoe DJ (2008) Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav Brain Res 192:106-113.
Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL (2007) Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci 27:2866-2875.
Shi LL, Yang WN, Chen XL, Zhang JS, Yang PB, Hu XD, Han H, Qian YH, Liu Y (2012) The protective effects of tanshinone IIA on neurotoxicity induced by beta-amyloid protein through calpain and the p35/Cdk5 pathway in primary cortical neurons. Neurochem Int 61:227-235.
Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P (2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8:1051-1058.
Song MS, Rauw G, Baker GB, Kar S (2008) Memantine protects rat cortical cultured neurons against beta-amyloid-induced toxicity by attenuating tau phosphorylation. Eur J Neurosci 28:1989-2002.
Sonkusare SK, Kaul CL, Ramarao P (2005) Dementia of Alzheimer's disease and other neurodegenerative disorders--memantine, a new hope. Pharmacol Res 51:1-17.
Sun X, Jin L, Ling P (2012) Review of drugs for Alzheimer's disease. Drug Discov Ther 6:285-290.
Texido L, Martin-Satue M, Alberdi E, Solsona C, Matute C (2011) Amyloid beta peptide oligomers directly activate NMDA receptors. Cell Calcium 49:184-190.
Yamada A, Gaja N, Ohya S, Muraki K, Narita H, Ohwada T, Imaizumi Y (2001) Usefulness and limitation of DiBAC4(3), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials regulated by recombinant large conductance Ca2+-activated K+ channels in HEK293 cells. Jpn J Pharmacol 86:342-350.