NEURITE OUTGROWTH STIMULATORY ACTIVITY OF AN EDIBLE MUSHROOM PLEUROTUS GIGANTEUS IN DIFFERENTIATING NEUROBLASTOMA-2A CELLS PHAN CHIA WEI THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2015 ABSTRACT Neurite outgrowth is an important process for the establishment of synaptic connections during development, as well as neuronal regeneration in neuropathological conditions or injury. With growing concerns over neurodegenerative diseases attributed to impairment of neurite outgrowth e.g. dementia and Alzheimer’s disease, identification of alternative therapeutics has become paramount. One way to prevent and/or delay the onset of such diseases is by discovering alternative therapeutic molecules from functional foods. One such candidate is the edible mushroom (higher Basidiomycetes). In this study, eight culinary-medicinal mushrooms were evaluated for neurite outgrowth stimulatory effects by using neuroblastoma-2a (N2a) cells as an in vitro model. The mushroom extracts were also subjected to in vitro neuro- and embryotoxicity tests using N2a and 3T3 fibroblasts cell lines. The preliminary results showed that the aqueous extract of Pleurotus giganteus significantly (p < 0.05) promoted neurite outgrowth in N2a cells by 33.4 ± 4.6%. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 hours exposure of N2a and 3T3 cells to the mushroom extract. The basidiocarps of P. giganteus were then analysed for various nutritional attributes. The mushroom composed of protein (15.4–19.2 g/100 g), polysaccharides, phenolics, and flavonoids as well as vitamins B1, B2, and B3. The antioxidant properties of the aqueous and ethanol extracts of P. giganteus were investigated. The results indicated that the aqueous extract of P. giganteus exhibited scavenging of 2,2-diphenyl-1- picrylhyd-razyl (DPPH) radical with an IC50 value of 21.46 ± 6.95 mg/mL. Based on the ferric reducing antioxidant power (FRAP) assay, the reducing power of the mushroom extracts was in the range of 1.17–3.88 µM FeSO·7H2O/g mushroom and the ethanol extract showed lipid peroxidation inhibitory activity of 49.58–49.80%. The efficacy of the chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, iii caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) for neurite outgrowth activity was investigated. Uridine (100 µM) increased the number of neurite bearing cells by 43.1 ± 0.5%, which was about 1.8-fold higher than NGF (50 ng/mL)- treated cells. In this study, we demonstrated that uridine of P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt); simultaneously promoting neurite outgrowth in N2a cells. Neurite outgrowth stimulatory activity was inhibited by the inactivation of mitogen-activated protein kinase (MEK)/ERKs and Akt signaling with specific inhibitors. Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43), tubulin alpha 4a (TUBA4A), and tubulin beta 1 (TUBb1); all of which promoted neurite outgrowth of N2a cells. In conclusion, these findings demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecule responsible for neurite outgrowth is uridine. iv ABSTRAK Pengunjuran neurit merupakan satu proses yang penting untuk penubuhan sambungan sinaptik semasa perkembangan, dan regenerasi sel saraf (neuron) selepas keadaan neuropatologi ataupun kecederaan. Memandangkan penyakit neurodegenerasi yang berkaitan dengan kemerosotan pengunjuran neurit (demensia dan penyakit Alzheimer’s) semakin ketara, pengenalpastian cara perubatan alternatif menjadi lebih penting. Satu cara untuk menghindari dan/atau melewatkan permulaan penyakit tersebut ialah melalui penemuan molekul terapeutik alternatif daripada makan fungsian. Satu calon makan fungsian ialah cendawan (Basidiomysit tinggi). Dalam kajian ini, sekoleksi cendawan masakan-ubatan telah dinilai dari segi aktiviti perangsangan pengunjuran neurit atas sel neuroblastoma-2a (N2a). Ekstrak cendawan juga dinilai secara in vitro dari segi ketoksikan saraf dan embrio. Hasil awalan menunjukkan bahawa ekstrak akueus Pleurotus giganteus meningkatkan pengunjuran neurit sel N2a secara signifikan (p < 0.05) sebanyak 33.4 ± 4.6%. Nilai perencatan pada 50% kepekatan (IC50) yang didapati daripada asai tetrazolium (MTT), pengambilan neutral red (NRU) dan pembebasan laktat dehidrogenase (LDH) menunjukkan ketidakhadiran kesan toksik selepas pendedahan sel N2a dan 3T3 fibroblas kepada ekstrak cendawan selama 24 jam. Komposisi nutrisi basidiokarp (tubuh buah) P. giganteus juga dianalisa. Cendawan ini didapati tinggi dari segi kandungan proteinnya (154–192 g/kg), begitu juga polisakarida, fenolik, flavonoid, vitamin B1, B2, dan B3. Sifat antioksidaan ekstrak akueus dan etanol P. giganteus turut dikaji. Ekstrak akueus P. giganteus menunjukkan pemulungan radikel 2,2-diphenyl-1-picrylhyd-razyl (DPPH) pada kepekatan IC50 21.46 ± 6.95 mg/mL. Kuasa antioksidaan penurunan ferik ekstrak cendawan adalah dalam lingkungan 1.17– 3.88 µM FeSO·7H2O/g cendawan dan ekstrak etanol menunjukkan aktiviti perencatan pengoksidaan lipid sebanyak 49.58–49.80%. Keberkesanan juzuk kimia daripada P. giganteus (asid linoleik, asid oleik, asid cinnamic, asid kafeik, asid p-coumaric, asid v succinic, asid benzoik dan uridin) dalam pengunjuran neurit turut dikaji. Uridin (100 µM) meningkatkan peratusan sel yang mengandungi neurit sebanyak 43.1 ± 0.5%, dan ini adalah kira-kira 1.8 kali ganda daripada sel yang dirawat dengan NGF (50 ng/mL). Dalam kajian ini, uridin daripada P. giganteus (1.796 ± 0.032 g/100g ekstrak cendawan) meningkatkan pemfosforilan kinase yang dikawalselia oleh isyarat luaran sel (ERKs) dan protein kinase B (Akt); dan pada masa yang sama ia menggalakkan pengunjuran neurit pada N2a. Penyahaktifan protein kinase yang diaktifkan oleh mitogen (MEK)/ERKs dan Akt dengan perencat khusus merencatkan aktiviti stimulasi pengunjuran neurit. Tambahan pula, pemfosforilan sasaran mamalia rapamycin (mTOR) juga meningkat. MEK/ERK dan PI3K-Akt-mTOR mendorong pemfosforilan protein pengikat elemen tindak balas cAMP dan membawa kepada pengungkapan protein yang berkaitan dengan pertumbuhan 43 (GAP 43), tubulin alfa 4a (TUBA4A), dan tubulin beta 1(TUBb1); semua ini menggalakkan pengunjuran neurite sel N2a. Secara keseluruhannya, kajian ini mencadangkan bahawa P. giganteus mungkin dapat meningkatkan kesihatan otak dan persembahan kognitif; dan salah satu molekul bioaktif yang memainkan peranan yang penting dalam aktivi pengunjuran neurit ialah uridin. vi ACKNOWLEDGEMENTS I owe my gratitude to all those who contributed in many ways to the success of my study and made it an unforgettable experience for me. Foremost, my supervisor, Prof. Dr. Vikineswary Sabaratnam deserves an acknowledgement for skilfully guiding me in this research project from conception up to publication. I was immensely moved by her passion and learnt from her invaluable advice, as well as constructive criticism. I am also indebted to my co-supervisor, Dr. Rosie Pamela David from the Anatomy Department, who kept an eye on my progress and provided valuable suggestions in shaping the course of my research and thesis. I would also like to thank the Institute of Biological Science and all the staffs in the Faculty of Science, University of Malaya. In addition, I also wish to extend my thanks to the Institute of Postgraduate Study for the postgraduate research grant (PV007/2012A) and for the UM scholarship for my first year of PhD. Also, thanks to Bright Sparks Scholarship Unit from the Chancellery Office for sponsoring my PhD study for two years. Special thanks go to all members of the Fungal Biotechnology Lab, B403 lab, microscopy lab of Anatomy Department, and HIR Functional Molecules Lab. Each colleague and staff of the mentioned laboratories has contributed uniquely in this PhD study of mine. I also want to acknowledge the contributions of Dr. Murali Naidu, Dr. Wong Kah Hui, Dr. Tan Yee Shin, Syntyche Seow, Eik Lee Fang, Priscilla Ann, Wong Wei Lun, and June Ha. Most importantly, none of this would have been possible without the love and patience of my family. I would like to express my heart-felt gratitude to my family for their unequivocal support throughout my life. vii CONTENTS ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii CONTENTS viii LIST OF FIGURES xiv LIST OF TABLES xvii ABBREVIATIONS AND SYMBOLS xviii CHAPTER 1 1.1 Introduction 1 1.2 Research Objectives 3 CHAPTER II LITERATURE REVIEW 2.1 Neurite outgrowth 4 2.2 Neurite outgrowth in development 6 2.3 Neurite outgrowth in neurodegenerative diseases and 8 neuroregeneration 2.4 Measurements of neurite outgrowth 10 2.4.1 Semi quantitative method 11 2.4.2 Quantitative method 11 2.4.3 Biochemical marker 13 2.5 In vitro cell line model for neurite outgrowth 15 2.6 Neuritogenic substances that promotes neurite 18 outgrowth 2.6.1 Neurotrophic factors 18 2.6.2 Neuritogenic substances from plant 20 viii 2.7 Medicinal mushrooms for neurite outgrowth 22 2.7.1 Sarcodon cyrneus Maas Geest and Sarcodon 22 scabrosus (Fr.) P. Karst 2.7.2 Hericium erinaceus (Bull.: Fr.) Pers. 27 2.7.3 Ganoderma lucidum (Fr) P. Karst,