阿茲海默症（Alzheimer’s disease）是漸進性的神經退化性疾病，其病徵包括漸進性的記憶能力損失以及在大腦皮質和海馬迴等區域有類澱粉斑塊（amyloid plaque）產生。膽鹼性假說（cholinergic hypothesis）說明乙醯膽鹼（acetylcholine）的濃度下降會導致認知與記憶能力的損失。先前的研究顯示，當抑制乙醯膽鹼酯酶（acetylcholinesterase），水解乙醯膽鹼的酵素，可以提升腦部乙醯膽鹼的濃度，而改善AD病人腦中膽鹼神經（cholinergic neuron）之間的訊息傳導。目前用於治療AD的藥物當中乙醯膽鹼酯酶抑制劑仍佔有一席之地，其可改善病徵以及認知功能。所以本篇研究目的在於鑑定黃芩萃取物NCT016是否可以有效抑制乙醯膽鹼酯酶的活性來保護神經細胞的新穎藥物。本實驗使用的細胞模式是Tet-On Aβ42-GFP SH-SY5Y人類神經瘤母細胞，以及動物模式APP/PS1/tau的基因轉殖鼠來進行實驗。得到的初步結果顯示，在細胞模式中NCT016可以降低乙醯膽鹼酯酶的活性；藉由降低β-secretase （β-site APP-cleaving enzyme 1，BACE1）、類澱粉前驅蛋白C端片段蛋白（APP β-C-terminal fragments，β-CTF）和類澱粉蛋白（Aβ42）的表現量以減緩類澱粉蛋白產生途徑（amyloidogenic pathway）；增加粒線體膜電位並降低Bax/Bcl-2 ratio以改善粒線體的功能；降低cleaved caspase-9 和cleaved PARP的表現量避免細胞走向細胞凋亡（apoptosis）；並且增加神經纖維（neurite）的長度和複雜度以改善神經纖維生長（neurite outgrowth）。在動物模式中，NCT016可以改善基因轉殖鼠在空間上的學習與記憶能力以及短期記憶能力；降低其大腦中類澱粉前驅蛋白（APP）、BACE1和p-tau的表現量以及減緩粒線體調控的細胞凋亡路徑。總結來說，NCT016可作為治療阿茲海默症的藥物來抑制乙醯膽鹼酯酶的活性、類澱粉蛋白產生途徑和細胞凋亡路徑，並增加粒線體功能等。

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by progressive memory loss and deposition of β-amyloid (Aβ) plaques in the cortical and hippocampal region of the brain. Previous studies emphasizes that the inhibition of acetylcholinesterase (AChE) activity increases the level of acetylchline in the brain and improves cholinergic functions in AD patients. Until now, AChE inhibitors are the major class of drugs approved for AD. Thus, we identified an effective novel compound NCT016 from Scutellaria baicalensis for protecting neurons via inhibiting AChE activity by using the AD cell model of Tet-On Aβ42-GFP SH-SY5Y neuroblastoma cells in vitro, and the triple-transgenic mice (3xTg-AD) harboring PS1M146V, APPSwe, and tauP301L in vivo. Our data showed that NCT016 isolated from Scutellaria baicalensis: (1) decreased significantly the AChE activity of the AD cells, (2) attenuated the amyloidogenic pathway by decreasing the expressions of BACE1, β-CTF and Aβ aggregation, (3) increased mitochondria membrane potential and rescued mitochondrial function, (4) protected AD cells from apoptosis, (5) improved neurite outgrowth by increasing neurite length and complexity of AD cells, (6) improved AD mice performance significantly better than vehicle-treated AD mice on Morris water maze test, Y-maze test and novel object recognition task at 24th week old, and (7) attenuated the expressions of APP, BACE1, phosphorylated tau protein and mitochondria-mediated apoptosis in the cerebra of AD mice. In conclusions, NCT016 could be a potentially therapeutic compound for the treatment of AD.

Anand P, Singh B (2013) A review on cholinesterase inhibitors for Alzheimer's disease. Archives of pharmacal research 36:375-399.
Azmi NH, Ismail N, Imam MU, Ismail M (2013) Ethyl acetate extract of germinated brown rice attenuates hydrogen peroxide-induced oxidative stress in human SH-SY5Y neuroblastoma cells- role of anti-apoptotic, pro-survival and antioxidant genes. BMC complementary and alternative medicine 13:177-188.
Badshah H, Kim TH, Kim MO (2014) Protective effects of Anthocyanins against Amyloid beta-induced neurotoxicity in vivo and in vitro. Neurochemistry international 80C:51-59.
Barbero-Camps E, Fernandez A, Martinez L, Fernandez-Checa JC, Colell A (2013) APP/PS1 mice overexpressing SREBP-2 exhibit combined Abeta accumulation and tau pathology underlying Alzheimer's disease. Human molecular genetics 22:3460-3476.
Capetillo-Zarate E, Gracia L, Tampellini D, Gouras GK (2012) Intraneuronal Abeta accumulation, amyloid plaques, and synapse pathology in Alzheimer's disease. Neuro-degenerative diseases 10:56-59.
Carvajal FJ, Inestrosa NC (2011) Interactions of AChE with Abeta Aggregates in Alzheimer's Brain: Therapeutic Relevance of IDN 5706. Frontiers in molecular neuroscience 4:19.
Chen X, Wehle S, Kuzmanovic N, Merget B, Holzgrabe U, Konig B, Sotriffer CA, Decker M (2014) Acetylcholinesterase inhibitors with photoswitchable inhibition of beta-amyloid aggregation. ACS chemical neuroscience 5:377-389.
Chu J, Li J, Hoffman NE, Madesh M, Pratico D (2014) Degradation of gamma secretase activating protein by the ubiquitin-proteasome pathway. Journal of neurochemistry.
Citron M (2010) Alzheimer's disease: strategies for disease modification. Nature reviews Drug discovery 9:387-398.
Clinton LK, Billings LM, Green KN, Caccamo A, Ngo J, Oddo S, McGaugh JL, LaFerla FM (2007) Age-dependent sexual dimorphism in cognition and stress response in the 3xTg-AD mice. Neurobiology of disease 28:76-82.
Costa RO, Ferreiro E, Oliveira CR, Pereira CM (2013) Inhibition of mitochondrial cytochrome c oxidase potentiates Abeta-induced ER stress and cell death in cortical neurons. Molecular and cellular neurosciences 52:1-8.
El-Malah A, Gedawy EM, Kassab AE, Salam RM (2013) Novel Tacrine Analogs as Potential Cholinesterase Inhibitors in Alzheimer's Disease. Archiv der Pharmazie 346:1-8.
Evans NA, Facci L, Owen DE, Soden PE, Burbidge SA, Prinjha RK, Richardson JC, Skaper SD (2008) Abeta(1-42) reduces synapse number and inhibits neurite outgrowth in primary cortical and hippocampal neurons: a quantitative analysis. Journal of neuroscience methods 175:96-103.
Farokhnia M, Shafiee Sabet M, Iranpour N, Gougol A, Yekehtaz H, Alimardani R, Farsad F, Kamalipour M, Akhondzadeh S (2014) Comparing the efficacy and safety of Crocus sativus L. with memantine in patients with moderate to severe Alzheimer's disease: a double-blind randomized clinical trial. Human psychopharmacology 29:351-359.
Ferreira IL, Ferreiro E, Schmidt J, Cardoso JM, Pereira CM, Carvalho AL, Oliveira CR, Rego AC (2014) Abeta and NMDAR activation cause mitochondrial dysfunction involving ER calcium release. Neurobiology of aging.
García-Ayllón M, Campanari M, Brinkmalm G, Rábano A, Alom J, Saura C, Andreasen N, Blennow K, Sáez-Valero J (2013) CSF Presenilin-1 complexes are increased in Alzheimer’s disease. Acta Neuropathologica Communications 1:46-59.
Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309-1313.
Han RW, Zhang RS, Chang M, Peng YL, Wang P, Hu SQ, Choi CL, Yin M, Wang R, Han YF (2012) Reversal of scopolamine-induced spatial and recognition memory deficits in mice by novel multifunctional dimers bis-cognitins. Brain research 1470:59-68.
Hellstrom-Lindahl E, Viitanen M, Marutle A (2009) Comparison of Abeta levels in the brain of familial and sporadic Alzheimer's disease. Neurochemistry international 55:243-252.
Henriques AG, Oliveira JM, Carvalho LP, da Cruz ESOA (2014) Abeta Influences Cytoskeletal Signaling Cascades with Consequences to Alzheimer's Disease. Molecular neurobiology.
Hroudova J, Singh N, Fisar Z (2014) Mitochondrial dysfunctions in neurodegenerative diseases: relevance to Alzheimer's disease. BioMed research international 2014:175062.
Huang HJ, Chen WL, Hsieh RH, Hsieh-Li HM (2014) Multifunctional Effects of Mangosteen Pericarp on Cognition in C57BL/6J and Triple Transgenic Alzheimer's Mice. Evidence-based complementary and alternative medicine : eCAM 2014:813672.
Inestrosa NC, Alvarez A, Dinamarca MC, Pérez-Acle T, Colombres M (2005) Acetylcholinesterase-amyloid-beta-peptide interaction: effect of congo red and the role of the Wnt pathway. Current Alzheimer research 2:301-306.
Jankowsky JL, Fadale DJ, Anderson J, Xu GM, Gonzales V, Jenkins NA, Copeland NG, Lee MK, Younkin LH, Wagner SL, Younkin SG, Borchelt DR (2004) Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Human molecular genetics 13:159-170.
Kazim SF, Blanchard J, Dai CL, Tung YC, LaFerla FM, Iqbal IG, Iqbal K (2014) Disease modifying effect of chronic oral treatment with a neurotrophic peptidergic compound in a triple transgenic mouse model of Alzheimer's disease. Neurobiology of disease 71:110-130.
Kontsekova E, Zilka N, Kovacech B, Skrabana R, Novak M (2014) Identification of structural determinants on tau protein essential for its pathological function: novel therapeutic target for tau immunotherapy in Alzheimer's disease. Alzheimer's research & therapy 6:45.
Kuhn PH, Wang H, Dislich B, Colombo A, Zeitschel U, Ellwart JW, Kremmer E, Rossner S, Lichtenthaler SF (2010) ADAM10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons. The EMBO journal 29:3020-3032.
Kwon SH, Lee HK, Kim JA, Hong SI, Kim HC, Jo TH, Park YI, Lee CK, Kim YB, Lee SY, Jang CG (2010) Neuroprotective effects of chlorogenic acid on scopolamine-induced amnesia via anti-acetylcholinesterase and anti-oxidative activities in mice. European journal of pharmacology 649:210-217.
LaFerla FM, Green KN, Oddo S (2007) Intracellular amyloid-beta in Alzheimer's disease. Nature reviews Neuroscience 8:499-509.
Lee YS, Kim HY, Youn HM, Seo JH, Kim Y, Shin KJ (2013) 2-Phenylbenzofuran derivatives alleviate mitochondrial damage via the inhibition of beta-amyloid aggregation. Bioorganic & medicinal chemistry letters 23:5882-5886.
Leuner K, Schulz K, Schutt T, Pantel J, Prvulovic D, Rhein V, Savaskan E, Czech C, Eckert A, Muller WE (2012) Peripheral mitochondrial dysfunction in Alzheimer's disease: focus on lymphocytes. Molecular neurobiology 46:194-204.
Lewczuk P, Mroczko B, Fagan A, Kornhuber J (2014) Biomarkers of Alzheimer's disease and mild cognitive impairment: A current perspective. Advances in medical sciences 60:76-82.
Liu X, Xu K, Yan M, Wang Y, Zheng X (2010) Protective effects of galantamine against Abeta-induced PC12 cell apoptosis by preventing mitochondrial dysfunction and endoplasmic reticulum stress. Neurochemistry international 57:588-599.
Madhusoodanan S, Ting MB (2014) Pharmacological management of behavioral symptoms associated with dementia. World journal of psychiatry 4:72-79.
Mansouri A, Haouzi D, Descatoire V, Demeilliers C, Sutton A, Vadrot N, Fromenty B, Feldmann G, Pessayre D, Berson A (2003) Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver. Hepatology 38:715-725.
Marcinkiewicz M, Seidah NG (2000) Coordinated expression of β-amyloid precursor protein and the putative β-secretase BACE and α-secretase ADAM10 in mouse and human brain. Journal of neurochemistry 75:2133-2143.
Mastrangelo MA, Bowers WJ (2008) Detailed immunohistochemical characterization of temporal and spatial progression of Alzheimer's disease-related pathologies in male triple-transgenic mice. BMC neuroscience 9:81.
McGleenon BM, Dynan KB, Passmore AP (1999) Acetylcholinesterase inhibitors in Alzheimer’s disease. The Journal of clinical pharmacology 48:471-480.
Miners JS, Palmer JC, Tayler H, Palmer LE, Ashby E, Kehoe PG, Love S (2014) Abeta degradation or cerebral perfusion? Divergent effects of multifunctional enzymes. Frontiers in aging neuroscience 6:238.
Moghul S, Wilkinson D (2001) Use of acetylcholinesterase inhibitors in Alzheimer's disease. Expert review neurotherapeutics 1:61-69.
Moubarak RS, Yuste VJ, Artus C, Bouharrour A, Greer PA, Menissier-de Murcia J, Susin SA (2007) Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis. Molecular and cellular biology 27:4844-4862.
Mowrer KR, Wolfe MS (2008) Promotion of BACE1 mRNA alternative splicing reduces amyloid beta-peptide production. The Journal of biological chemistry 283:18694-18701.
Murray AP, Faraoni MB, Castro MJ, Alza NP, Cavallaro V (2013) Natural AChE inhibitors from plants and their contribution to Alzheimer’s Disease therapy. Current Neuropharmacology 11:388-413.
Nair S, Traini M, Dawes IW, Perrone GG (2014) Genome-wide analysis of Saccharomyces cerevisiae identifies cellular processes affecting intracellular aggregation of Alzheimer's amyloid-beta42: importance of lipid homeostasis. Molecular biology of the cell 25:2235-2249.
Nalivaeva NN, Belyaev ND, Kerridge C, Turner AJ (2014) Amyloid-clearing proteins and their epigenetic regulation as a therapeutic target in Alzheimer's disease. Frontiers in aging neuroscience 6:235.
Obulesu M, Lakshmi MJ (2014) Apoptosis in Alzheimer's disease: an understanding of the physiology, pathology and therapeutic avenues. Neurochemical research 39:2301-2312.
Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM (2003) Triple-Transgenic Model of Alzheimer's Disease with Plaques and Tangles. Neuron 39:409-421.
Oliveira JM, Henriques AG, Martins F, Rebelo S, da Cruz ESOA (2015) Amyloid-beta Modulates Both AbetaPP and Tau Phosphorylation. Journal of Alzheimer's disease : JAD.
Ordonez-Gutierrez L, Torres JM, Gavin R, Anton M, Arroba-Espinosa AI, Espinosa JC, Vergara C, Del Rio JA, Wandosell F (2013) Cellular prion protein modulates beta-amyloid deposition in aged APP/PS1 transgenic mice. Neurobiology of aging 34:2793-2804.
Penke B, Toth AM, Foldi I, Szucs M, Janaky T (2012) Intraneuronal beta-amyloid and its interactions with proteins and subcellular organelles. Electrophoresis 33:3608-3616.
Prinz M, Parlar S, Bayraktar G, Alptuzun V, Erciyas E, Fallarero A, Karlsson D, Vuorela P, Burek M, Forster C, Turunc E, Armagan G, Yalcin A, Schiller C, Leuner K, Krug M, Sotriffer CA, Holzgrabe U (2013) 1,4-Substituted 4-(1H)-pyridylene-hydrazone-type inhibitors of AChE, BuChE, and amyloid-beta aggregation crossing the blood-brain barrier. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences 49:603-613.
Scheff SW, Price DA, Schmitt FA, Mufson EJ (2006) Hippocampal synaptic loss in early Alzheimer's disease and mild cognitive impairment. Neurobiology of aging 27:1372-1384.
Selkoe DJ (2002) Alzheimer’s Disease Is a Synaptic Failure. science 298:789-792.
Selvatici R, Marani L, Marino S, Siniscalchi A (2013) In vitro mitochondrial failure and oxidative stress mimic biochemical features of Alzheimer disease. Neurochemistry international 63:112-120.
Stachlewitz RF, Arteel GE, Raleigh JA, Connor HD, Mason RP, Thurman RG (1997) Development and Characterization of a New Model of Tacrine-Induced Hepatotoxicity: Role of the Sympathetic Nervous System and Hypoxia-Reoxygenation. The Journal of phatmacology and experimental therapeutics 282:1591-1599.
Sterniczuk R, Antle MC, Laferla FM, Dyck RH (2010) Characterization of the 3xTg-AD mouse model of Alzheimer's disease: part 2. Behavioral and cognitive changes. Brain research 1348:149-155.
Stoddard SV, Hamann MT, Wadkins RM (2014) Insights and ideas garnered from marine metabolites for development of dual-function acetylcholinesterase and amyloid-beta aggregation inhibitors. Marine drugs 12:2114-2131.
Stoppelkamp S, Bell HS, Palacios-Filardo J, Shewan DA, Riedel G, Platt B (2011) In vitro modelling of Alzheimer's disease: degeneration and cell death induced by viral delivery of amyloid and tau. Experimental neurology 229:226-237.
Sun Q, Peng DY, Yang SG, Zhu XL, Yang WC, Yang GF (2014) Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Abeta aggregation, and beta-secretase. Bioorganic & medicinal chemistry 22:4784-4791.
Tanzi RE (2012) The genetics of Alzheimer disease. Cold Spring Harbor perspectives in medicine 2.
Tarozzi A, Bartolini M, Piazzi L, Valgimigli L, Amorati R, Bolondi C, Djemil A, Mancini F, Andrisano V, Rampa A (2014) From the dual function lead AP2238 to AP2469, a multi-target-directed ligand for the treatment of Alzheimer's disease. Pharmacology research & perspectives 2:e00023.
Thal DR, Walter J, Saido TC, Fandrich M (2014) Neuropathology and biochemistry of Abeta and its aggregates in Alzheimer's disease. Acta neuropathologica.
Tu S, Okamoto S, Lipton SA, Xu H (2014) Oligomeric Aβ-induced synaptic dysfunction in Alzheimer’s disease. Molecular Neurodegeneration 9:48-60.
Wang CY, Zheng W, Wang T, Xie JW, Wang SL, Zhao BL, Teng WP, Wang ZY (2011) Huperzine A activates Wnt/beta-catenin signaling and enhances the nonamyloidogenic pathway in an Alzheimer transgenic mouse model. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 36:1073-1089.
Zempel H, Luedtke J, Kumar Y, Biernat J, Dawson H, Mandelkow E, Mandelkow EM (2013) Amyloid-beta oligomers induce synaptic damage via Tau-dependent microtubule severing by TTLL6 and spastin. The EMBO journal 32:2920-2937.
Zhao LN, Long H, Mu Y, Chew LY (2012) The Toxicity of Amyloid beta Oligomers. International journal of molecular sciences 13:7303-7327.