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In vitro analysis of the biochemical pathways activated by cholesteryl glucoside in a motor neuron hybrid… Ly, Philip Tuan Thanh 2007

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In vitro ana lys i s of the b i o c h e m i c a l pathways act ivated by cho les tery l g l u c o s i d e in a motor n e u r o n hybr id m o d e l of a m y o t r o p h i c lateral s c l e r o s i s - p a r k i n s o n i s m dement ia c o m p l e x by PHILIP TUAN THANH LY B.Sc, The University of British Columbia, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA October 2007 ©Philip Tuan Thanh Ly, 2007 Abstract Steryl g lycosides are a family of compounds commonly found in the environment with unclear biological roles. Cycad seeds , a dietary link to the etiology of the Guamanian amyotrophic lateral sc lerosis-Parkinsonism dementia complex ( A L S - P D C ) have an abundant amount of steryl g lycosides. Previously, our laboratory demonstrated that several members of the cycad-derived steryl g lycosides have toxic properties. Cholesteryl glucosides (CG) , a variant form of the cycad-derived steryl glycosides have been found to induce cell death in primary rat cortical neurons. However, other groups have demonstrated that C G can protect fibroblast cells from heat shock. In the present study, we showed that the motor neuron-derived cell line, N S C 3 4 cells, exposed to C G resulted in a concentration- and time-dependent reduction in cell viability. However, a brief exposure of C G for one hour to N S C 3 4 cells induced cytoprotection against serum deprivation stress. The Kinetworks™ K P S S - 1 . 3 phosphosite screen was used to examine phosphorylation changes during C G preconditioning and indicated elevated A k t S e r " 4 7 3 phosphorylation. Suppression of C G -stimulated Akt phosphorylation with pharmacological inhibitors abol ished C G preconditioning. Furthermore, the results indicated A k t S e r " 4 7 3 phosphorylation via a PI3K-dependent and independent way. Erk1, but not Erk2 phosphorylation at its activation site was inhibited with C G treatment. The role of Erk1 inhibition in C G toxicity remains unclear. J N K and p38 M A P K were activation were temporally delayed, but were found to not involved in mediating cell death. A sub-population of N S C 3 4 cells treated with C G for at least 4 days displayed a differentiated phenotype with enlarged soma and extended neurites. Moreover, focal neurite swell ings with accumulated cytoskeletal proteins and d isease-associated phospho-Tau were observed. CG-treatment did not induce abnormal cytoplasmic accumulation of T D P 4 3 , as seen in sporadic A L S and Guamanian A L S and P D C cases . Trypan blue staining indicated that the treated-cells with abnormal morphology were viable. Taken together, these results demonstrated that C G is toxic to N S C 3 4 cells. A s a cellular response to stress, these cells upregulated survival signals and/or undergo differentiation to resist C G toxicity. A better understanding of the biochemical pathways triggered by C G in N S C 3 4 cells may provide insights to a common aberrant mechanism underlying the cause of neuronal death in A L S - P D C . i i Table of contents Abstract Table of contents List of tables List of figures Acknowledgement CHAPTER 1. Introduction 1.1 A unique disease complex—ALS-PDC 1.1.1 History of ALS-PDC 1.1.2 Clinical observations 1.1.3 Neuropathology of ALS-PDC 1.2 Etiology and environmental causes of ALS-PDC 1.2.1 The cycad hypothesis 1.2.2 A mouse model of ALS-PDC 1.2.3 Cycad toxins 1.2.4 Steryl glycosides are common in the environment 1.2.5 Steryl glycoside as a "lipid stress" mediator 1.3 Signal transduction 1.3.1 Phosphatidylinositol 3-kinase (PI3K) signaling 1.3.2 Protein kinase B (PKB/Akt) signaling 1.3.3 Mitogen Activated Protein Kinase (MAPK) signaling 1.3.3.1 Erk1 and Erk2 signaling 1.3.3.2 Stress-activated protein kinase and p38 MAPK signaling 1.4 Rationale and research objectives 1.4.1 Rationale 1.4.2 Objectives CHAPTER 2. Materials and methods 2.1 Materials 2.1.1 Chemical reagents 2.1.2 Laboratory supplies 2.1.3 Primary and secondary antibodies 2.2 Methods 26 2.2.1 Neuroblastoma x spinal cord 34 (NSC34) cell line culture 26 2.2.2 Cholesterol glucoside stock solution and kinase inhibitor preparation 26 2.2.3 Cell viability assays 26 2.2.4 Treatment protocols 27 2.2.5 Homogenization and protein preparation 28 2.2.6 SDS-polyacrylamide gel electrophoresis 28 2.2.7 Western blot analysis 29 2.2.8 Kinetworks™ KPSS-1.3 phosphosite screen 30 2.2.9 Lipid extraction and TLC analysis of steryl glucoside 30 2.2.10 Immunocytochemistry and quantification of cell morphology 31 2.3 Statistics and data analyses 32 CHAPTER 3. Cholestervl glucoside preconditioning of NSC34 Cells for serum deprivation stress 3.1 Results 33 3.1.1 C G preconditioning for serum deprivation stress 33 3.1.2 Signaling pathways underlying C G preconditioning in NSC34 cells... 34 3.2 Discussion 36 3.2.1 Preconditioning as an intrinsic property of the cell 37 3.2.2 Protective mechanisms of C G 38 3.2.3 Phosphorylation changes in CG preconditioning 38 3.2.4 Akt/Src/GSK3 signaling 39 3.2.5 Erk1/2 signaling 41 3.2.6 Adducin and the cytoskeleton 42 3.3 Conclusion 43 CHAPTER 4. Cholestervl glucoside activates the Akt and MAPK pathways 4.1 Results 53 4.1.1 CG-induced activation of Akt in NSC34 cells 53 4.1.2 CG-induced suppression of Erk1 but not Erk2 in NSC34 cells 54 4.1.3 Effects of kinase inhibitors on CG-induced activation of Akt 55 4.1.4 Effect of pharmacological inhibitors on CG-suppression of Erk1/2 ... 56 4.1.5 Late activation of stress-activated protein kinases 57 iv 4.1.6 Effect of k inases inhibitors on CG- induced reduction in cell viability.. 58 4.2 Discussion 59 4.2.1 CG- induced Akt activation 59 4.2.2 Erk1/2 suppression 61 4.2.3 J N K and p38 activation 63 4.3 Conclusion 64 CHAPTER 5. NSC34 cells differentiate to resist cholesteryl glucoside toxicity 5.1 Results 78 5.1.1 Quantitative assessment of cell morphology 78 5.1.2 Qualitative assessment of cell morphology 79 5.2 Discussion 80 5.2.1 Morphological changes in CG-t reated N S C 3 4 cel ls 81 5.2.2 Neurite swell ings and cytoskeletal disruption 83 5.2.3 Stress- induced differentiation for protection 84 5.2.4 T D P 4 3 proteinopathy and neurodegeneration 85 5.3 Conclusion 86 CHAPTER 6. General discussion and future studies 6.1 General discussion 95 6.1.1 Cytotoxicity triggers cholesteryl glucoside (CG) synthesis 95 6.1.2 Activating the PI3K/Akt pathway for neuroprotection 96 6.1.3 Stress differentiation: an adaptive process 96 6.2 Future studies 97 v List of tables Table 1 Summary of CG's modulatory role on survival and stress kinases List of figures Figure 1 PI3K and MAPK signaling pathway 22 Figure 2 Time and concentration-dependent effect of C G on NSC34 cell viability.... 44 Figure 3 Treatment of C G prevented cell death caused by serum deprivation 45 Figure 4 NSC34 cells pretreated with C G are resistant to cell death induced by serum deprivation 46 Figure 5 Prolonged C G treatment exacerbates cell death induced by serum deprivation 47 Figure 6 Kinetworks™ KPSS-1.3 phospho-sitemulti-immunoblot analysis of C G treated and serum deprived NSC34 cell lysates 48 Figure 7 Protein phosphorylation changes in NSC34 cells during C G preconditioning against serum deprivation 49 Figure 8 Time course of phosphorylation changes during serum deprivation during C G preconditioning 50 Figure 9 Effect of Erk1/2 and Akt inhibition on C G preconditioning against serum deprivation in NSC34 cells 51 Figure 10 Serum withdrawal induces C G synthesis in NSC34 cells 52 Figure 11 C G suppresses serum stimulation of Akt activity 65 Figure 12 C G treatment induces Akt activation in NSC34 cells maintained in complete media 66 Figure 13 CG-induced activation of Akt independent of PDK1 activity 67 Figure 14 C G interferes with serum stimulation of Erk1/2 activity 68 Figure 15 C G treatment induced Erk1 suppression, but not Erk2 in NSC34 cells grown in the presence of serum 69 Figure 16 Role of Raf1 and Mek1/2 in C G suppression of Erk1 71 Figure 17 Role of Mek1/2 and PI3K in CG-induced Akt phosphorylation 72 Figure 18 CG-induced Akt phosphorylation independent of mammalian target of rapamycin (mTOR) 73 Figure 19 Role of Mek1/2 and PI3K in CG-induced Erk1/2 phosphorylation 74 vii Figure 20 C G treatment induced late activation of stress-activated protein kinases 75 Figure 21 Effect of Mek1/2, PI3K, JNK1/2, and p38 MAPK inhibition on CG-treated NSC34 cell viability 76 Figure 22 Morphological quantification of CG-induced cytopathology in NSC34 cells 87 Figure 23 Abnormal processes and axonal blebs in CG-treated NSC34 cells are immunoreactive with B-tubulin III and NFH 88 Figure 24 CG-treated NSC34 cells are immunopositive for disease-associated phospho-Tau 90 Figure 25 TDP43 pathology is not present in CG-treated NSC34 cells 92 Figure 26 Viability and morphology of CG-treated NSC34 cells 94 Figure 27 Protective and toxic effects of cholesteryl glucoside 100 viii Acknowledgments I would like to thank my supervisory committee, Drs. Christopher Shaw, Steven Pelech, and Vincent Duronio for their support and guidance throughout my graduate degree in the Experimental Medicine Graduate Program at the University of British Columbia. You have allowed me to work on a topic that I am extremely passionate about and develop a thesis that I am truly proud of. To Dr. Chris Shaw, I am grateful that you have been an excellent supervisor by allowing me to make mistakes and learn from my own mistakes. From this, I have learned to work independently. To Dr. Vince Duronio, I am very thankful that you taught me cell culture techniques, which was the basis of all my experiments. Without this basic, yet essential skill, I would not be able to do any of these experiments. To Dr. Steve Pelech, I extend my profound gratitude for being an excellent advisor on experimental designs and data analysis. You have taught me a lot of things in the past two years. One thing in particular that I will never forget is: how to grow to become a better scientist. As part of a collaborative study, you have allowed me to use the Kinetworks™ KPSS-1.3 phospho-site screen to study signaling pathways triggered by steryl glycosides. I would also like to thank the staff at Kinexus Bioinformatics Corp. for all their help and guidance as well as some much needed laughs. To Reyniel, our friendship has grown tremendously over the past two years. Thank you for all your help. To everyone else in the Shaw laboratory, you have made my experience in graduate school a positive one—thank you. To my family, I am truly grateful for all the support you have provided me in so many ways. And to Carmie, you have been the source of logic and moral support through this whole experience. I thank you from the bottom of my heart. I X CHAPTER 1. Introduction 1.1 A unique disease complex—ALS-PDC 1.1.1 History of ALS-PDC The amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC) endemic to the island of Guam represents a spectrum of neurological disorders characterized by features of ALS, Parkinsonism dementia, or a combination (Kurland, 1988). Neuropathological features of ALS-PDC resemble in many aspects classical forms of ALS, and atypical forms of Parkinson's disease (PD) and Alzheimer's disease (AD) (Kurland, 1988). In the 1950s, the island of Guam was recognized for having a remarkably high incidence of neurodegenerative diseases. Shortly after the Second World War, a US Navy pathologist serving on Guam presented the first formal report of a high incidence of ALS among the natives indigenous to Guam (Hirano et al., 1966). Initially, only ALS was recognized to be highly prevalent on Guam. A similiarly high number of reported cases predominantly with Parkinsonism symptoms and dementia were later identified ( Hirano et al., 1961; Kurland, 1988). Later on that decade, a significant population of Guamanians was reported to have both ALS and PDC. Kurland and colleagues referred to the collection of these symptoms as ALS-PDC (Hirano et al., 1961). There are some basic similarities and differences in the clinical and neuropathological persepectives of Guamanian ALS-PDC and ALS, PD, AD seen elsewhere in the world. This makes the nosology of ALS-PDC very difficult. For clarity in this report, the Guamanian ALS-PDC will be distinguished from ALS, PD, and AD found in the Western world. The latter three will be termed classical forms of neurodegenerative diseases. Researchers (including neurologists, pathologists, and epidemiologists) have since been interested in understanding these Guamanian cases as they provide a unique paradigm for studying the etiopathogenesis of neurodegenerative diseases worldwide. It was important to understand the etiopathogenesis of ALS-PDC, because this disease complex once accounted for nearly 25% of adult deaths among the Guamanians during 1950's and 1980's (Plato er al., 2003; Waring er al., 2004). Since the 1980's the incidences of Guamanian ALS has declined to 3/100,000/year (Plato ef al., 2003). PDC incidences dropped since the 1980's but is still higher than ALS cases with about 32/100,000/year (Plato ef al., 2003). 1 1.1.2 Clinical observations ALS is a fatal paralytic disease characterized by loss of lower and upper motor neurons resulting in progressive muscle weakness and atrophy. From a clinical perspective, classical ALS and Guamanian ALS are nearly identical. As in classical ALS, the most common diagnostic features are muscle weakness, muscle atrophy, hyperreflexia, and fasiculations (Mukai eta/., 1982; Kurland and Mulder, 1987). Disease onset is insidious; an increasing paralysis leads to death on average 3 to 4 years after diagnosis due to failure of the respiratory muscles (Hirano era/., 1966; Mukai eta/., 1982; Kurland and Mulder, 1987). Conversely, age of onset differs between Guamanian ALS and classical ALS. The onset age observed in Guamanian ALS cases are typically 10 years younger (Mukai et al., 1982). PDC is characterized by Parkinsonism features with cognitive decline that is reminiscent of AD. Signs of Parkinsonism include tremor, rigidity, gait disturbances, and bradykinesia. PDC is also associated with speech difficulties (Coates and Bakheit, 1997) and olfactory dysfunction (Ahlskog ef al., 1998). Fine motor movements are impaired and there is marked facial masking with reptilian stare and infrequent blinking (Elizan et al., 1966; Rodgers-Johnson ef al., 1986). It should be noted that unlike classical PD patients, PDC patients do not respond to L-deoxyphenyl alanine (L-DOPA) treatments. Virtually all PDC patients present with varying degrees of dementia, characterized by memory deficits, disorientation with regards to place and time, difficulty with reasoning, and personality changes including aggressivity (Lessell ef al., 1962; Elizan ef al., 1966; Doty ef al., 1991). PDC patients usually develop Parkinsonism signs followed by dementia. However a study by Elizan and colleague showed that in 22 out of 79 PDC patients, cognitive impairment preceded appearance of Parkinsonism signs (Elizan ef al., 1966). Therefore, dementia is considered to be a ubiquitous feature of PDC, eventually appearing in all PDC patients (Elizan ef al., 1966). As noted earlier, ALS-PDC represents a spectrum of neurological disorders. Patients often express features of ALS and PDC, in varying severities. In a study of 104 Guamanian ALS cases, Elizan and colleagues (1966) reported 5 patients subsequently develop PDC. Furthermore, the authors observed that 27 of 72 PDC cases developed ALS. Taken together, this lends support to the speculation that the Guamanian ALS and PDC are not distinct disease entities, but rather points towards a common etiopathology. 2 1.1.3 Neuropathology of ALS-PDC Similar to clinical presentations, the spinal cord neuropathology of Guamanian ALS and classical ALS spinal cords is very similar (Hirano ef al., 1966). The typical neuropathological features of ALS are loss of spinal and cortical motor neurons resulting in muscle weakness and atrophy. The notable differences in Guamanian ALS include an abundance of neurofibrilliary tangles (NFT) composed of the microtubule-asociated protein Tau throughout the central nervous system (Rodgers-Johnson et al., 1986). Tau regulates the assembly and stability of microtubles in a manner dependent on the level of phosphorylation (Brich et al., 2003). Although NFT are not present in classical ALS pathology, abnormally high levels of hyperphosphorylated Tau are associated with various neurological disorders including AD (Alonso ef al., 1996; Iqbal et al., 2005) and to some degree PD (Sperfeld etal., 1999; Rosso and van Swieten, 2002; Ishizawa etal., 2003). Further differences are present in other regions of the central nervous system of Guamanian ALS patients. In a study of ALS patients lacking PDC symptoms, 46% showed significant neuronal loss with increased NFTs in the hippocampus, which is normally associated with AD and dementia (Rodgers-Johnson ef al., 1986). Furthermore, a more recent study found that Guamanian ALS patients have reduced uptake of 6-fluorodopa in the striatum. This finding indicates a decreased number of dopaminergic cells in the substantia nigra, which is the hallmark pathology of PD (Snow ef al., 1990). Whiles these changes are not features found in classical ALS, their presence could indicate preclinical, concomitant PDC that has not progressed to a clinically detectable point (Snow ef al., 1990). This agrees with numerous studies of neurodegenerative disease demonstrating that significant neuron loss occurs before clinical symptoms appear. For example, the symptoms of PD only become apparent when more than 80% of the dopaminergic neurons in the substantia nigra are lost (McGeer et al., 1988). Moreover, it has been estimated that up to a 70% reduction in the number of alpha motor neurons occurs before the ALS can be diagnosed (Arasaki and Tamaki, 1998). The neuropathological changes in PDC are also similar to those observed in PD and AD. For example, there is a loss of dopaminergic neurons in the substantia nigra and reduced 6-fluorodopa uptake in the striatum of patients with classical PD and PDC upon post-mortem analysis (Hirano et al., 1961; Lessell etal., 1962; Snow etal., 1990). However, one of the hallmark pathologies of PD is the presence of intracellular inclusions known as Lewy bodies that consist of aggregated synuclein protein. The number of Lewy bodies 3 observed in PDC patients are far fewer than in PD patients (Calne and Eisen, 1989; Forman et al., 2002; Winton et al., 2006). The neuropathology of PDC also includes marked cortical atrophy and NFTs in the hippocampus and entorhinal cortex (Hirano et al., 1961). These are neuropathological features of classical AD. The ultrastructure and immunohistochemical profile of the NFTs appears to be identical to that in AD (Hirano et al., 1961; Forman et al., 2002; Winton ef al., 2006) but there are differences in NFT distribution (Kurland, 1988). In Guamanian PDC, there is a preference for NFTs to be found in the cortical layer 3, whereas NFTs are typically found the cortical layer 5 of classical AD patients (Hirano ef al., 1961). It should be noted that there are many cases in which Guamanians who died without any apparent neurological disease yet displayed significant number of NFTs at post-mortem analysis (Chen, 1981). Chen and colleagues (1981) documented that 95% of unsymptomatic Guamanians over the age of 60 have extensive NFTs in various regions of their central nervous system. It is possible that these cases are preclinical ALS-PDC and did not show overt signs of neurological deficits to be clinically detectable. Also, we cannot rule out the possibility that NFTs are merely a background feature in the Guamanian population and is unrelated to the Guamanian ALS-PDC. The coexistence of Guamanian ALS and PDC within individual patients supports the speculation of a common underlying etiolopathology. Furthermore, the clinical and pathological similarities between Guamanian ALS-PDC and classical forms of ALS, PD, and AD indicate that an understanding of the etiopathogenesis of the Guamanian disease complex might shed light on neurodegenerative diseases throughout the world. 1.2 Etiology and environmental causes of ALS-PDC During the initial investigations of the etiology of ALS-PDC, researchers hoped that the straightforward causual factors would be readily unearthed. The native population on Guam was relatively homogeneous in genetic background (Plato etal., 2003), indicating that genetic inheritance is the likely cause of this disorder. Despite in-depth genetic surveys and analyses, researchers failed to identify the disease gene (Reed ef al., 1975; Chen, 1995; Ince and Codd, 2005). Moreover, natives living on Saipan, an island 80 miles north of Guam, had virtually the same genetic background as the Guamanian natives, yet there was no evidence of ALS-PDC pandemic (Yanagihara ef al., 1984; Garruto ef al., 1985). The apparent lack of genetic basis of the disorder is in keeping with observations regarding the classical forms of ALS (Shaw, 2005; Ly etal., 2007), PD (Jenner, 2001; Schulz et al., 2006), 4 and AD (Monteagudo et al., 1989; Rocchi et al., 2003; Klein ef al., 2004) in which the majority of cases do not show a familial pattern of inheritance. The lack of a clear genetic cause of ALS-PDC strongly indicates an environmental contribution to the etiology. Investigators began screening hundreds of potential environmental factors on Guam, including the levels of various minerals and heavy metals in the soil and ground water, native food products, military wastes, industrial materials, amongst others (Chen, 1995; Durlach ef al., 1997; Spencer ef al., 2005). For various reasons, most of these potential environment factors were discounted as being causal to ALS-PDC. Amongst the many proposed hypotheses, consumption of seeds from Cycas micronesica (cycad) appeared to be etiologically relevant to ALS-PDC (Kurland, 1988). The incidence of ALS-PDC has mirrored changes in cycad consumption among the natives that inhabited Guam. Cycad seeds have been consumed by the Guamanians as a dietary staple for many centuries. However, due to food shortages during World War II, Guamanians were forced to increase their consumption of cycad seeds (Kurland, 1988; reviewed in Shaw and Wilson, 2003; Miller, 2006). This coincided with a spike increased of ALS-PDC incidence. Since then, the natives adopted a more Westernized diet that does not include cycad, and the incidence of ALS-PDC has decreased dramatically (Zhang ef al., 1990; Miller, 2006). Moreover, supporting this hypothesis is the finding that Saipan natives genetically identical to the Guamanians, rarely consume cycad and were not affected by ALS-PDC. Therefore, Kurland (1988) argued that the dramatic differences in ALS-PDC incidence on these two neighboring islands could be a result of cycad consumption. Following this observation, cycad was considered by other researchers to be the key factor in the etiopathogenesis of ALS-PDC. 1.2.1 The cycad hypothesis Epidemiological studies point to a strong dietary link between the etiology of ALS-PDC and consumption of seeds from the cycad plant. A variety of cycad species of cycad are found throughout the world. For as many as several hundred years, cycad seeds, stems, roots, and leaves have been exploited as a traditional source of dietary starch by various native populations around the globe. Since the seeds are a more renewable source than stems, these are the most commonly ingested part. Traditional processing renders the seeds into flour, which was then made into food products such as tortillas. The cycad plant was found to contain many toxic compounds that must be removed before eating (Kurland, 1972; Kurland, 1988; Spencer etal., 1991). Processing techniques 5 involving extensive washes and cooking have been taught to the Guamanian natives by early European travelers to remove these toxic substances. While this removes toxins that are water-soluble, water-insoluble toxins remain. S ince the former are associated mainly with acute i l lness, it was assumed that washing and cooking cycad prevent toxicity from such resistant toxins. However, the clinical effects were not seen until many years after exposure. It is not surprising that even consumption of the processed cycad products is still associated with the development of Guam's unique d isease complex (Kurland, 1988). 1.2.2 A mouse model of A L S - P D C A s descr ibed above, epidemiological studies point to a strong dietary link between cycad seed consumption and A L S - P D C . Our laboratory developed a mouse model of A L S -P D C which linked cycad consumption to A L S - P D C . Dietary exposure of processed cycad seeds to mice on a daily basis produced outcomes that mimic human A L S - P D C . The amount of cycad exposed to the mice was calculated to be equivalent to the amount the Guamanian natives consumed between 1950 to 1980. This approximately constitute one quarter of the animals' daily intake by weight. Control mice were fed an identical amount of normal mouse chow. There were no weight differences between the two groups indicating no interference with systemic metabolism (Wilson ef al., 2002). Neurological deficits were assessed using a battery of behavioral tests for progressive deficits in motor, cognitive, and olfactory functions. For example, mice exposed to cycad were impaired on the leg extension reflex and wire hang, indicating compromised motor neuron integrity. Reduced gait length, a feature of P D , was also observed (Wilson ef al., 2002). Cognitively, the animals were impaired in their ability to perform on the Morris water maze (a test for spatial memory) and the radial arm maze (a test for working and reference memory) (Wilson ef al., 2002; Schu lz ef al., 2003). The behavioral deficits induced by cycad exposure were associated with neuropathological changes in the central nervous system. Histological analysis of the lumbar spinal cord revealed a dramatic reduction in motor neurons, implicating ALS- l i ke neuropathology (Wilson ef al., 2002). Furthermore, decreased tyrosine hydroxylase immunoreactivity and dopaminergic cell loss were observed in the substantia nigra, which are neuropathological features of P D (Schulz ef al., 2003). Extensive activated caspase-3 immunoreactivity was observed in the olfactory bulb (Schulz ef al., 2003). Neuronal loss was observed in the cortex and hippocampus, possibly associated with cognitive deficits (Cruz-Aguado ef al., 2006). Magnetic resonance microscopy analysis found decreased volumes of 6 various parts of the central nervous systems of mice exposed to cycad (Wilson et al., 2004). A downregulation of the glial-specific glutamate transporters, gliosis (measured by increased glial fibrillary acidic protein, G F A P ) , and microgliosis (measured by ionized calcium binding protein 1, Iba1) were observed in mice exposed to cycad (Wilson ef al., 2002, 2003, 2004). 1.2.3 Cycad toxins The original epidemiological description of a dietary link between cycad and A L S -P D C provoked research into identifying the toxin in cycad that might cause the d isease. Previous studies revolved around toxic amino acids, including cycasin, methyl-azoxymethanol-p -D-glucoside, p-N-oxalylamino-L-alanine (BOAA) , and p -N-methylamino-L-alanine (BMAA) (Ross and Spencer , 1987). Recent studies have refuted the idea that these compounds are causal to A L S - P D C (Wilson ef al., 2002; Wilson and Shaw, 2003; ; Cruz -Aguado ef al., 2006). The reasons are two fold. First, these compounds are extremely water-soluble and are completely eliminated during the traditional cycad seed washing procedures employed by the natives of G u a m . Secondly, when treated in their pure forms, these compounds fail to reproduce any behavioral deficits and pathology associated with A L S - P D C (cited in Wi lson and Shaw, 2003). For example, monkeys treated with pure B O A A develop a neurological condition called lathyrism, a d isease completely different from A L S -P D C (Spencer ef al., 1986). Further, a recent study found that mice fed B M A A showed neither deficits in motor skil ls, muscle strength, spinal reflexes, or cognition and have no histopathological changes (Cruz-Aguado ef al., 2006). S ince cycad processing effectively removes all water-soluble compounds, yet consumption of processed cycad seeds still induced the pathological outcomes of A L S -P D C , is strongly suggestive that the causal toxin(s) must remain in the seeds . In addition, the toxin(s) must be water-insoluble, heat-resistant, and sufficiently lipophilic to be transported across the blood brain barrier (reviewed in Wi lson and Shaw, 2003). Through a series of chemical extractions of washed cycad flour, Khabaz ian and col leagues isolated three sterol p - D - g l u c o s i d e s that had neurotoxic properties in vitro (Khabazian et al . , 2002). The isolated steryl glycosides were identified as campesterol glucoside, stigmasterol glucoside, and p-sitosterol-D-3-glucoside ( B S S G ) , with the latter being least toxic, but most abundant. Severa l experiments in our laboratory found that these steryl g lycosides and synthetic cholesterol glucoside (CG) were capable of inducing depolarizing field potentials, activating various protein k inases, and inducing lactate dehydrogenase (LDH) release in rat cortical cultures (Khabazian ef al., 2002). Moreover, we recently demonstrated that a mouse 7 motoneuron-derived cell line, NSC-34, treated with synthetic B S S G showed a dose-dependent decreased in cell viability (Ly ef al., 2007). These findings were supported by data from postnatal rat spinal cord organotypic culture slices treated with B S S G for 3 weeks, which showed significant motor neuron loss as compared to the controls, as do organotypic hippocampal slices and organotypic striatum culture (Ly ef al., 2007). The neurotoxic role of steryl glycosides is further supported by mice exposed to B S S G showed significant motor neuron loss six months after initial B S S G feeding. Taken together, these findings lead us to believe that steryl glycosides are at least some of the neurotoxins in cycad that may be involved in initiating ALS-PDC (reviewed in Ly et al., 2007). 1.2.4 Steryl glycosides are common in the environment The phytosterol glycosides isolated from the washed cycad flour belong to a larger family of molecules known as steryl glycosides (SGs). Members of this family are characterized by a carbohydrate unit attached to a tetracyclic carbon backbone. The nature of the sugar moiety can vary greatly and can be glucose, xylose, galactose, fructose, amongst others. Typical well-known sterols sharing this basic structure include cholesterol, campesterol, stigmasterol, brassicasterol, and sitosterol. SGs are peculiar glycoplipid constituents of the plant cell membrane (Wojciechowski, 1983; Misiak ef al., 1991), but their biological functions have not been well characterized. Previously, B S S G was found to act as a primer for de novo cellulose biosynthesis (Peng ef al., 2002). Despite their unclear functions, bacterial cell membranes are relatively abundant in steryl glycosides. Moreover, epidemiological studies have associated various chronic bacterial infections with neurodegeneration in ALS, PD, AD, amongst other neurological disorders (reviewed in Ly ef al., 2007). In view of the steroidal structure, it is also probable that steryl glycosides serve as hormones to mediate intercellular signaling. Furthermore, B S S G may have possible immunomodulary roles in mammals (van Rensburg etal., 2000). The effect of phytosterols and phytosteryl glycosides in mammals is highly controversial. Agricultural industries have claimed beneficial effects of plant-derived SGs in patients with hypercholesterolemia (Bouic, 2002; Nestel, 2002). This view ultimately hinges on the issue of the type and concentration of SGs. However, some of the better known SGs have well-documented toxic actions. For example, ouabain, a poisonous compound derived from the seeds of several native African plants is a potent blocker of essential ion pumps in heart muscle cells and neurons (Gao ef al., 2002). Another group of related toxic S G includes digitalin, which is a mixture SGs derived from the leaves and seeds of the common 8 foxglove (Wasserstrom and Aistrup, 2005). A number of similar examples are known in the medical literature, including scillirosidin which is found in a semi-arid desert plant that causes paralysis when ingested (Azoyan et al . , 1991), and solanine from unripe potato skin which can cause neurological symptoms after ingestion (Bodart et al . , 2000; Bodart and Noirfalise, 2003). Cycad S G s are found in many plants, albeit not in the same concentrations. The total S G concentration in a number of plants or plant products was recently published for the same three types of S G s isolated in cycad seeds (Phill ips et al . , 2005). The amount of S G s in cycad seeds is several magnitudes higher as compared to other plants or their derivatives (Marler et al., 2005). All of these observations lead to the well-known fact that dose can be critically important in toxicity. Most humans may be exposed to only relatively low doses of S G s that may never reach the nervous system or will do so only at levels that will do no harm. However, amongst the Guamanian natives, a higher concentrated exposure of such molecules could have deleterious consequences. 1.2.5 Steryl glycoside as a "lipid stress" mediator Membrane steryl glycosylation is considered to be an early and crucial event in stress-response signaling (Kunimoto et al., 2000). S G s , in particular, have been suggested to play key roles in mediating a stress signaling cascade (Kunimoto ef al., 2002, 2003). Endogenous synthesis of S G has been documented in human fibroblasts under heat stress (Kunimoto ef al., 2000) and the true sl ime mold Physarum polycephalum, was also found to rapidly induce S G during nutrient starvation and in a high salt environment (Murakami-Murofushi ef al., 1997; Kunimoto ef al., 2002). Bacterial synthesis of S G serves an entirely different purpose, namely to maintain pathogenicity and avoid bacterial exclusion from the body. For example, the bacterial HP0421 enzyme responsible for converting cholesterol to C G was later found to be critical for H. pylori to escape immune cells (Wunder ef al., 2006). A n increased S G level is correlated with increased expression of heat shock protein (HSP) 70 under stressful conditions (Kunimoto ef al., 2000, 2002, 2003). Kunimoto ef al. (2002) reported that heat stress treatment of human fibroblast culture results in elevated production of C G and H S P 7 0 . Furthermore, this group showed that the former is a crucial molecule in stress signaling and regulates the expression level of H S P 7 0 . Treatment of N S C 3 4 cells with the phytosteryl glycoside, B S S G also induces a significant elevation in H S P 7 0 expression, indicating that the plant-derived S G may also be involved in the same stress signaling cascade (Ly et al., 2006). The possibility that H S P 7 0 upregulation is a 9 consequence of other damage (i.e. protein misfolding or oxidative stress) caused by S G s cannot be excluded (reviewed in Ly etal., 2007). 1.3 Signal transduction Signals initiated from the cell surface receptors are transmitted through the plasma membrane to stimulate specif ic intracellular biochemical pathways causing a specif ic response (Figure 1). These signals mediate growth, survival, differentiation, proliferation, or apoptosis of the cell . Many different signals are integrated which ultimately regulate gene expression through altering the phosphorylation state of various proteins. Reversible protein phosphorylation is a major mechanism of intracellular signaling that modulates a variety of enzymatic activities and physiological processes such as cell survival, neurite outgrowth, intracellular protein trafficking, amongst others (Krebs, 1985; Downward, 2001; Dent ef al., 2003). These processes are governed by the activity of various protein kinases and protein phosphatases, which form a highly integrated network (Downward, 2001). Extracellular signals such as growth factors, neurotransmitters and hormones bind to their cell surface receptors and the resulting signal is transduced via protein signaling cascades . The receptors can relay the signals via associat ions with serine/threonine-specif ic protein k inases, tyrosine-specific protein k inases, small guanine nucleotide binding (G) proteins, adapter proteins, or lipid kinases (Downward, 2001). Protein-protein interactions occur through conserved protein modules that contain specif ic sequences which are recognized by other proteins. The recognition domains contain related sequences of 50-100 amino acids in length which are present in may signaling molecules which then build up a complex network of protein interaction (Pawson, 1995; Blundell et al . , 2000). S o m e of these conserved sequences include the Src-homology 2 (SH2) and Src-homology 3 (SH3) domains which were found originally in the non-receptor protein-tyrosine kinase Src (Schlessinger and Lemmon, 2003). The S H 2 domain recognizes and binds specif ic phosphorylated tyrosine residues where as S H 3 domains interact with proline rich sequences in their binding partner (Anderson ef al., 1990; Koch ef al., 1991; Kavanaugh and Wil l iams, 1994). The pleckstrin homology (PH) domain is another conserved sequence originally found in pleckstrin and in the C 2 domain of protein kinase C ( P K C ) (Hirata et al . , 1998). P H domains are believed to associate with phospholipids and promote re-localization of the PH-domain containing protein to the plasma membrane (Harlan ef al., 1994). C 2 domains found in classical and novel P K C isoforms binds both calcium ions and phospholipids (Sutton etal., 1995). 10 Different protein-protein interactions are used to recruit specif ic signaling modules to the appropriate subcellular location where the modules are allowed to interact with specif ic substrates or downstream effectors. For instance, growth factors binding to a specif ic protein-tyrosine kinase receptor induce receptor dimerization, autophosphorylation on certain tyrosine residues, and recruitment of a series of proteins with S H 2 , S H 3 , or P H domains. The signal is further transmitted downstream in the cytoplasm and eventually gets relayed in the nucleus for gene transcription, which translates into a physiological response (Downward, 2001). The signaling pathways of phosphatidylinositol 3-kinase (PI3K), Akt (also known as protein kinase B, P K B ) , and mitogen-activated protein kinase (MAPK) , will be d iscussed below. 1.3.1 Phosphatidylinositol 3-kinase (PI3K) signaling Lipids are no longer considered as only structural components of membranes, but a lso as intracellular messengers that regulate cellular activities and survival. Inositol phospholipids were first proposed to have important roles as secondary messengers in signal transduction pathways in the 1980's (Dent et al., 2003). This family of lipids is phosphorylated at the D3 position of their inositol ring by a lipid kinase called PI3K. The PI3K enzyme consists of two subunits, a catalytic p110 subunit and a regulatory p85 subunit. The p85 subunit contains an S H 2 domain which is required for membrane re-localization whereas the p110 subunit mediates the catalytic functions (Ching et al., 2001). The resulting lipid products are phosphatidylinositol 3-phosphate (PI3-P), phosphatidylinositol (3,4)-bisphosphate (PI3,4-P 2 ) and phosphatidylinositol (3,4,5)-trisphosphate (PI3,4,5-P 3 ) (Dent e ra / . , 2003). PI3K also possesses intrinsic protein-serine/threonine activity in addition to its lipid kinase activity (Carpenter ef al., 1993). Autophosphorylation of the p85 subunit at Ser-608 results in 80% decrease in PI3K activity (Dhand ef al., 1994). The discovery of specif ic inhibitors of PI3K has improved our understanding of the functions of PI3K, as well as the downstream signaling cascades . Wortmanin is a fungal metabolite that binds covalently to the p110 catalytic subunit and inhibits the activity of PI3K when used at nanomolar concentrations (Yano ef al., 1993). Another commonly used PI3K inhibitor is LY294002, which competes for the A T P binding site in the p110 subunit. This compound functions in the micromolar concentrations to inhibit PI3K activity (Vlahos ef al., 1994). The biological functions of the PI3K pathway include promoting cell survival, membrane trafficking, rearrangement of actin cytoskeleton, membrane blebbing, lymphocyte development and activation, amongst others (reviewed in Vanhaesebroeck and Waterfield, 11 1999; Cantrel l , 2001; Okkenhaug and Vanhaesebroeck, 2003). In the nervous system, PI3K plays an essential role in transducing neurotrophin mediated survival signals (Kaplan and Miller 2000). Furthermore, PI3K activity was found to be required for growth factor dependent survival. The activity of PI3K appears to be sufficient in promoting cell survival in the absence of trophic support (Crowder and Freeman, 1998). Neurons transfected with a constitutively active PI3K, with receptor mutants in which PI3K is exclusively activated in response to receptor ligand binding were found to be resistant to apoptosis due to lack of trophic factors (Crowder and Freeman. 1998; Datta et al., 1999). This was supported by another finding that a constitutively active R a s mutant that exclusively binds to and activates PI3K can also prevent apoptosis induced by lack of trophic signaling (Philpott et al., 1997; Ulrich etal., 1998). There are increasing numbers of downstream targets of PI3K. The 3-phophoinosit ide-dependent kinase 1 (PDK1) was discovered as a kinase that phosphorylates Akt in a PI3K-dependent manner. PDK1 can only phosphorylate the Thr-308 site of P K B in the presence of PI(3,4,5)P 3 (Alessi et al., 1997b; Walker ef al., 1998; A less i et al., 1996). This mechanism will be d iscussed in further detail in Sect ion 1.3.2. PDK1 has also been found to activate the protein kinases p70 S 6 K (Zhang ef al., 2001), p90 ribosomal S 6 kinase (p90 R S K ) (Jensen ef al., 1999), cyclic AMP-dependent protein kinase (PKA) (Cheng ef al., 1998; Biondi ef al., 2000), and P K C (Le Good ef al., 1998; Wil l iams ef al., 2000). These k inases are activated through a similar mechanism as the phosphorylation of the equivalent Thr-308 residue of Akt. There has also been evidence that PI3K can activate the extracellular regulated kinases 1 and 2 (Erk1/2), which are members of the mitogen-activated protein kinase family (reviewed in Vanhaesebroeck and Waterfield, 1999; Ruffels ef al., 2004). In the nervous system, PI3K signaling is primarily involved in mediating survival signals (Brunet ef al., 2001; Leinninger ef al., 2004). The importance of the pro-survival functions of PI3K signaling in the nervous system has been highlighted in acute neurodegenerative conditions induced by epileptic seizures (Henshall et al., 2002), ischemia/reperfusion brain injury (Sakurai ef al., 2001), or axotomy (Murashov et al., 2001) and chronic neurodegenerative d iseases such as A D (Rickle ef al., 2004) and A L S (Wagey ef al., 1998). Human post mortem analysis of A L S spinal cord samples showed an increased in PI3K activity, but not in its downstream target Akt, in the particulate fraction (Wagey ef al., 1998). This indicates an impaired signaling cascade mediated by PI3K in A L S . However, other groups have observed a reduction in the PI3K signaling in a mouse 12 model of familial ALS with a low expressor mutant line (Warita et al., 2001). Since the PI3K is involved in transducing survival signals, a decreased in PI3K activity is likely to render motor neurons vulnerable to cell death. More recently, Bendotti and colleagues found that PI3K activity was not altered in the familial ALS mouse line with the high expressor mutant gene (Peviani ef al., 2007). The authors argued that since motor neurons degeneration involves many pro-apoptotic protein kinases (Tortarolo ef al., 2003), the lack of increased changes in Akt activity indicates that these cells were incapable of balance the mechanisms leading to cell death (Peviani ef al., 2007). Furthermore, the authors proposed that enhancing this pathway may hold a therapeutic possibility to protect from motor neuron degeneration. 1.3.2 Protein kinase B (PKB/Akt) signaling PKB is also known as Rac (Related to protein kinase A and protein kinase C) or Akt (cellular homologue of the viral oncogene, v-Akt). PKB is a protein-serine/threonine protein kinase downstream of PI3K and exist in three isoforms: P K B a (Akt1), PKBp (Akt2), PKBy (Akt3) in mammals. Of the three isoforms, only PKBa (Akt1) is expressed at high levels in the central nervous (Burgering and Coffer, 1995). For consistency in the remainder of this text unless stated otherwise, PKBa will be referred to as Akt. Akt contains a pleckstrin homology (PH) domain at its amino-terminus that binds phospholipids, a glycine rich motif, a catalytic domain, and a small regulatory carboyx-terminus extension (reviewed in Kandel and Hay, 1999a and Datta ef al., 1999). The three isoforms of Akt have conserved serine and threonine residues which play critical roles in the activation of this kinase. Akt activation is highly dependent on the activity of PI3K. Inhibitors, such as wortmannin and LY294002, that targets PI3K diminishes Akt activation by growth factors (Burgering and Coffer, 1995; Chaudhary and Hruska, 2001). Akt activity was reported to be regulated in a PI3K-dependent manner. The lipid product of PI3K, PI(3,4,5)P2 binds directly to the PH domain of Akt allowing the kinase to dimerize for subsequent activation events (Kandel and Hay, 1999b). Upon insulin stimulation, Akt activity is phophorylated at Thr-308 and Ser-473. Phosphorylation at both sites is required to achieve full activation of Akt (Alessi and Cohen, 1998). The PH domain is important in translocating Akt to the cell membrane where phosphorylation of Akt occurs, leading to its full activation. Upstream kinases responsible for phosphorylating Akt at Thr-308 and Ser-473 include PDK1 and PDK2, respectively. PDK1 was reported to be a constitutively active kinase. However, PDK1 will phosphorylate Akt only if a phospholipid is bound to it. Downward (1998) proposed a model of Akt regulation that requires PI(3,4,5)P3 13 to bind to the P H domain of Akt, which causes Akt translocation to the plasma membrane, where Akt expose the Thr-308 site for PDK1 phosphorylation. The Ser-473 site of Akt is phosphorylated by P D K 2 , which causes Akt to translocate to the cytosol or the nucleus (Downward, 2001). The specif ic mechanism of P D K 2 phosphorylation of Akt is still unclear. However, PDK1 was found to interact with the carboxy-terminal region of P K C related kinase (PRK2) through a PDK1 interacting fragment (PIF) region (Balendran et al., 1999). This interaction causes PDK1 to phosphorylate both the Thr-308 and Ser-473. Furthermore, another study showed that Thr-308 phosphorylation triggers autophosphorylation of Ser-473 indicating that P D K 2 is possibly not required for Akt phosphorylation at Ser-473 (Toker and Newton, 2000). However, the integrin-linked kinase (ILK) was found to phosphorylate Akt on Ser-473, indicating that ILK could possibly be the same P D K 2 enzyme (Delcommenne et al., 1998; Persad et al., 2001). Another kinase that may act as P D K 2 is the mammalian target of rapamycin (mTOR). m T O R attracted a lot of attention because it phosphorylates a hydrophobic site of p70 S 6 K similar to the Ser-473 site of Akt (Burnett et al . , 1998). However, phosphorylation of Ser-473 of Akt by m T O R could not be confirmed (Balendran et al . , 1999; Chan and Tsichl is, 2001). Recent work has demonstrated that m T O R forms a complex with two regulatory proteins, raptor and rictor. The mTOR-raptor complex is rapamycin sensit ive, but the mTOR-r ictor complex is rapamycin-insensit ive. The former complex was found to mediate phosphorylation of p70 S 6 K and Akt at Ser-473 (Pearse ef al., 2007; Proud, 2007). Akt can be negatively regulated via several mechanisms (Kandel and Hay, 1999a). For example, the P H domain in this kinase can act as both a positive and negative regulator. In the inactive form, the P H domain hinders the ability of PDK1 to phosphorylate Thr-308. The actions of specif ic phosphatases including protein phosphatase 2 A (PP2A) , S H 2 domain-containing inositol 5 phosphatase (SHIP) and the phosphatase and tensin homologue (PTEN) on the conserved phosphorylated Thr-308 and Ser-473 can negatively regulate Akt activity (Kandel and Hay, 1999a; Datta ef al., 1999). The latter two lipid phosphatases can hydrolyze PI(3,4,5)P 3 i and prevent the recruitment of Akt to the plasma membrane. A well characterized downstream target of Akt is glycogen synthase kinase 3 (GSK3) . Upon stimulation with insulin or insulin-like growth factors, Akt phosphorylates G S K 3 on a serine residue near its N-terminus in a PI3K-dependent manner. This results in G S K 3 inhibition and dephosphorylation of glycogen synthase, which in turn facilitates an activation of this anabol ic enzyme (Cross ef al., 1995; Shaw et al., 1997; Ueki ef al., 1998; 14 van Weeren et al., 1998). Another important action of Akt is to promote cell survival by inhibiting apoptosis. Akt phosphorylates Bad (a member of the Bcl2 oncogene family that promotes cell death) at Ser-136 and prevents Bad-mediated cell death (Datta ef al., 1997; del P e s o ef al., 1997). Phosphorylation of Bad allows associat ion of Bad with the adaptor protein 14-3-3, thereby interfering the interaction between Bad and Bcl-xL (the death inhibiting member of the Bcl2 family) that would promote cell survival (Zha ef al., 1996; Henshal l ef al., 2002). Akt can regulate cell death by phosphorylating caspase 9, which plays a pivotal role in promoting apoptosis (Cardone et al., 1998). Moreover, phosphorylation of forkhead transcription by Akt could in part prevent cell death. It has been shown that overexpression of the forkhead transcription factor F K H R L 1 triggers apoptosis, but this is prevented by Akt phosphorylation of F K H R L 1 (Guo ef al., 1999; Kops ef al., 1999). Akt has also been found to synergize with the Raf/Mek/Erk pathway to cause transformation of fibroblasts in culture. Other downstream effectors in the PI3K/Akt pathway include m T O R and the elongation initiation factor binding proteins, which are involved in regulating protein translation (Proud, 2007). In the nervous system, Akt appears to respond to a variety of trophic factors and transduce survival signals in neurons. In phencycl idine-induced excitotoxity, Akt phosphorylated and inhibited G S K 3 a/(3. The latter phosphorylates microtubule-associated Tau proteins resulting in microtubule instability (Lei et al . , 2007). This condition was also observed in A D pathogenesis, where Akt inhibits G S K 3 a/p activity via phosphorylation on a specif ic serine residue. Inhibition of G S K 3 a/p by Akt prevents abnormal Tau phosphorylation, thereby conferring neuronal survival (Gervitz ef al., 2002; Ks iezak-Red ing ef al., 2003). 1.3.3 Mitogen activated protein kinase ( M A P K ) signaling 1.3.3.1 Erk1 and Erk2 signaling The M A P K cascade is a major intracellular signaling network that plays a prominent role in mediating signals for cell proliferation, differentiation, synaptic plasticity in neurons, and insulin production, amongst other functions. Members of this cascade have the characteristic of being activated upon phosphorylation on both threonine and tyrosine residues. In mammals, the best characterized members of the M A P K family are the 44 kDa Erk1 (extracellular signal regulated kinase 1) and the 42 kDa Erk2 (extracellular signal regulated kinase 2). Erk1 and Erk2 are activated following stimulation with growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor ( P D G F ) and are 15 currently known to be activated by many other extracellular stimuli (Pelech and Charest , 1995; Widmann et al., 1999). Erk1 and Erk2 classical ly play a vital role in cell survival and proliferation. There are abundant evidence that show Erk1 and Erk2 have other essential roles in the nervous system such as , regulating long-term potentiation induced by N M D A type glutamate receptor (Cobb, 1999; Platenik et al., 2000; Li ef al., 2006). Erk1 and Erk2 activation by receptor tyrosine kinases involves the recruitment a series of docking proteins. Once the receptor is activated, it will recruit S H 2 containing adaptor proteins such as insulin receptor substrate 1 (IRS1) which itself is phosphorylated by the receptor tyrosine kinase at specif ic tyrosine sites (Biondi and Nebreda, 2003). Another adaptor protein, growth receptor binding protein 2 (Grb2) interacts with the phosphorylated tyrosine residue and IRS1 and recruits other proteins through two of its S H 3 domains (Schlessinger and Lemmon, 2003). S H 3 domains recognize and bind proline rich motifs in the target protein. A protein with proline rich regions that is known to interact with Grb2 is the guanine exchange protein S o s (Son of sevenless) (Shields et al., 2000). Interaction of S o s with Grb2 results in re-localization of the former to the plasma membrane where it can activate Ras by catalyzing the release of G D P and the subsequent binding of G T P (Shields ef al., 2000; Downward, 2001). R a s belongs to a family of guanosine nucleotide binding proteins (G proteins) characterized by activation upon binding of a G T P molecule (Aronheim ef al., 1994b; Wedegaertner ef al., 1995; Sears and Nevins, 2002). Important effectors of Ras signaling is the Raf protein-serine/threonine kinase family. Interaction of Raf1 (or RafA and RafB) with Ras recruits Raf1 to the plasma membrane where it is subjected a conformational change in structure and phosphorylation at several sites in the C-terminus to become activated (reviewed in Dhillon and Kolch, 2002; Baccar ini , 2005; Frebel and Wiese , 2006). The activation of Raf1 leads to the phosphorylation and activation of its downstream effector, the M A P K kinases 1 and (MEK1/2) (Wellbrock etal., 2004). Activation of M E K 1 / 2 is bel ieved to activate Erk1 and Erk2 through phosphorylation of tyrosine and threonine residues in a conserved T E Y motif (Ahn ef al., 1992; Nishida and Gotoh, 1993). Downstream targets of Erk1 and Erk2 in the M A P K signaling cascade include ribosomal S 6 kinase (RSK) 1, R S K 2 , and the M A P K activating protein kinase (Frodin and Gammeltoft, 1999). The activated forms of Erk1 and Erk2 can also translocate to the nucleus and phosphorylate a series of transcription factors including Elk-1 (Cahill et al., 1996; Hodge ef al., 1998). This results in transcription of growth associated genes such as c-fos (Hodge ef al., 1998). The actual signaling events in the M A P K cascade may be more complex than 16 described and may involve cross talk from other cascades such as the PI3K pathway (Dent et al., 2003; Ruffels ef al., 2004). While Erk1 and Erk2 signaling are classical ly known to promote cell survival and proliferation, many recent data chal lenges this view. Luo and DeFranco (2006) describe two phases of Erk1 and Erk2 activation in embryonic rat cortical primary neurons subjected to excitotoxic stress with glutamate. The authors describe an initial phase of Erk1 and Erk2 activation (within min of excitotoxic stress) in response to the stress for cytoprotection. A late phase activation of Erk1 and Erk2 (within h) appears to mediate apoptosis induced by oxidative stress. They argue that Erk1 and Erk2 play dual roles in neurons acting as part of a cellular adaptive response during early phases and contributing to toxicity at later stages of stress (Luo and DeFranco, 2006). Another study also reported that Erk1 and Erk2 may play a role in hydrogen peroxide induced cell death in the human S H - S Y 5 Y neuroblastoma cell line (Ruffels ef al., 2004). More recently, two individual groups reported that two Alzheimer 's d isease associated genes, presenilin 1 and the amyloid precursor protein (APP) can enhance the activity of Erk1 and Erk2 in vitro and in vivo (Venezia ef al., 2006; Nizzari ef al., 2007). Furthermore, A P P seems to cause Erk1 and Erk2 to relocalize to the centrosome of the cell . The meaning of these findings are currently unclear, but lead to the hypothesis that Erk1 and Erk2 may be involved in phosphorylating cytoskeletal proteins such as tau and promoting tangle formation (Nizzari etal., 2007). 1.3.3.2 Stress-activated protein kinase and p38 M A P K signaling Another member of M A P K superfamily is the c-Jun amino terminal k inases (JNKs) , also known as the stress-activated protein k inases ( S A P K s ) . A s the name implies, these kinases are activated in response to stress factors, such as heat shock, osmotic shock, D N A damage, U V irradiation, cytotoxic drugs, and various inflammatory cytokines (Sluss ef al., 1994; Dent ef al., 2003). J N K s respond to stress stimuli and phosphorylate the transcription factor c-Jun at two conserved serine residues (ser-63 and ser-73) in the amino-terminal region (Davis, 1999). Ser ine phosphorylation of c-Jun increases its ability to bind the activator protein 1 (AP1) enhancer elements that activate transcription of many stress inducible genes (Davis, 1999). This pathway is distinct from the Erk1 and Erk2 cascades , s ince J N K s are activated in response to completely different stimuli. However similar to the Erk1 and Erk2, J N K activities are regulated by dual threonine and tyrosine phosphorylation through recognition of a T P Y motif and are modulated by an upstream protein kinases such as stress activated extracellular regulated kinase 1 (SEK1) , also known as M A P K kinase 17 kinase (MKK) 4, and another isoform M K K 7 (Yang et al., 1998; Marinissen et al., 1999). Ras and Raf1 do not activate the J N K pathway, whereas the Ras-related small G T P binding proteins, Cdc42 and R a c 1 , have been shown to activate members of the J N K family (Minden ef al., 1995; Yang ef al., 1998). A n increasing number of reports are showing that the J N K pathway can be activated by a multitude of protein k inases that act upstream of M K K 4 and M K K 7 (Fanger ef al., 1997). These new protein k inases include T G F - p activated kinase, germinal center kinase, tumor progression locus 2, specif ic mixed l ineage k inases, the p21-activated protein kinase (PAK) , amongst others (Fanger et al., 1997; Schmitz et al., 1998; Hocevar ef al.,., 1999; X u ef al., 2001; Kim ef al., 2004). The activated form of J N K s undergo nuclear translocation where they phosphorylate transcription factors such as c-Jun and A T F 2 , resulting in gene transcription that mediates growth arrest and/or apoptosis (Dent ef al., 2003). Considerable attention is devoted to studying the dysregulation of J N K signaling underlying various human d iseases for the possibility of therapeutic interventions (Johnson and Nakamura, 2007). For example, J N K knock-out mice develop neural tube defects due to excess ive apoptosis in the developing forebrain and abnormally less apoptosis in the hindbrain (Kuan etal., 1999). Deregulated levels of J N K activity have been observed in mice injected with the neurotoxin 1-methyl-4-pheny-1, 2, 4, 6- tetrahydropyridine (MPTP), . which induces Parkinsonism behavioral deficits with selective loss of dopaminergic neurons. M P T P injected mice show an abnormal level of phosphorylated c-Jun implicating an increased level of J N K activity. Pharmacological inhibitors against J N K and J N K knockout animals show variable degrees of protection against M P T P administration (Hunot et al . , 2004). In p -amyloid mouse model of Alzheimer 's d isease, J N K was found to induce neuronal apoptosis via activation of AP1-dependent Fas- l igand. J N K transgenic knockout animals are completely resistant to p-amyloid induced apoptosis (Morishima etal., 2001). A third member of the M A P K superfamily is the p38 M A P K . This protein kinase was originally descr ibed as a mammal ian homologue to the yeast high osmolarity sensing protein (HOG1) . The p38 M A P K pathway is activated by many forms of cellular stresses and involves the activity of several M K K kinase enzymes, eg. P A K , which then activate M K K 3 and M K K 6 (Fanger ef al., 1997; Dent ef al., 2003). There are several direct downstream effectors of p38 M A P K which include the M A P K activated protein kinase2 ( M A P K A P K 2 ) and mitogen and stress-activated protein k inase l and 2 (MSK1/2) (Maizels etal., 2001; Wiggin ef al., 2002). Upon activation, M A P K A P K 2 phosphorylates and activates heat shock protein 18 27 (Alford et al., 2007), and M S K 1 / 2 phosphorylates the transcription factor cycl ic A M P response element binding protein ( C R E B ) (Swart etal., 2000; Maizels etal., 2001). The role of p38 M A P K signaling in cellular responses is diverse, depending on the cell type and stimulus. For instance p38 M A P K activity is required for chrondrocyte survival and differentiation in culture (Yosimichi et al., 2001). Conversely, ethanol-induced oxidative stress is mediated by p38 M A P K signaling, which leads to neuronal death in a mouse hippocampal cell line, HT22 (Ku ef al., 2007). Furthermore, activation of p38 M A P K is frequently correlated with neuronal degeneration and has been shown to occur in human cases of Alzheimer 's d iseases and other tauopathies (Horstmann ef al., 1998; Skaper and Wa lsh , 1998; Atzori ef al., 2001a; Zhu ef al., 2000; Tortarolo et al., 2003). Several groups have independently reported activation of p38 M A P K in the motor cortex (Holasek ef al., 2005) and lumbar spinal cord of a transgenic murine model of A L S (Wengenack ef al., 2004; Tortarolo et al., 2003). p38 M A P K is implicated in various functions such as phosphorylating cytoskeletal elements, biosynthesis of cytokines, and nitric oxide production (Lee ef al., 1994; Mielke and Herdegen, 2000; Ono and Han, 2000). These mechanisms are supposedly involved in neurodegenerative events seen in A L S and Alzheimer 's d isease (Cleveland and Rothstein, 2001; Atzori etal., 2001a). Other members of the M A P K superfamily include Erk3, Erk5, and Erk7. Limited information is available on these M A P K s and they will not be d iscussed in further detail. The three M A P K cascades d iscussed above reflect a diversity of stimuli that converge on a complex network of regulatory pathways that selectively converge to mediate a physiological event. 1.4 Rationale and research objectives 1.4.1 Rationale Both epidemiological and experimental studies have indicated a strong link between the pathogenesis of the Guamanian neurological disorder A L S - P D C with dietary consumption of seeds from the cycad plants (Hirano ef al., 1966; Kurland, 1988; Wi lson ef al., 2002; Wi lson et al., 2004). Cycad seeds were found to contain a family of bioactive compounds called steryl g lycosides (Khabazian ef al., 2002). Var ious members of the family of steryl g lycosides were found to have toxic properties and may be the putative neurotoxin involved in the etiopathogenesis of the Guamanian A L S - P D C and classical cases of A L S , P D , and A D . Given that most people are not exposed to cycad and its toxins, it is important to determine if similar compounds are present in our environment. A n interesting link 19 between cycad toxins, bacterial infections, mammalian synthesis of steryl glycosides, and the etiopathogensis of age-related neurodegenerative diseases has recently been reviewed (Ly ef al., 2007). This link provides a more relevant identification and mechanistic action of cycad toxins. The precise roles of this class of compound and their mechanisms of action have yet to be determined. Living systems have the ability to regulate cell death pathways as a mean for protection from stressful challenges. An example is a rapid up-regulation of survival pathways such as the PI3K/Akt and/or Raf/Mek/Erk pathways to counteract cell death caused by the stress stimuli. The inability to up-regulate these pathways renders the cell vulnerable to stress stimulation, which likely leads to cell death. This effect was characterized in a mouse model of familial ALS, where the lack of PI3K/Akt activity is a contributing factor to motor neuron degeneration (Warita ef al., 2001; Peviani ef al., 2007). Another example of this form of cytoprotection is ischemic preconditioning, where a brief ischemic episode protects against the potentially lethal effect of subsequent ischemic events. However, the key mediators for this phenonmenon are unclear. Ischemic preconditioning has received much attention as a protective strategy against ischemic damage to the heart, brain, liver, amongst other organs. However ischemia-induced oxidative stress has been associated with many forms of pathological conditions. Particularly, oxidative stress in neurons induces neuronal apoptosis and is believed to be a contributing cause to neurodegenerative diseases such as ALS. Likewise, cholesteryl glucoside (CG) is a member of the S G family and has toxic effects in rat cortical slice cultures. However, a brief C G exposure can protect mammalian fibroblasts from stress induced by heat shock and heat shock can also up-regulate endogenous synthesis of C G in fibroblasts (Kunimoto ef al., 2000, 2002). This indicates that C G has both toxic and protective properties. Although neurodegeneration observed in Guam can be considered a rare and geographically isolated disorder, it can share a common etiological basis with the rest of the neurodegenerative diseases. SGs are present in excessive quantities in cycads seed on Guam, but this family of compounds can also be found in a wide range of natural settings. However, the physiological role of SGs in the mammalian system remains unclear. On one hand, it fits the notion of a neurotoxin that acts negatively on neurons leading to various neurological conditions. On the other hand, brief exposures of SGs appear to temporarily protect cells against various stress stimuli. Futhermore, the mechanism of action by which SGs kills and confer protection is unclear. It is imperative that we understand how C G 20 differentially regulates cell death and cell survival pathways, and the mechanisms underlying this regulation. This will further our understanding on the physiological actions of SGs and better describe the molecular events leading to neurodegeneration. The complete description of events leading to neuronal death will suggest potential stages of therapeutic intervention targeting specific stages of disease progression. 1.4.2 Objectives The objectives of this study were two-fold. First, one objective was to evaluate if C G treatment will confer preconditioning for serum deprivation stress in a neuroblastoma x spinal cord cell line (NSC34 cells) and to determine which signaling pathways are involved. A Kinetworks™ phosphosite screen was used to indicate potential candidate signaling molecules may be involved in C G preconditioning for serum deprivation. Specific pharmacological inhibitors were also used to study the role of the tracked signaling molecules during preconditioning. Secondly, the aberrant regulation of certain protein kinases in NSC34 cells exposed to C G was investigated. Specifically, Western blot analysis of Akt, Erk1/2, p38 MAPK, and JNK were performed on NSC34 cells exposed to C G under various experimental parameters. C G induced reduction of NSC34 cell viability was also evaluated in the presence or absence of specific pharmacological inhibitors that block the Akt, Erk1/2, p38MAPK, and JNK pathways. In addition, C G treatment to NSC34 cells led to morphological changes that were not observed in control cells. These morphological changes were characterized using immunocytochemistry and determined the relationship to CG-mediated toxicity. This study aimed to contribute to the long term goal of identifying the causative molecule(s) in ALS-PDC which may share a common basis with the rest of the neurodegenerative diseases. SGs and related compounds are commonly found in the environment and can cause neurodegenerative changes in vulnerable individuals. The information obtained from these three approaches may be used to establish the mechanistic action of SGs, a family of compounds that may contribute to the etiopathogenesis of ALS-PDC. The ultimate goal of this work was to understand the molecular changes induced by this putative neurotoxin, which may provide new insights into potential therapeutic interventations for ALS, PD, and AD. 21 Figure 1. PI3K and M A P K signal ing pathways. Binding of a growth factor to its receptor could activate various signaling pathways including the activation of PI3K, Ras, and their downstream effectors. CHAPTER 2. Materials and Methods 2.1 Materials 2.1.1 Chemical reagents Chemicals and abbreviations Companies Acetic acid Sigma Acrylamide BioRad Bicinochoninic acid protein assay kit Pierce Bis-acrylamide BioRad BlokHen blocking reagent Aveslab Bovine serum albumin fraction V (BSA) Sigma Bromophenol blue Fisher Chloroform Sigma Cholesterol (water soluble) Sigma Collagen (0.1%) solution Sigma Dulbecco's modified Eagle medium Sigma Enhanced chemiluminence kit Pierce Ethanol Vancouver General Hospital Ethylene bis (oxyethylenenitrilo) tetra-acetic acid (EGTA) Fisher Ethylene diamine tetra-acetate disodium salt (EDTA) Sigma Fetal bovine serum Sigma Glutamine Invitrogen Glycerol Sigma p-glycerophosphate Fisher Glycine Sigma LY294002 ((2-(4-morpholinyl)-8-phenyl-4H-143enzopyran-4-one) EMDBiosciences Magnesium sulphate heptahydrate Fisher p-mercaptoethanol Fisher Methylene chloride (DCM) Fisher MOPS 3-[N-morpholino]ethanesulfonic acid Fisher MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Promega Paraformaldehyde Sigma 23 Phenylmethylsulphonyl fluoride (PMSF) Sigma Ponceaus S concentrate Sigma Potassium chloride (KG) Fisher Potassium phosphate (K 2 HP0 4 ) Fisher Prestained protein standards Fermentas Protease inhibitor cocktail tablets Roche Rapamycin S. Pelech Lab SB230580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)- EMD Biosciences 5-(4-pyridyl)1 H-imidazole) Skim milk Fisher Sodium azide Fisher Sodium bicarbonate (NaHC0 3 ) Fisher Sodium chloride (NaCI) Fisher Sodium dodecylsulphate (SDS) Fisher Sodium fluoride (NaF) Fisher Sodium orthovanadate (Na 3 V0 4 ) Fisher Sodium phosphate (NaHP0 4 ) Fisher Sodium pyrophosphate (Na4P2C>7) Fisher SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one) EMD Biosciences Sulfuric acid Fisher Tris hydroxy I methyl methylamine (Tris) Fisher Tris hydroxylmethylaminomethane hydrochloride (Tris-HCI) Fisher Triton X-100 Fisher Trypan blue dye Sigma Trypsin (0.25% with EDTA) Invitrogen Tween 20 (polyoxyethylene-20-sorbitan monolaurate) Fisher U0126 (1,4-diamino-2,3-dicyano-1,4- EMD Biosciences bis[2-aminophenylthio] butadiene) Vanillin 4-hydroxy-3methoxybenzaldehyde Sigma 24 2.1.2 Laboratory supplies Laboratory supplies 3 M M filter paper Cel l culture coversl ips Cel l culture d ishes (sterile) Nitrocellulose Reflection autoradiography film Thin layer chromatography plates (NanoSi l NH2) 8-well multichamber glass sl ides Companies V W R Fisher Sarstedt B ioRad Pierce Macharey-Nagel B D Biosc iences 2.1.3 Primary and secondary antibodies Antibody Company Application Dilution Actin S igma W B 1 1 5000 Goat anti-chicken IgY-fluorescein Aves lab I C C 2 1 1000 isothiocyanate conjugate Goat anti-mouse IgG-horse radish Jackson W B 1 10,000 peroxidase conjugate ImmunoResearch Goat anti-rabbit lgG-Alexa548 conjugate Invitrogen ICC 1 500 Goat anti-rabbit IgG-horse radish Jackson peroxidase conjugate ImmunoResearch W B 1 2000 Heavy neurofilament antibody Chemicon ICC 1 500 Pan specif ic Akt Cel l signaling W B 1 2000 Pan specif ic Erk Cel l signaling W B 1 2000 Phospho-Tau (AT8 clone) Pierce ICC 1 500 P h o s p h o - A K T S e r " 4 7 3 Cel l signaling W B 1 1000 P h o s p h o - A K T T h r ' 3 0 8 Cel l signaling W B 1 1000 Phospho -ERK1 /2 Cel l signaling W B 1 1000 Phospho -MEK1 /2 Cel l signaling W B 1 1000 P h o s p h o - P D K 1 S e r " 2 4 1 Cel l signaling W B 1 1000 P h o s p h o - R a f 1 S e r " 2 5 9 Cel l signaling W B 1 1000 T A R DNA-binding protein 43 (TDP43) ProteinTech group ICC 1 500 p-Tubulin III Aves lab ICC 1 1000 1 WB is the abbreviation for western blot analysis 2 ICC is the abbreviation for immunocytochemistry 25 2.2 Methods 2.2.1 Neuroblastoma x spinal cord 34 (NSC34) cell line culture The mouse-derived neuroblastoma x spinal cord cell line, NSC34 cells (a kind gift from Dr. Neil Cashman, Brain Research Center, University of British Columbia) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2 mM L-glutamine and 10% (v/v) fetal bovine serum. Cells were maintained at 37°C in a humidified 5% C 0 2 atmosphere and subcultured at 90% confluency. For every experiment, NSC34 cells were seeded at a density of 1500 cells/mm 2 onto collagen coated plates and were allowed to grow for another 5 days before treatment. Cell culture medium was replaced every 3-4 days. Collagen coating was achieved by pre-incubating culture plates with 0.1% collagen solution (500 ul/mm2) for 3 h at room temperature and washed once with sterile distilled water. Cell number was estimated by diluting an aliquot of cells in trypan blue dye (1:10) and counting cells in 9 squares of a hemacytometer. 2.2.2 Cholesterol glucoside stock solution and kinase inhibitor preparation Cholesterol-p-D-glucoside (MW=548.8 g/mol) was synthetically made in the laboratory of Dr. Stephen Withers, Department of Chemistry, University of British Columbia. Cholesterol glucoside (CG) stock solutions were prepared in 100% dimethyl sulfoxide (DMSO) to give concentrations of 30 mM and 50 mM. C G stock solutions were stored at 4°C protected from light. The final concentrations of C G were diluted in DMEM to give the highest experimental concentration under 0.5% DMSO. Inhibitors were initially dissolved in 100% DMSO at various stock concentrations: LY294002 (20 mM), U0126 (20 mM), rapamycin (0.2 mM), SB230580 (30 mM), and SP600125 (50 mM). Inhibitors were stored at -20°C protected from light. Inhibitors were either pretreated or co-treated with C G to NSC34 cells. The final concentration of DMSO during co-treatment experiments did not exceed 0.5%. Increasing doses of DMSO up to 0.5% were previously found to have no toxic effects on cell viability (Ly et al., 2007). Appropriate DMSO vehicle solutions were made for the kinase activity studies. 2.2.3 Cell viability assays Cell death was determined using a trypan blue dye exclusion test. To determine the percentage of dead cells in each well, the medium (1 mL per well) was first removed and the cells were trypsinized. The medium was returned to the well to halt further trypsinization. To allow dead cells to take up the trypan blue dye, each well received a 1:10 dilution of the dye 26 for 10 min followed by quantification in 9 counting areas on a hemacytometer. To be consistent in data presentation with other experiment, results from trypan blue dye exclusion tests are presented as the percent of live cells, determined by substracting the percentage of dead cells from 100%. Cell viability was determined using the MTT assay. This method is based on the ability of viable cells, but not dead cells, to convert a tetrozolium salt (MTT) to a coloured formazan product. Treated cells were replenished with fresh media with MTT (1 ug/ml) and allowing reduction of MTT to occur at 37°C for 4 h. The media were removed and replaced by 100% DMSO to solublize the formazan product. Optical density at 540 nm was measured using an ELx808™ absorbance microplate reader (Biotek Instrument). Viable cell values of treated cells were expressed a percentage of control cells, the latter being defined as 100%. 2.2.4 Treatment protocols NSC34 cells were subjected to various treatment paradigms in order to study the effects of C G . For the serum deprivation experiment, the complete media was removed followed by two washes with serum free media, and the cells were cultured in serum free media. Serum free media did not have 10% FBS, but was supplemented with 2 mM L-glutamine. For experiments with C G treatment and serum deprivation, NSC34 cells were either pretreated or co-treated with C G . C G pretreatment was performed in the presence of serum followed by two washes with serum free media, and culturing in serum free media. For co-treatment experiments, NSC34 cells were washed twice with serum free media and cultured in serum free media in the presence of C G . For serum stimulation experiments, NSC34 cells were deprived of serum for 12 h and stimulated with media containing serum and C G for varying times. NSC34 cells were exposed to increasing concentrations of C G (0 - 250 uM) for 5 min to study the dose relationship on various kinase activities and cell viability. 0.3% DMSO was used as a treatment control to show that DMSO has no effect on kinase activities. For a time course study on kinase activities and cell viability, NSC34 cells were exposed to 250 uM of C G for varying times (0 to 1440 min). For the experiments using kinase inhibitors, NSC34 cells were pretreated with the inhibitors at the appropriate concentrations for 1 h. The cells were then washed once with cell culture media and cultured in media containing C G . For studying the role of PI3K, mTOR, and Mek1/2 in modulating Akt and Erk1/2 activity, cells were pretreated with the inhibitors LY294002 (30 uM), rapamycin (200 nM), and U0126 (10 uM) for 1 h. The inhibitors 27 were removed by one wash with complete media and incubated in complete media with or without C G for 10 min. For studying the role of PI3K, Mek1/2, p38 M A P K , and J N K 1 / 2 on cell viability during C G treatment, N S C 3 4 cells were pretreated with the inhibitors LY294002 (30 pM), U0126 (10 uM), SB203580 (10 uM), and S P 6 0 0 1 2 5 (10 uM) for 1 h. Afterwards, complete media with C G and the inhibitor were added for 20 h. After 20 h of treatment, the inhibitor was removed by a wash with complete media and the cells were cultured in complete media with C G for up to 48 h total before assessment of cell viability. For inhibitor controls, N S C 3 4 cells were treated with inhibitors only without C G . Abnormal morphology was observable at least 3 days after 100 uM of C G treatment. N S C 3 4 cells were treated under these conditions to induce abnormal morphology, followed by immunocytochemistry. 2.2.5 Homogenzat ion and protein preparation N S C 3 4 cells were washed once with ice cold phosphate buffered saline ( P B S , 137 m M NaCI, 10 m M sodim phosphate, 2.7 m M KCI, pH 7.4) and harvested in lysis buffer containing 0.5% Triton X-100, 2 m M E G T A , 5 m M E D T A , 20 m M M O P S , 200 m M sodium vanadate, 25 m M p-glycerophosphate, 20 mM sodium pyrophosphate, 30 m M sodium fluoride, 1 m M P M S F , and 1 complete mini protease inhibitor cocktail tablet (Roche Diagnostics, Germany) for every 10 mL of homogenizing buffer, pH 7.0. Lysed cells were transferred to microfuge tubes and were then subjected to sonication for 15 sec on ice followed by centrifugation at 12,000 x g for 30 min to remove impurities such as nucleic acids and lipids. A n aliquot of the protein lysate was diluted in homogenizing buffer (1:7) and was used to assess protein concentration. Bicinchoninic acid protein determination kit was used to determine the concentration of the protein lysate by following the manufacteurer's instructions (Pierce, USA) . 2.2.6 SDS-polyacry lamide gel electrophoresis Samples containing proteins were separated using sodium dodecylsulphate polyacrylamide gel electrophoresis ( S D S 4 3 A G E ) . Proteins were diluted with 4 X concentrated S D S - s a m p l e buffer (125 mM Tris4HCI pH 6.8, 4 % S D S (w/v), 2 0 % glycerol (v/v), 10% p-mercaptoethanol, 0 .01% bromophenol blue (w/v)), boiled for 5 min then loaded onto an S D S - P A G E gel . Twenty micrograms of proteins were subjected to electrophoresis on 1.0 mm thick polyacrylamide gels with 5% stacking gels and 12% separating gels. The 28 proteins were separated for 2 h at 200 mV in running buffer (25 m M Tris, 192 mM glycine, 3.5 m M S D S ) in a mini Protean 3 electrophoresis system (BioRad). After electrophoresis, the separating gel was equilibrated in transfer buffer (20 m M Tris, 120 m M glycine, 1% S D S (w/v), 2 0 % methanol (v/v), pH 8.6) for 5 min. Nitrocellulose membrane was hydrated in transfer buffer for 5 min and assembled with the gel into a sandwich between 2 p ieces of 3 M M filter paper and 2 fiber pads. The protein on the gel was electrophoretically transferred to the nitrocellulose membrane for 55 min at 500 mA in a Mini Trans-Blot® electrophoretic transfer cell (BioRad). 2.2.7 Western blot analysis Transferred proteins were stained using Ponceau S dye by incubating the membrane in the stain for 5 min in order to visual ize the proteins on the nitrocellulose membrane. Excess ive staining was removed by rinsing the membrane in distilled water. The nitrocellulose membrane was blocked in 1% B S A (w/v) in Tris-buffered saline (TBS, 20 m M Tris and 137 m M NaCI, pH7.4) containing 0 .1% Tween-20 (v/v) (TBST) for 1 h. The membrane was then probed with various commercial ly available primary antibodies diluted in 1% B S A T B S T for 18 h at 4°C with gentle shaking. After primary antibody incubation, membranes were washed three times 10 min each with T B S T followed by incubation with appropriate horse radish-conjugated secondary antibodies diluted in T B S T containing 5% skim milk (w/v) for 1 h at room temperature. The membrane was washed again to remove excess secondary antibodies. The Western blots were incubated with enhanced chemi luminecence detection system (Pierce) for 1 min and exposed to film to visual ize the immunoreactive protein. The immunoreactive protein band was quantified by scanning the film into a TIFF format file and densitometry analysis of band densit ies were quantified in arbitrary intensity units using the NIH Image J 1.37 software. To reuse the nitrocellulose membrane for antibody detection, the nitrocellulose membranes were stripped using a commercial ly available 10X stripping solution (Chemicon). The stripping solution was dilute to 1X in distilled water. The membrane was incubated in the stripping solution for 10 min at room temperature with gentle shaking. After stripping, the membrane was rinsed with T B S T three times to remove the stripping solution. The membranes were re-blocked with 1% B S A in T B S T for 1 h and the Western blot procedures were followed as previously descr ibed. 29 2.2.8 Kinetworks™ KPSS-1.3 phosphosite screen NSC34 cells were pretreated with 100 uM of C G for 1 h, washed with serum free media and cultured in serum free media for 4 h. The cells were harvested as described above and diluted in 4X SDS-sample buffer to give a final protein concentration of 1 ug/ul. The samples were then boiled and sent to Kinexus Bioinformatics Corp., Vancouver, BC. for Kinetworks™ KPSS-1.3 phospho-site screen. The KPSS-1.3 screen tracks the phosphorylation levels of 35 kinases (eg. Erk1/2, Akt, GSK3 a/p) as well as phosphoproteins. For a full list of phosphoproteins tracked in the K P S S 1.3, please refer to the company's information at www.kinexus.ca. The KPSS-1.3 analysis was a collaborative project with Dr. Steven Pelech (Kinexus Bioinformatics Corp.) to study the phosphorylation changes during C G preconditioning. A list of phosphorylation changes was provided by the company. Akt phosphorylation changes were the most dramatic so the changes of Akt and its downstream targets and Erk1/2 were confirmed using Western blot analysis in my home laboratory. The protocols for confirming these changes are described above. 2.2.9 Lipid extraction and TLC analysis of steryl glucoside TLC analysis of steryl glucoside requires a large amount of starting material. Therefore, NSC34 cells (6x106 cells/ml) were grown in 10 cm collagen-coated culture dishes prior to serum deprivation. After serum deprivation, the medium was removed and the cells were washed once with ice cold PBS. The cells were then harvested in 1 ml of ice cold Krebs-Henseleit buffer (118 mM NaCI, 47 mM KCI, 1.19 mM K H 2 P 0 4 , 1.19 mM M g S 0 4 -7H 20, 25 mM NaHC0 3 ) with a cell scraper. The cells were transferred to a microfuge tube and sonicated on ice for 15 sec. The lipids were extracted with the mixture of chloroform/methanol (1:2, 1:1, and 2:1) successively by vortexing and centrifuging at 1500 x g for 10 min. The supernatants were combined from 3 successive extractions per group and evaporated to dryness with nitrogen gas. The lipid extract was resuspended in 100 pi of chloroform/methanol (1:1) and the entire lipid extract was analyzed by thin layer chromatography (TLC) using Nanosil NH 2 based TLC plates (Macharey-Nagel). C G standards (10 uM and 100 uM) were also spotted to show the migration pattern of steryl glucosides on the TLC plate. After spotting the lipid samples and standards, the TLC plate was allowed to dry in an oven at 40°C for 10 min followed by developing with dichloromethane/methanol/water (84:15:1) for 20 min. The TLC plate was allowed to air dry for 30 min and was sprayed with a visualization reagent (1% vanillin (w/v), 1% sulphuric acid (v/v), and pure ethanol). The plate was allowed to dry for a 30 few min followed by heating to 90°C. A purple band with a specif ic Rf value (0.33) were observed on the T L C plates and compared with the C G standards. The T L C plates were scanned into TIFF files for densitometry analysis. Band densit ies were quantified in arbitrary intensity units using the NIH Image J 1.37 software and expressed as the percentage of control. 2.2.10 Immunocytochemistry and quantification of cell morphology N S C 3 4 cells were grown on a col lagen coated multi-chamber glass slide at the indicated density prior to treatment. N S C 3 4 cel ls were then washed twice with ice-cold P B S and fixed in 4 % paraformaldehyde (w/v in P B S ) for 30 min at room temperature. Fixed cells were rinsed twice with P B S and permeabl ized with 0.3% Triton-X 100. The cells were blocked with either 10% normal goat serum or the BlokHen blocking reagent (Aveslab) diluted in P B S . BlokHen blocking reagent is the manufactuerer-recommended blocking reagent for a chicken-derived primary antibodies. Primary antibodies were diluted in the same blocking buffer and incubated with gentle shaking overnight at 4°C. After incubating with the primary antibody, the cells were washed three times for 5 min each with P B S and incubated with an Alexafluor 548 conjugated or FITC conjugated secondary antibody as appropriate, for 30 min at room temperature. For double labeling of two different proteins in N S C 3 4 cel ls, the above descr ibed procedures were performed subsequently to avoid cross reaction of the antibodies. The cells were mounted with DAPI mounting medium (Vector Labs) and the sl ides were sealed using nail polish. Images were taken using a Ze iss Axiovert florescent microscope and a focal high performance C C D camera. Cel ls were viewed under a 20X or 40X objective lens in at least six different areas on the slide. To a s e s s cell morphology, treated N S C 3 4 cel ls were stained with p-Tubulin III and quantified using the Ze iss AxioVis ion 4.5 imaging software. S o m a size was determined tracing the edge of the cell body. The automated calculation function from the software provides a value of the outline cell perimeter in micrometer (um). With the same outline indicating the cell perimeter, any cellular components protruding outside of this boundary is considered a process. The number of p rocesses as manually counted and expressed as the number of processes per cell . P rocesses length was determined by tracing the observed processes. The automated calculation function from the software provides a value for processes length in um. The sum of all measured processes length per cel ls was presented. 31 2.3 Statistics and data analyses Statistical signif icance was determined by either one way A N O V A followed by Tukey 's honest post hoc test or unpaired Student's t-test if the data were normally distributed and the var iances were homogenous. The non-parametric Krustal-Wall is two sample test was used when the data were not normally distributed and/or the var iances were not homogenous. Statistical signif icance was defined as p<0.05. All data are presented as mean±S.E.M from experiments that were performed at least in triplicate. 32 CHAPTER 3. Cholesteryl glucoside preconditioning of NSC34 cells for serum deprivation stress Hypothesis: Cholesteryl g lucoside ( C G ) exposure preconditions N S C 3 4 cel ls for serum deprivation stress. Specific aims: 1. To determine if C G can precondition the motor neuron-derived cell line, N S C 3 4 cells, for serum deprivation. 2. To determine the mechanisms underlying C G preconditioning for serum deprivation. The mechanism of action will be determined using a multi-immunoblot phospho-site screen. 3.1 Results 3.1.1 C G preconditioning for serum deprivation stress To investigate whether C G can confer neuroprotection against serum deprivation in motor neurons, we developed a preconditioning paradigm in a motor neuron-derived cell line, N S C 3 4 cells. This cell line possess properties of motor neurons, including the generation of action potentials, acetylcholine production, expression of neurofilament triplet proteins, and formation of neuromuscular junctions with myotubes in vitro (Cashman et al., 1992). The main advantage of using this cell line is to avoid contamination of the motor neuron culture with glia, which has been speculated to contribute to neuronal death (Holden, 2007). Therefore, the responses observed from C G exposures are attributed to these motor neuron-derived cells only. Prior to studying the preconditioning effect, the cytotoxicity of C G was first estabil ished for the N S C 3 4 cel ls. These cells were exposed to increasing concentrations of C G for 1 to 7 days and cell viability was assessed using the MTT reduction assay . A two way A N O V A analysis showed that N S C 3 4 cell viability is dependent on the concentration and the duration of C G exposure (Concentration: F 4,217=52.3 p<0.001; Duration: F 3 | 2 17=33.3 p<0.001). A Tukey 's honest post hoc analysis indicated at which time point the treatment group was different from the control group (p<0.05). Significant cell death was observed only after 24 h of C G treatment at 50-250 uM range (Figure 2A). Treatment with cholesterol only (without the g lucose moiety) did not induce any significant effect on N S C 3 4 cell viability over increasing dose and time (Figure 2B). 33 Serum deprivation of the NSC34 cells for 24 h induced approximately 25% reduction in cell viability (unpaired Student's t-test, p<0.05). The NSC34 cells were pretreated with 50 uM of C G for 1 h prior to serum deprivation. The purpose of the C G pretreatment is to allow the cells to upregulate proteins involved in preconditioning (Andoh ef al., 2000) and hypothetical^ confer protection against serum deprivation. Cell viability was then determined by the trypan blue dye exclusion test. Serum deprivation alone induced roughly 25% cell death, but this effect was slightly protected by C G preconditioning (Figure 3). To assess whether C G can precondition for serum deprivation over a range of concentrations, NSC34 cells were pretreated with increasing doses of C G for 1 h, followed by serum deprivation for 24 h. Cell viability was assessed using the MTT reduction assay. One way ANOVA (F5i3o=4.78 p<0.05) followed by Tukey's honest post hoc (p<0.05) indicated that 25-100 uM C G treatment induced cytoprotection against serum deprivation for 24 h (Figure 4). To examine if C G can continuously protect NSC34 cells from serum deprivation, 100 uM of C G was administered simultaneously with serum deprivation for 24 h, 48 h, and 72 h (Figure 5). Prolong C G exposure could not protect NSC34 cells from cell death induced by serum deprivation (unpaired Student's t-test p<0.05). 3.1.2 Signaling pathways underlying C G preconditioning in NSC34 cells The previous experiment clearly demonstrated that C G preconditions NSC34 cells for serum deprivation, so the following studies focused on investigating the signaling pathways involved in C G preconditioning. To approach this question, the Kinetworks™ KPSS-1.3 phospho-site screen was employed to track the activity of 35 different signaling proteins at specific phosphorylation sites. The phospho-site specific antibodies used in the screens have been rigorously validated in-house at Kinexus. Figure 6 shows an example of the multi-immunoblot generated from the phospho-site screen of NSC34 cell serum deprived for 4 h not treated with 100 uM C G (Figure 6A) or pretreated with 100 uM of C G for 1h (Figure 6B). In Figure 6A, all the phosphorylation sites detected by the phospho-site screen were marked with a red arrow. Those phospho-sites that were not detected were not indicated in the figure can be deduced by comparison with the lists of all the target phospho-proteins found in the Kinetworks™ KPSS-1.3 phospho-site screen at the Kinexus Bioinformatics Corporation website (http://www.kinexus.ca). In Figure 6B, the percentage change from control (%CFC) greater than 25% were indicated by dotted circles. These proteins include: A k i ^ " 4 7 3 , Adducina S e r - 7 2 6 , Adduciny S e r" 6 9 3, B 2 3 S e M , P K R T h M 5 1 , G S K 3 a T y r ' 2 7 9 , GSK3f3 Y 2 1 6 , G S K 3 a S e r " 2 1 , GSK3p S e r " 9 , S rc T y r - 4 1 8 , Mek6 S e r - 2 0 7 , Msk1 S e r " 3 7 6 , Mek1 S e r - 2 1 7 + S e r - 2 2 1 , 34 E r k 2 Thr-i85*Tyr-i87 j a n d J N K ThM83 + Tyr- i85 T h e r e g r e v a r j o u s unidentified immunoreactive protein bands that were detected by the phospho-site screen. These unidentified protein bands were arbitrary labeled A ? to E ? . The % C F C for each unclassified protein was indicated below the arbitrary names on the multi-immunoblot. Figure 7 is a summary graph of the % C F C for the signaling proteins indicated in Figure 6B. To study the time course of phosphorylation changes induced by C G preconditioning, N S C 3 4 cells were pretreated with 100 uM of C G for 1 h and serum deprived for up to 8 h. Previous studies have suggested that the PI3K/Akt pathway is important in modulating preconditioning against various stress stimuli (Alvarez-Tejado ef al., 2001; Han ef al., 2001; Ruffels ef al., 2004; Zhuang ef al., 2007). Therefore, it was of particular interest to investigate the role of the PI3K/Akt pathway in C G preconditioning for serum deprivation in N S C 3 4 cells. The Raf/Mek/Erk pathway is another survival pathway that was investigated to compare with the PI3K/Akt pathway. The phosphorylation levels of A k t 3 ^ " 4 7 3 , A d d u c i n g 7 2 6 , Adduc iny S e r - 6 9 3 , G S K 3 a T y r " 2 7 9 , G S K 3 ( 3 Y 2 1 6 , S r c T y r " 4 1 8 , E r k 1 T h r - 2 0 2 + T y r - 2 0 4 , and E r k 2 T h M 8 5 + T y r " 1 8 7 were determined using phospho-site specif ic antibodies (Figure 8A-I). The activity levels from treated or control groups over time were analyzed using a two way A N O V A analysis. Amongst the tracked phosphorylation changes, E r k 2 T h M 8 5 + T y M 8 7 (group: F 1 ] 1 6 =0.44 p>0.05; time F 3 , 1 6=7.68 p<0.05), A d d u c i n S 6 9 3 (group: F 1 i 1 6 =3.71 p<0.05; time F 3,16=30.0 p<0.001), and A k t S e M 7 3 (group: F 1 , 1 6 =4.63 p<0.05; time F 3 , 1 6 =15.7 p<0.001) showed significant changes. A follow-up analysis using the unpaired Student's t-test (p<0.05) showed at which time point the treated and control groups were significantly different from each other (Figure 8). The blots were stripped and reprobed with actin. There were no differences in the level of actin throughout the groups, which indicated equal protein loading. The activity level of each protein is represented as the intensity of the phosphorylated protein over the intensity of actin. To determine the role of the PI3K/Akt pathway and Raf /Mek/Erk pathway in C G preconditioning, pharmacological inhibitors that individually inhibit PI3K and Mek were used (Figure 9). N S C 3 4 cells were subjected to C G preconditioning in the presence or absence of the inhibitors for 1 h and serum deprived for 24 h followed by cell viability measurements using the MTT reduction assay. Treatment of 100 uM of C G preconditions N S C 3 4 cells for cell death-induced by serum deprivation (unpaired Student's t-test, p<0.05). Treatment with the Mek1/2 inhibitor U0126 (10 u M , Mek1/2) alone to inhibit the Mek/Erk pathway did not have any effect on C G preconditioning. Inhibition of the PI3K/Akt pathway with the PI3K inhibitor LY294002 (30 uM) during C G preconditioning prevented cytoprotection as 35 compared to C G treatment alone (unpaired Student's t-test p<0.05). Simultaneously inhibiting the pathways using U0126 and LY294002 during C G preconditioning also prevented cytoprotection (unpaired Student's t-test p<0.05). The loss of cytoprotection during preconditioning in the presence of U0126 and LY294002 is mainly attributed to the effect of LY294002, since treatment with LY294002 alone and LY294002 with U0126 equally abolished the C G preconditioning (unpaired Student's t-test, p<0.05). U0126 treatment alone did not have any effect on C G preconditioning. The JNK members of the MAPK family have not been found to be involved in cellular preconditioning to stress stimuli (Nakano et al., 2000). To show that the JNKs do not play a role in C G preconditioning, a pharmacological JNK inhibitor SP600125 (10 uM, JNK) was used to block the JNKs during C G preconditioning. Blocking the JNKs with the SP600125 compound did not have any effect on this form of cytoprotection. Living cells have ways to prevent cell death mediated by various stress stimuli. Previously, the process of cholesterol glycosylation to form cholesteryl glucoside has been reported to be upregulated during heat shock of human fibroblasts in culture (Kunimoto ef al., 2000). To investigate if NSC34 cells were capable of endogenous synthesis of cholesteryl glucoside during stress induced by serum deprivation, NSC34 cells were maintained in serum free media for 0-18 h followed by analysis of the lipid profiles by thin layer chromatography. The C G band intensities were then quantified (Figure 10A). One way ANOVA shows a time dependent increase in endogenous C G synthesis with increasing duration of serum deprivation (F 3 8=4.23 p<0.05). Tukey's honest post hoc analysis (p<0.05) showed that 18 h of serum deprivation significantly upregulated endogenous C G content in NSC34 cells by two-fold. 3.2 Discussion Brief exposures of C G can trigger intercellular signaling within physiological levels, but prolonged exposures are cytoxic (Figure 2A). This cytotoxic potential indicates that cells may have intrinsic resistance mechanisms that prevent cell death mediated by C G and possibily other stress stimuli. The purposes of this study were to examine if C G treatment can prevent cell death induced by serum deprivation and the mechanisms underlying C G preconditioning. The finding that endogenous steryl glycoside synthesis in NSC34 cells occurs during serum deprivation indicates that C G may function at physiological levels as a response mechanism to stress (Figure 10). TLC analysis of endogenously synthesized C G was based on the same Rf value as a C G standard. It should be noted that further work with 36 mass spectrometry or high performance liquid chromatography will be required to definitely show that C G is indeed endogenously-synthesized during serum deprivation. Brief exposure of exogenous C G was found to upregulate survival pathways to counteract cell death induced by serum deprivation (Figures 3 and 4). Using a multi-immunoblot phospho-site screen, we identified various protein kinases that appear to be involved in C G preconditioning (Figures 6 and 7). Likewise to other types of preconditioning conditions (Han ef al., 2001; Alvarez-Tejado ef al., 2001; Ruffels et al., 2004), Akt activity was upregulated during C G preconditioning (Figure 8E), which further supports the notion that the PI3K/Akt pathway plays a central role in preconditioning. The activities Erk1/2 were also examined and Erk2 activity was found to be upregulated (Figure 8D). However, blocking the activation of Erk1/2 with pharmacological inhibitors did not interfere with C G preconditioning for serum deprivation (Figure 9). In contrast, inhibiting the PI3K/Akt pathway completely abolished the cytoprotection (Figure 9). 3.2.1 Preconditioning as an intrinsic property of the cell Murray ef a/.(1986) were first to describe a preconditioning phenomenon in the heart myocardium, where the tissue exposed to brief episodes of ischemia developed protection against irreversible damage during subsequent, sustained ischemia. This type of preconditioning is known as ischemic preconditioning and has been found to extend its effect in noncardiac tissues and organs, such as the brain (Kitagawa ef al., 1990; DeFily and Chilian, 1993; Heurteaux ef al., 1995; Peralta ef al., 1996; DeFily and Chilian, 1993; Heurteaux et al., 1995; Peralta etal., 1996). Moreover, the concept has also been extended to preconditioning triggered by non-ischemic stresses, such as reactive oxygen species, hypoxia, stretch, and various chemicals (Heurteaux et al., 1995; Pang etal., 1995; Verdouw ef al., 1996; de Zeeuw ef al., 1999). Preconditioning appears to protect tissue against injury under many conditions. Pang ef al., (1995) reported that acute ischemic preconditioning protects against skeletal muscle infarction in the pig heart. Defily and Chilian (1993) found that preconditioning protects coronary arteriolar endothelium from sustained ischemic insults. Transient ischemia in distal organs (such as the kidney) has been shown to precondition the myocardium for ischemia (de Zeeuw ef al., 1999). The outcome of of preconditioning is reduced cell death, which results in improved function at the tissue and at organ levels (Han etal., 2001). The preconditioning effects described above are produced namely at the organ level and could involve multiple tissue and other factors, including hormonal readjustment, 37 homestatic adaptation, nutrient demands, amongst others. Preconditioning was later observed in cells maintained in culture. For example, Han e ra / . , (2001) found that hydrogen peroxide preconditions rat fibroblast L-cells for subsequent hydrogen peroxide exposures. This finding was extended to two other cell l ines, the human embryonic kidney cell line (HEK293 cells) and a rat ventricular cell line (H9c2) (Han et al., 2001). Hypoxia also preconditions the rat pheochromocytoma cell line (PC12 cells) to serum deprivation and various pharmacological agents that induce apoptosis (Alvarez-Tejado e ra / . , 2001). Bishop et al. (1999) reported that motor neurons develop adaptive resistance to nitric oxide treatment. The group found that treatment of sublethal doses of nitric oxide (NO) to the motor neuron-derived cell line (NSC34 cells) upregulated adaptive mechanisms that counteract cytotoxic levels of N O (Bishop et al., 1999). 3.2.2 Protective mechanisms of C G C G has been rapidly induced after exposure of various cell types of a wide range of organisms to heat shock (Murakami-Murofushi et al., 1997; Kunimoto et al., 2000, 2002, 2003). Particularly, C G has been suggested to be a mediator in the early stage stress response by upregulating heat shock protein expression (Kunimoto ef al., 2003). In the present study, brief C G treatment was found to confer preconditioning against cell death induced by serum deprivation. Moreover, serum deprivation can induce N S C 3 4 cel ls to upregulate endogenous C G synthesis (Figure 10), which supports a physiological role of C G in mediating intercellular stress signaling. Continuous C G exposure cannot prevent cell death mediated by serum deprivation. Prolonged or continuous treatment of C G with serum deprivation resulted in a greater reduction in cell viability than serum deprivation alone (Figure 5). This indicates that the role of C G is dependent on the duration of exposure, where brief exposures confer cytoprotection and prolonged exposures induce cytotoxicity. 3.2.3 Phosphorylat ion changes in C G preconditioning The Kinetworks™ K P S S - 1 . 3 multi-immunoblot phospho-site screen proved to be a relatively inexpensive, efficient, and more sensitive approach to study broad spectrum phosphorylation changes during C G preconditioning. The Kinetworks™ screens typically vary by 5-20% depending on the signal intensity of each immunoreactive protein (Pelech ef al., 2004). S ince the biggest change detected by the phospho-site screen was A k t 8 6 ' " 4 7 3 phosphorylation, the activities of Akt and several related protein kinases were investigated in a more in-depth time course study. The Kinetworks™ K P S S - 1 . 3 phospho-site screen has 38 detected many other changes in the activities of other protein kinases (Figures 6 and 7). In this study, only the pathways of two survival kinases Akt and Erk1/2 have been studied in greater detail. However, the possibility of other kinases such as the double-stranded dependent protein kinase (PKR) and the mitogen- and stress-induced protein kinase 1 (MSK1) involved in C G preconditioning cannot be excluded. Further work will be required to study the roles of these kinases in C G preconditioning. One of the collateral results of the Kinetworks™ KPSS-1.3 phospho-site screen is the detection of unknown cross-reative proteins. These unidentified immunoreactive proteins that change in expression or phosphorylation in response to C G preconditioning can be purified or at least tracked with the cross-reactive antibodies that detected them in the first place (Pelech ef al., 2004). This permits the identification of these proteins by mass spectrometry or by direct sequencing by standard Edman degradation methodology (Pelech et al., 2004). 3.2.4 Akt/Src/GSK3 signaling The increase in A k i ^ " 4 7 3 phosphorylation was the greatest of the observed changes as detected by the phospho-site screen during C G preconditioning. However, this effect was not reproducible in the time course evaluation of Akt S e r " 4 7 3 phosphorylation, where C G preconditioning did not significantly upregulate Ak t S e r ' 4 7 3 phosphorylation at the 4 h time point. However, Ak t S e r _ 4 7 3 phosphorylation was significantly increased at earlier time points (0 h and 2 h of serum deprivation). This apparent inconsistency is likely due to biological variability, since Akt S e r~ 4 7 3 phosphorylation at the 4 h time point did show an increasing trend, albeit statistically insignificant (Figure 8). Akt signaling is believed to have a protective role in response to neuronal injury by phosphorylating and inhibiting downstream regulators of apoptosis (Kandel and Hay, 1999a). Furthermore, increased Akt activity parallels various preconditioning conditions. For example, Akt is activated during oxidative (Han et al., 2001) and hypoxia (Alvarez-Tejado ef al., 2001) preconditioning against various various stress stimuli. Inhibition of Akt by blocking its upstream activator, PI3K prevented this cytoprotective effect. Inhibition of PI3K with LY294002, which leads to inhibition of Akt abolished C G preconditioning against serum deprivation, indicating that CG-induced Akt activation plays a role cytoprotection against the serum deprivation induced stress (Figure 9). Akt activity was increased without any changes in the phosphorylation level of PDK1 as detected by the Kinetworks™ KPSS-13. phospho-site screen. However, an increase g r c Tyr -4 i8 phosphorylation was detected. The non-receptor protein-tyrosine kinase, Src is 39 activated upon phosphorylation of the Tyr-418 site (Thomas and Brugge, 1997). Src has been reported to activate Akt via tyrosine phosphorylation independent of the PI3K/PDK1/Akt pathway (Chen et al., 2001; Conus ef al., 2002). S rc is a proto-oncogene originally believed to only promote cell survival (Stehelin et al . , 1976). However, S rc functions to regulate voltage-gated ion channels such as potassium (Fadool ef al., 1997), calcium (Cataldi ef al., 1996), y-aminobutyric acid (GABA) (Moss ef al., 1995; W a n ef al., 1997), and nicotinic acetylcholine receptors (Wang ef al., 2004) in differentiated neurons. The activation of S rc during C G preconditioning may imply that S rc induced activation of Akt may be involved in cytoprotection, but tyrosine phosphorylation of Akt was not detected. The Src phosphorylation was not reproduced in a followup time course analysis by Western blotting of phosphorylation changes of S r c 1 7 ' " 4 1 8 during C G preconditioning. The discrepancy between the Kinetworks™ K P S S - 1 . 3 phospho-site screen and the classical Western blot analysis results is likely due to the different dilutions of primary antibody used for S r c T y r _ 4 1 8 detection, variability between different lots of primary antibody used for each experiment, and possibly cell preparation variation. A lso , inhibition of the PI3K/Akt pathway with LY294002 abol ished C G preconditioning, which indicates that the PI3K/Akt is involved in this form of cytoprotection. S ince PI3K and Src are both involved in cell survival and proliferation, it is tempting to speculate that the activities of these kinases converged to enhance Akt activation, which contributes to C G preconditioning against serum deprivation. Glycogen synthase kinase (GSK) 3 is activity has been associated with cytoskeletal disruption and regulating commitment to apoptosis during neuronal injury (Chen ef al., 2001; Hu ef al., 2003; Endo ef al., 2006). There are two isoforms of G S K 3 : G S K 3 a and G S K 3 p . G S K 3 activity is regulated by serine (inhibition) and tyrosine (activation) phosphorylations (Grimes and Jope, 2001). Akt phosphorylates at the serine inhibition site (Datta ef al., 1999; Gr imes and Jope, 2001), whereas Mek phosphorylates at a tyrosine activation site (Takahashi -Yanaga ef al., 2004). There is an increase in G S K 3 p S e r ~ 9 phosphorylation during C G preconditioning as detected by the phospho-site screen. The increased in G S K 3 p S e r " 9 phosphorylation parallels A k t S e r " 4 7 3 phosphorylation. Therefore, it is possible that during C G preconditioning, upregulation of Akt suppresses the activity of G S K 3 and prevents cell death. In contrast, tyrosine phosphorylation of G S K 3 a T y r 2 7 9 / p T y r " 2 1 6 and Mek phosphorylation were decreased as determined by the phospho-site screen. S ince Mek1 was found to phosphorylate G S K 3 al p on tyrosine residues (Takahashi -Yanaga ef al., 2004), it was not surprising to see the activities of these two kinases coincidentally decreased. However, the 40 role of Mek1 phosphorylation of GSK3 a/p remains unknown (Takahashi-Yanaga et al., 2004). This observation supports the previous finding that GSK3 al p activities are suppressed during C G preconditioning against serum deprivation. In a time course analysis of C G preconditioning against serum deprivation G S K 3 a T y r " 2 7 9 / p T y r " 2 1 6 phosphorylation level was unchanged. This finding further supports that GSK3a/ p is not activated during C G preconditioning, since tyrosine phosphorylations are required for GSK3a / p activity (Grimes and Jope, 2001). 3.2.5 Erk1/2 signaling Erk1/2 signaling is classically known to promote cell survival and is activated by dual phosphorylation on a threonine and tyrosine residue (Pelech ef a/., 1993). During C G precondioning, the Kinetworks™ KPSS-1.3 phospho-site screen detected no changes in E r k 1 T h r " 2 0 2 + T y r 2 0 4 phosphorylation, but only a modest decrease in phosphorylation of E r k 2 T h r i 8 5 + T y r i 8 7 j h j s , a t t e r fj n cjj ng w a s n o f reproduced in a time course analysis on C G preconditioning and phosphorylation level of Erk1/2. E r k 1 T h r 2 0 2 + T y r 2 0 4 did not show any phosphorylation changes during C G preconditioning (Figure 8C). However, serum deprivation alone appears to induce phosphorylation of E r k 2 T h M 8 5 + T y r ' 1 8 7 in a time-dependent manner. C G pretreatment followed by serum deprivation induced more Erk2 phosphorylation, particularly at the 4 h time point (Figure 8D). This finding contradicts the Kinetworks™ KPSS-1.3 phospho-site screen result, which was a decrease in Erk2 T h r " 1 8 5 + T y r ~ 1 8 7 phosphorylation at this time point. This inconsistency may be caused by the different lots of antibodies used for detecting Erk1/2 phosphorylation, the different antibody dilutions used for each analysis, and cell preparation variation. In addition, the Kinetworks™ phospho-site 1.3 screen and the Western blot time course analysis used different methods to quantify band intensities, which might also account for the discrepancies (Leroy et al., 2007). Inhibition of Mek1/2 with U0126, which leads to suppressed Erk1/2 phosphorylation, did not interfere with C G preconditioning against serum deprivation (Figure 9). Co-treatment with U0126 and LY294002 was abolished C G preconditioning for serum deprivation. However, the loss of cytoprotection during C G preconditioning is primarily due to inhibition of the PI3K/Akt pathway, since LY294002 treatment alone sufficiently produced the same effect. The observed increase Erk2 phosphorylation in the time course experiment is likely caused by serum deprivation and is not involved in C G preconditioning. 41 3.2.6 Adducin and the cytoskeleton Adducins are a family of heteromeric membrane-asociated cytoskeletal proteins composed of a , p, and y subunits. Previous studies have demonstrated that adducin colocal izes with another cytoskeletal protein spectrin (Kaiser ef al., 1989), at sites of epithelial contact junctions and is involved in the assembly of the spectrin-actin network in erythrocytes (Derick ef al., 1992). In neurons, adducin is one of major cytoskeletal protein found in dendritic spines of neuron and axonal growth cones (Matsuoka ef al., 2000). Functions of adducin include regulating cell motility and cell shape (Kuhlman ef al., 1996; Matsuoka et al., 1998). Adducin contains a myristoylated alanine-rich C kinase substrate (MARCKS)- re la ted domain that is targeted by protein kinase C , which are phorbol 12-myristate 13-acetate (PMA)-activated protein kinases (Matsuoka ef al., 2000). Upon phosphorylation of a conserved serine residue in this domain, adducin loses the ability to promote actin capping and recruit spectrin to actin filaments in dendritic spines of neurons, which may play an essential role in the dynamics of synaptoplasticify (Matsuoka ef al., 1998). Serum deprivation alone without C G preconditioning appears to suppress adducin (a,y) phosphorylation over time. S ince adducin phoshorylation has been found to be involved in cytoskeletal rearrangements, it is not expected that the N S C 3 4 cells will rearrange their cell morphology during serum deprivation stress. Rather, it is logical that N S C 3 4 cells counteract the stress by laying down stress fiber, which correlates with a decrease in adducin phosphorylation (Matsuoka et al., 2000; Larsson, 2006). C G preconditioning did not have any significant effect on adducin a phosphorylation at the Ser-726 site during C G preconditioning (Figure 8A). On the contrary, C G preconditioning tends to increase adducin y phosphorylation at the Ser-693 site during t imes of serum deprivation (Figure 8B). It is unclear why the phosphorylation state of these two isoforms would differ, given their identical function in regulating cell motility. The y subunit of adducin has been suggested to be multi-functional and may be critical to preserve the integrity of cells (Yang ef al., 2004). It is tempting to speculate that the a and y subunits are differentially regulated by extracellular signals, where the latter is specifically affected by C G treatment. Furthermore, the upregulation of adducin y phosphorylation may be a toxic side-effect of C G and pretreatment with C G prior to serum deprivation may have already sensit ized the N S C 3 4 cells. 42 Increased phosphorylation state of adducin has been previously linked to the pathogenesis of ALS. Using various Kinetworks™ phospho-site screen, Hu and colleagues (Hu et al., 2003) reported an increased phosphorylation of adducin (a,y) in the spinal cord homogenates of patients who died from ALS. In another study, Shan et a/.( 2005) showed increased phopho-adducin immunoreactivity in a murine model of familial ALS. The increase in phosphorylation of adducin was correlated with an increase in PKC activity and cell death. Therefore, it is conceivable that PKC-mediated adducin phosphorylation plays an important role in neuronal death in ALS by affecting the basic structural scaffolding of neuronal dendritic spines (Hu ef al., 2003). Although the role of adducin phosphorylation in C G preconditioning is unknown, an increased in adducin y phosphorylation was not accompanied by increased PKC activity as determined by the Kinetworks™ phospho-site 1.3 screen. Other possible that kinases have been reported to phosphorylate adducin in the MARCKS domain, include PKA (Matsuoka etal., 1996), camodulin-Ca 2 + kinase (Matsuoka ef al., 1996; Larsson, 2006), and Rho kinase (Yagita ef al., 2007). These kinases were not tracked with the Kinetworks™ KPSS-1.3 phospho-site screen. 3.3 Conclusion C G has both toxic and protective sides. Brief exposure of C G can mediate a protective effect, whereas prolong exposure of C G contributes to cell death. The multi-immunoblot identified Akt was one of the protein kinases activated during C G preconditioning. Inhibition of the PI3K/Akt pathway abolished preconditioning against serum deprivation. GSK3 a / p, downstream of Akt is not activated during C G preconditioning. The Mek1/2/Erk1/2 pathway is not involved in C G toxicity, since inhibition of this pathway with U0126 did not prevent C G preconditioning. The cytoskeletal proteins adducin a / y were differentially regulated, implying that the neuronal cytoskeleton may undergo rearrangements during C G preconditioning. 43 Figure 2. Time and concentration-dependent effect of CG on NSC34 cell viability. (A) NSC34 cells were treated with increasing concentrations of C G for up to 7 days and cell viability was monitored using the MTT reduction assay. A two-way ANOVA (Time: F 3 2 1 7 =33.3 p<0.001 and Dose: F 4 | 2 1 7=52.3 p<0.001) followed by Tukey's honest post hoc (*p<0.05) indicated significant decrease in cell viability with increasing concentrations and duration of C G treatment. (B) Cholesterol without the glucose moiety did not show any significant effect on cell viability. Cell viability is expressed as a percentage of control cells without any treatment (defined as 100%) and represented as mean±S.E.M (n=12). 44 C G __- -*- - •+-No se rum S e r u m deprivation deprivation for 24h Figure 3. Treatment of CG prevents cell death caused by serum deprivation. NSC34 cells deprived of serum for 24 h resulted in 25% cell death (unpaired Student's t test *** p<0.001). NSC34 cells pretreated with 50 uM of C G for 1 h significantly prevented cell death induced by serum deprivation. Cell death was assessed by the trypan blue dye exclusion test and represented as the percentage of live cells calculated by (100% - number of dead cells) I total number of cells counted. Data are represented as the mean±S.E.M (n=12). 45 100-, 0 2.5 25 50 100 250 C G concentration (JJM) Figure 4. NSC34 cells pretreated with CG are resistant to cell death induced by serum deprivation. NSC34 cells were pretreated with increasing concentrations of C G for 1 h followed by serum deprivation for 24 h. The MTT reduction assay was used to monitor cell viability followed by C G preconditioning against cell death induced by serum withdrawal. One way ANOVA followed by Tukey's honest post hoc showed significant increase in cell survival in NSC34 cells pretreated with 25-100 uM of C G prior to serum deprivation (*p<0.05). Cell viability is represented as a percentage of control cells (untreated and not serum deprived). Data are represented as the mean±S.E.M (n=12). 46 100 -, CG + _; f _ __• +_ 24 48 72 Duration of serum deprivation (h) Figure 5. Prolonged CG treatment exacerbates cell death induced by serum deprivation. Simultaneous C G treatment with serum deprivation for the indicated times resulted in increased cell death as monitored by the MTT reduction assay (unpaired Student's t test * p<0.05). Cell viability is represented as a percentage of control cells (untreated and not serum deprived). Data are represented as the mean±S.E.M (n=12). 47 net mm * • OS'S MKK« - * -B2f & B Mafcl (3oJSK3o Ert; » * 5 Untreated serum deprived NSC34 cells C G treated serum deprived NSC34 cells Figure 6. Kinetworks™ KPSS-1.3 phospho-site multi-immunoblot analysis of C G treated and serum deprived NSC34 cell lysates. NSC34 cells were pretreated with 100 uM of C G for 1 h and serum deprived for 4 h. Control cells were serum deprived for 4 h only. Cell lysates were subjected to Kinetworks™ phospho-site 1.3 broad range multi-immunoblot analysis to screen for phosphorylation changes during C G preconditioning. (A) A multi-immunoblot of serum deprived NSC34 cells with the tracked phospho-proteins at the indicated phosphorylation sites. The arrows marked the locations of the tracked phospho-proteins bands. (B) A multi-immunoblot of NSC34 cells pretreated with 100 uM and serum deprived for 4 h. Circled bands indicate those target phospho-sites where the observed percent change from control (%CFC) was 25% or greater. Protein bands that are labeled A? to E? show significant changes in band intensity but are currently not identified. The % C F C for these proteins is presented under the corresponding letter. 48 500 1000 Normalized Counts Per Minute 1500 2000 2500 3000 3500 4000 Adducin alpha [S726] Adducin gamma [S693) Double-stranded RNA-dependent protein-serine kinase [T451] Extracellular regulated protein-serine kinase 1 [T202+Y204] Extracellular regulated protein-serine kinase2rj185+Y187] Jun N-terminus protein-serine kinase (46)[T183/Y185] Jun N-terminus protein-serine kinase (41)[T183/Y185] Glycogen synthase-serine kinase 3 alpha [Y279] Glycogen synthase-serine kinase 3 beta [Y216] Protein-serine kinase B alpha (Akt1) [S473] Protein-serine kinase B alpha (Akt1) [T308] Rafl proto-oncogene-encoded protein-serine kinase [S259] Raf1 proto-oncogene-encoded protein-serine kinase [S259] Src proto-oncogene-encoded protein-tyrosine kinase [Y418] Control Control Figure 7. Protein phosphorylation changes in NSC34 cells during CG preconditioning against serum deprivation. The intensities (counts per minute) of the immunoblot enhanced chemiluminence signals for target phosphoproteins were quantified for serum deprived NSC34 cells pretreated with 100 uM C G (black bars) and without pretreatment (white bars). 49 A 0.20 « 0 15 s 2 0J0-o 1 0.05-ooo Time (h> CG treated Control) •-124K •12* i • • • 0 2 4 8 lime of serum deprivation (h) B = 0 3 0 •I 0 25-1 | 0 20 ? 0 15-| S3 •s 0.10-1 -o 1 o.os 000-Time (hi C G treated Controf I 1 Control H i Treated AAA Time of serum deprivation (h) 1.00 c 1| 0.75 jjfj 0.50' as & 0.25 0.00 Time at serum deprivation (h) Time (ti) o CG treateoflHl Contrail 0 2 4 8 T ime uf berui i i deprivation (h) Time of swrurn deprivation (h) me of serum deprivation (h) Figure 8. Time course analysis of phosphorylation changes during serum deprivation during CG preconditioning. NSC34 cells were either pretreated with 100 pM of C G for 1 h (black bars) or replenished with complete media without C G (white bars). The cells were then serum deprived for 0-8 h. The phosphorylation of ratio calculated by the intensity of the phospho-protein was divided by the intensity of actin, a loading control. A representative Western blot is presented on top of each graph. Two way ANOVA indicated significant changes in Adducin y (Time F 3 j 6=3.70 p<0.05; Group F 1 1 6=30.8 p<0.001), Erk2 (Time F 3 1 6=9.41 p<0.001), and Akt (Time F 3 1 6=15.7 p<0.001; Group F 1 1 6=4.63 p<0.05). For follow-up analysis of the significant changes indicated above, an unpaired Student's t-test was used to indicate changes between C G pretreated and untreated groups (* p<0.05; ** p<0.01; ***p<0.001). Data are represented as mean±S.E.M (n=3). 50 Figure 9. Effect of Erk1/2 and Akt inhibition on CG preconditioning against serum deprivation in NSC34 cells. NSC34 cells were pretreated with either 10 uM of U0126 (Mek1/2 inhibitor), 30 uM of LY294002 (PI3K inhibitor), a combination of U0126 and LY294002, or 10 uM of SP600125 (JNK1/2 inhibitor) for 1 h. These cells were then treated with 100 uM of C G in the presence or absence of the indicated inhibitors for 1 h followed by 2 washes and serum deprivation for 24 h. Cell viability was monitored using the MTT reduction assay and is expressed as a percentage of control (untreated cells not serum deprived). Inhibition of Mek/Erk pathway alone did not interfere with C G preconditioning. Inhibition with LY294002 to suppress the PI3K/Akt pathway prevented the cytoprotective effect of C G preconditioning against serum deprivation (unpaired Student's t-test *p<0.05). Simultaneously inhibiting the Mek1/2/Erk1/2 pathway and the PI3K/Akt pathway also ameliorated the preconditioning effect of C G (unpaired Student's t-test **p<0.001). Inhibition of JNK1/2 with SP600125 did not interfere with C G preconditioning. Data are represented as mean±S.E.M (n=12). 51 CG Standard Duration of serum deprivation (h) Figure 10. Serum withdrawal induces steryl glycoside synthesis in NSC34 cells. NSC34 cells were deprived of serum for 0, 2, 6, or 18 h and were subjected to lipid extraction and TLC analysis for steryl glycoside content. (A) C G standards (10 pM and 100 pM) were resolved in parallel with serum deprived samples. The serum deprived samples have bands with the same Rf value as the C G standards and is suggestive that steryl glucosides are endogenously synthesized. The arrow indicates the steryl glycoside band in serum deprived NSC34 cell lysates. (B) Quantification of the band intensity showed a two-fold increase in steryl glucoside content after serum deprivation for 18 (One way ANOVA F 3 8=4.23 p<0.05 followed by Tukey's honest post* hoc * p<0.05). Data are represented as the mean±S.E.M (n=3). 52 CHAPTER 4. Cholestervl glucoside activates the Akt and MAPK pathways Hypothesis: Cholesteryl glucoside (CG) activates both survival pathways and related stress-activated pathways for cell death in a t ime-dependent manner. Specific aims: 1. To determine the mechanist ic actions of C G on N S C 3 4 cells maintained in complete media, specifically examining the activities of Akt, Erk1/2, J N K and p38 M A P K . 2. To determine the role of these kinases in CG-media ted cell death with the use of pharmacological inhibitors. 4.1 Results 4.1.1 CG- induced activation of Akt in N S C 3 4 cells In the previous study, C G preconditioning resulted in upregulated Akt phosphorylation and presumably activation which paralleled the cytoprotection against serum deprivation-induced cell death. Therefore, it is likely that one of the effects of C G is to cause stimulation of Akt activity. To determine the effect of C G on Akt activation, N S C 3 4 cells were serum-deprived for 12 h and stimulated with 10% fetal bovine serum. Akt activity as indicated by its phosphorylation was assessed by Western blotting with anti-Akt and anti-phospho-Ak t S e r " 4 7 3 antibodies. Akt phosphorylation was expressed as the ratio of phospho-A k t s e r - 4 7 3 o v e r t o t g | A k t w h e n serum-deprived N S C 3 4 cells were stimulated with serum, Akt was phosphorylated in a t ime-dependent manner (Figure 11, white bars). In the presence of 250 uM of C G , serum-stimulation of Akt phosphorylation was ablated (unpaired Student's t-test p<0.05) (Figure 11, black bars). C G induced phosphorylation and presumably activation of Akt was observed in the time course analysis of C G preconditioning against serum deprivation (Figure 8E). In this case , C G treatment was in the presence of serum for preconditioning. It is possible that treatment of C G in the presence/absence of serum may have different effects. To assess the effect of C G on Akt phosphorylation in N S C 3 4 cells supplemented with serum, these cells were grown in complete media and treated with C G . Akt phosphorylation was a s s e s s e d as previously descr ibed. Stimulation of N S C 3 4 cells with 250 uM C G produced a marked increase in A k t S e r " 4 7 3 phosphorylation (Figure 12A). CG- induced phosphorylation of Akt was t ime-dependent (One way A N O V A F 5 , 29=5.18 p<0.01; Tukey 's honest post hoc test 53 p<0.05) and maximal increase was achieved after 30 min of exposure. C G exposure induced approximately 2-fold increase in Akt phosphorylation which was sustained for up to 480 min of C G exposure then insignificantly dropped below basal level. Furthermore, C G -mediated increases in A k t S e r " 4 7 3 phosphorylation were concentration-dependent (Figure 12C). N S C 3 4 cells were exposed to increasing concentrations of C G (0-250 uM) for 5 min. Maximal Akt phosphorylation was observed for the 250 uM C G treatment group (One way A N O V A F 5 i 3 o=2.63 p<0.05; Tukey's honest post hoc p<0.05). Treatment with 0.3% D M S O used as a control did not have any effect of Akt phosphorylation at this site. PDK1 is a direct upstream activator of Akt, which catalyzes Akt phosphorylation at Thr-380 (Kandel and Hay, 1999a). Therefore, increased Akt activation may be caused by increased PDK1 activity. To assess the effect of C G exposure on PDK1 activity, N S C 3 4 cells were treated with 250 uM of C G for 0 to 32 min. PDK1 activity was determined by Western blot analysis using an an t i - phospho -PDK1 S e r " 2 4 1 antibody and expressed as the ratio of p h o s p h o - P D K 1 S e r " 2 4 1 over actin. C G treatment did not affect PDK1 phosphorylation during Akt activation. 4.1.2 CG- induced suppression of Erk1 but not Erk2 in N S C 3 4 cells Erk1 and Erk2 activities as reflected by phosphorylation at their activation sites were analyzed in parallel with Akt phosphorylation. The Erk1/2 signaling pathway is believed to promote cell survival but has been found to be aberrantly regulated in various neuropathological conditions (Nizzari ef al., 2007). Furthermore, two phases of Erk1/2 activation during oxidative stress have been proposed, where an early activation is protective and a late activation mediates cell death. Erk1/2 activity was monitored by Western blotting using p h o s p h o - E r k 1 T h r - 2 0 2 + T y r - 2 0 4 / E r k 2 T h r - 1 8 5 + T y M 8 7 antibody. To determine the effect of C G on serum stimulation of Erk1/2 phosphorylation, N S C 3 4 cells were serum-starved for 12 h and stimulated with serum in the presence or absence of 250 uM of C G . Serum stimulation of Erk1/2 phosphorylation in serum-starved N S C 3 4 cell was maximal from 2 to 4 min and then returned to basal level (Figure 14). The presence of C G ablated serum stimulation of Erk1/2 phosphorylation. Quantification of two independent experiments showed a two-fold inhibition of Erk1/2 activity as compared to serum stimulation alone (unpaired Student's t-test p<0.05). The effect of C G treatment on Erk1/2 activity in N S C 3 4 cells maintained in complete medium was also determined. Erk1 and Erk2 activity appears to be differentially regulated by C G treatment. Erk1 activity (Figure 15A) was rapidly suppressed by 250 uM C G 54 exposure from 2 to 32 min (One way ANOVA F5,30=10.2 p<0.001; Tukey's honest post hoc p<0.05). The phosphorylation of Erk1 returned to basal level after 1 h exposure. There were no significant changes in Erk1 phosphorylation with longer C G exposures up to 1440 min (One way ANOVA F 8 1 8=4.06 p>0.05). The phosphorylation of Erk2 (Figure 15B.D) was not significantly affected by 250 pM C G treatment over time (One way ANOVA F8,18=1-18 p>0.05). C G inhibition of Erk1 phosphorylation (Figure 15E) was also concentration-dependent (One way ANOVA F5,30=3.77 p<0.001; Tukey's honest post hoc p<0.05), whereas Erk2 phosphorylation (Figure 15F) was not affected by increasing concentrations of C G (One way ANOVA F3,30=0.49 p>0.05). Treatment with 0.3% DMSO which served as a control did not have any notable effect on Erk1 phosphorylation. Mek1 and Mek2 (also known as MAPK kinases) are direct upstream activators of Erk1 and Erk-2 (Ahn et al., 1992; Nishida and Gotoh, 1993). Therefore, reduced Erk1 phosphorylation may be caused by decreased Mek1/2 activity. The activity of Mek1/2 was indirectly assessed by its phosphorylation using an anti-phospho-Mek1 S e r" 2 1 7 + S e r" 2 2 1 antibody and expressed as the ratio of phospho-Mek1/2 over actin. NSC34 cells treated with 250 pM of C G have a decreased Mek1/2 phosphorylation level as compared to control. Inhibition of Mek1/2 phosphorylation was observed as early as 2 min of C G exposure. Raf1 is a direct upstream kinase, which phosphorylates and activates Mek1/2 (Wellbrock et al., 2004). Therefore, the observed inhibition of Mek1/2 and Erk1/2 phosphorylation may be a consequence of a decreased Raf1 activity. To indirectly assess Raf1 activity, an anti-phospho-Raf1 S e r" 2 5 9 antibody was used. Ser-259 is an inhibitory site of Raf1 and upon phosphorylation induces a conformation change to Raf1, rendering it inactive (Frebel and Wiese, 2006). Furthermore, it has been previously suggested that there is a crosstalk between the PI3K/Akt and the Raf1/Mek1/2/Erk1/2 pathways. Moreover, Akt has been proposed to phosphorylate Ra f1 S e r " 2 5 9 and inhibit its activity during growth factor stimulation (Moelling ef al., 2002). A decrease in Raf1 S e r " 2 5 9 phosphorylation may be therefore be indicative of an enhanced Raf1 activity, whereas an increase in Raf1 S e r " 2 5 9 phosphorylation should be reflected by decreased Raf1 activity. However, NSC34 cells treated with 250 pM C G did not show any significant changes in Raf1 S e r " 2 5 9 phosphorylation (Figure 16). 4.1.3 Effects of kinase inhibitors on CG-induced activation of Akt To further study the effect of CG-induced Akt activation, NSC34 cells were pretreated with the PI3K inhibitor LY294002 (30 pM) and the Mek1/2 inhibitor U0126 (10 pM) for 1 h. The inhibitors were then removed and the cells were incubated in complete 55 medium in the presence or absence of 250 uM of C G for 10 min. Akt phosphorylation was determined as previously described. As demonstrated before, C G treatment alone induced Akt phosphorylation. Inhibition of PI3K/AW pathway followed by stimulation with 250 uM C G for 10 min resulted in a three-fold increase in Akt phosphorylation (unpaired Student's t-test p<0.01; LY294002 +CG vs LY294002 only) (Figure 17). U0126 treatment resulted in a compensatory increase in Akt phosphorylation. C G stimulation following U0126 treatment resulted in a slight increase in Akt phosphorylation compared to U0126 treatment alone, albeit without statistical significance. C G stimulation of Akt phosphorylation following LY294002 treatment is suggestive of an alternative mechanism for Akt activation independent of PI3K. A PDK2 enzyme that mediates Akt phosphorylation have been proposed (Datta ef al., 1999; Chan and Tsichlis, 2001). Recent work has indicated that the mammalian target of rapamycin (mTOR), a downstream kinase of Akt may mediate positive feedback activation of Akt (Jacinto ef al., 2006). To determine if mTOR mediates an alternative mechanism for C G activation, NSC34 cells were pretreated with the mTOR inhibitor rapamycin (200 nM) for 1 h and stimulated with 250 uM C G . C G stimulation following mTOR inhibition did not suppress Akt phosphorylation (Figure 18A). Furthermore, rapamycin treatment alone was not capable of suppressing Akt phosphorylation. Phosphorylation of Akt T h r ' 3 0 8 is also critical for Akt activation and is mainly mediated by PDK1 (Koul ef al., 2005). Similar to Ak t^ " 4 7 3 , C G treatment alone increased Akt T h r " 3 0 8 phosphorylation three-fold as compared to the control (unpaired Student's t-test p<0.05) (Figure 18B). C G stimulation following LY294002 treatment resulted in a 30-fold increased in Akt T h r " 3 0 8 phosphorylation as compared to LY294002 treatment alone (unpaired Student's t-test p<0.01). Rapamycin treatment did not cause any significant changes in Ak t ^ " 4 7 3 phosphorylation. However, C G stimulation following rapamycin treatment may have increased Akt T h r ' 3 0 8 phosphorylation, although this was not statistically significant. 4.1.4 Effect of pharmacological inhibitors on CG-suppression of Erk phosphorylation Erk1 and Erk2 were found to be differentially regulated by C G treatment. Furthermore, previous studies have indicated that Akt may suppress Erk1/2 activity. To further understand these effects, NSC34 cells were pretreated with the PI3K inhibitor LY294002 (30 uM) and the Mek1/2 inhibitor U0126 (10 uM) for 1 h. The inhibitors were then removed and the cells were incubated with complete media in the presence or absence of 250 uM C G for 10 min. Erk1/2 phosphorylation was determined by Western blotting analysis 56 with anti-phospho-Erk1/2 antibody and anti-Erk1/2 pan-specific antibodies and presented as the ratio phospho-Erk/ total Erk. Inhibition with U0126 completely abolished Erk1/2 phosphorylation and C G suppression of Erk phosphorylation could not be further observed (Figure 19). LY294002 treatment resulted in a compensatory increase in Erk1 phosphorylation and C G stimulation following LY294002 treatment resulted in selective inhibition of Erk1 phosphorylation (unpaired Student's t-test p<0.05) (Figure 19A). Erk2 phosphorylation was not affected by LY294002 treatment. Erk2 phosphorylation levels insignificantly decreased with C G treatment in the presence of absence of U0126 treatment. Taken together, these findings indicate that C G specifically regulated inhibition of Erk1 and not Erk2. 4.1.5 Late activation of stress-activated protein kinases The activities of stress activated protein kinases (SAPKs, also known as c-Jun N-terminal protein kinases (JNK)) and the p38 MAPK were found be to upregulated under conditions of cytotoxic stress, and can commit cells to apopotosis (Aroor and Shukla, 2004). Results from other laboratories as well as ours have found that C G is a lipid molecule that has been found to confer temporary cytoprotection during stressful conditions (Kunimoto ef al., 2002), but mediates cell death during prolonged exposures (Khabazian ef al., 2002). Therefore, it is conceivable that JNK and p38 MAPK activities are upregulated during C G mediate toxicity and lead to cell death. To study the time course of CG-induced JNK and p38 MAPK activition, NSC34 cells were treated with 250 uM of C G for over extended periods. The activities of JNK and p38 MAPK were assessed indirectly by their phosphorylations at stimulatory sites as determined by Western blotting using an anti-phospho-JNK and anti-phospho-p38 MAPK antibodies. In contrast to the rapid response from Akt and Erk1/2, both JNK and p38 MAPK showed a delayed stimulation (Figure 20A). JNK phosphorylation was first observed after 480 min of C G treatment and persisted until 1440 min. The phospho-JNK antibody used in this study recognized two isoforms, JNK1 (46 KDa) and JNK2 (41 KDa). The phosphorylation of p38 MAPK was not evident until after several h of C G treatment. The blots were stripped and probed with an anti-actin antibody to show that all lanes contained proteins and were equally loaded. The CG-induced stimulations of JNK and p38 MAPK phosphorylation were also concentration-dependent (Figure 20B). NSC34 cells treated with increasing concentrations of C G for 18 h were assessed for the JNK and p38 MAPK phosphorylation. Twenty-five to 100 uM of C G induced a slight increase in JNK phosphorylation, whereas 250 pM exposure substantially 57 upregulated the phosphorylation of J N K . The phosphorylation of p38 M A P K was concentration-dependent within a concentration range of 5-250 p M . Treatment of 0 .3% D M S O to N S C 3 4 cell served as a control to show that D M S O at the highest concentration used had no effect on J N K and p38 M A P K activation. 4.1.6 Effect of kinases inhibitors on CG- induced reduction in cell viability. Having demonstrated that C G differentially modulates the PI3K/Akt pathway and Raf/Mek/Erk pathways in N S C 3 4 cells, we next explored the role of these kinase pathways in CG- induced cell death using pharmacological inhibitors. A s shown in Figure 21 , N S C 3 4 cells exposed to 250 pM C G for 48 h induced over 2 5 % reduction in cell viability (unpaired Student's t-test p<0.05). To investigate the role of Akt and Erk1/2 in CG-med ia ted cell death, N S C 3 4 cells were pre-treated for 1 h with LY294002 (30 p M , PI3K inhibitor) and U0126 (10 pM, Mek1/2 inhibitor), followed by 250 p M C G with or without these inhibitors for another 20 h. The inhibitors were then removed and complete media with or without 250 pM of C G were added for a total of 48 h. Cel l viability was then evaluated by the MTT reduction assay. Although Erk1/2 has been found to mediate cell survival, Ruffels and col leagues have demonstrated that a delayed Erk1/2 activation mediates cell death of the human S H - S Y 5 Y neuroblastoma cell line stimulated with hydrogen peroxide (Ruffels et al., 2004). Inhibition of the Mek/Erk pathway did not protect or exacerbate CG-treated N S C 3 4 cel ls from cell death (Figure 21A). This indicates that the Mek1/2/Erk1/2 pathway was not involved in cell death mechanisms in CG-treated N S C 3 4 cel ls. Hypoxia and oxidative stress have been shown to increase upregulation of Akt phosphorylation which correlated with cytoprotection against stress stimuli (Ruffels ef al., 2004; Zhuang ef al., 2007). In this study, C G exposure also induced Akt phosphorylation and was found to protect against serum deprivation (Chapter 3). Therefore, it is possible that N S C 3 4 cel ls respond to C G exposures by activating Akt. Inhibition of the PI3K/Akt pathway with LY294002 in the presence of C G resulted in more cell death as compared to C G treatment alone (unpaired Student's t-test p<0.05) (Figure 21 A). Inhibitor treatment alone for 21 h (pretreatment 1 h + co-treatment with C G 20 h) did not have any significant effect on cell death as compared to control untreated cells. Unlike the rapid induction of Akt, C G mediated a slow and delayed activation of J N K and p38 M A P K . The latter two kinases are activated by stress stimuli and are believed to mediate pro-apoptotic signals (Aroor and Shukla, 2004). Therefore, it is likely that the late activation of these kinases is involved in mediating cell death. To investigate the role of p38 58 MAPK and JNK in CG-mediated cell death, NSC34 cells were pre-treated for 1 h with the p38 MAPK inhibitor SB203580 (10 uM) and and the JNK inhibitor SP600125 (10 uM), followed by 250 pM C G with or without these inhibitors for another 20 h. The inhibitors were then removed and complete media with or without 250 uM C G was added for a total of 48 h. Inhibition of p38 MAPK and JNK with these compounds had no significant effect on the loss of cell viability induced by 250 pM of C G treatment (Figure 21B). Inhibitor treatments alone did not have any significant effects on cell viability as compared to control untreated cells. 4.2 Discussion Previous studies demonstrated that a brief exposure of C G conferred cytoprotection against various stress stimuli (Kunimoto ef al., 2002). Our previous study confirmed that C G treatment for a brief duration protected NSC34 cells from serum deprivation. However, we also found that prolonged exposure of C G is toxic to NSC34 cells. No information was available on the signaling pathways triggered by C G in this motor neuron-derived cell line before the present study. This study demonstrated that C G treatment mediated concentration- and time-dependent activation of Akt (Figure 12). The molecular mechanisms underlying CG-induced Akt activation remain unclear, but may involve, in addition to PI3K, other upstream kinases that activate Akt. However, Akt activation during CG-treatment occurs independently of mTOR activity (Figure 18). Erk1, but not Erk2 phosphorylation is suppressed during C G treatment, but this does not contribute to the toxic effects of C G on NSC34 cells (Figure 15). JNK and p38 MAPK activities were delayed as compared to the Akt or Erk1 (Figure 20). Pharmacological inhibitors that target either JNK or p38 MAPK were not able to prevent CG-induced cell death, which indicated that both these stress-induced kinases were not critical in mediating cell death (Figure 21B). 4.2.1 CG-induced Akt activation C G treatment increased Akt phosphorylation in a concentration- and time-dependent manner, but did not affect total Akt expression levels. Akt is an important anti-apoptotic protein, whose signaling mechanisms suppressed cell death triggered by various cytotoxic stress stimuli (Alvarez-Tejado ef al., 2001; Han ef al., 2001; Ruffels ef al., 2004). Furthermore, the loss of Akt signaling has been correlated with neuronal death in various models of neurodegeneration. For example, loss of Akt signaling has been found to be the underlying cause of cell death in motor neurons deprived of trophic support (Newbern ef al., 2005). Furthermore, an early decrease in PI3K/Akt signaling was found in presymptomatic 59 stages of a mouse model of familial amyotrophic lateral sclerosis (ALS) (Warita ef al., 2001). It is unclear whether C G stimulates a membrane receptor which in turn stimulated Akt signaling or Akt activation was a cellular response used for countering the toxic effects of C G . The activation of Akt has been found to delay CG-media ted cell death (Figure 21) and is also an underlying mechanism for C G preconditioning against serum deprivation (Chapter 3). Inhibition of the PI3K/Akt pathway rendered N S C 3 4 cells more susceptible to C G exposure and completely abol ished C G preconditioning against serum deprivation. Akt activation ceased after several h of C G treatment (Figure 12B). This may be caused by an auto-inhibitory feedback mechanism that turns off CG-media ted Akt signaling, which lead to cell death. The presence of C G suppressed serum stimulation of Akt in N S C 3 4 cells deprived of serum for 12 h (Figure 11). This contradicted the finding that CG- induced Akt activation mediated cytoprotection. N S C 3 4 cells were found to endogenously synthesize C G during serum deprivation with a two-fold increase in C G content after 18 h of serum deprivation. The endogenously synthesized C G may have sensit ized the N S C 3 4 cel ls, which became unresponsive to the addition of exogenous C G and did not increase Akt phosphorylation. Furthermore, serum stimulation of Akt phosphorylation in serum-deprived N S C 3 4 cells does not necessari ly follow on the same mechanism as CG-media ted Akt activation. In any case , PI3K was found to be important in Akt activation and delayed the toxic effect of C G on N S C 3 4 cell viability. The PI3K inhibitor LY294002 (30 pM for 20 h) exacerbated the toxic effect of C G and caused a significant reduction in N S C 3 4 cell viability as compared to C G exposure alone (Figure 21A). At this concentration and duration of exposure, LY294002 alone did not have any effect on N S C 3 4 cell viability. However, when the cells were treated with LY294002 alone for 48 h, cell viability was significantly decreased. The upstream activator of Akt is PDK1 (Datta ef al., 1999) and its phosphorylation was not significantly upregulated by C G exposure during the times of Akt activation. However, a previous finding showed that unlike Akt, PDK1 was constitutively active, and its kinase activity within cells was not affected by extracellular stimuli that activated Akt (Alessi ef al., 1997a). Therefore, it is possible that C G stimulated PI3K activity and increased the levels of its phospholipid product, P I3 ,4 ,5P 3 , which is necessary for Akt relocation to the plasma membrane and where it becomes phosphorylated by the constitutively active PDK1 (Datta ef al., 1999). CG- induced upregulation of Akt activity was monitored at two phosphorylation sites, Thr-308 and Ser-473 (Figure 18). Both sites must be phosphorylated for maximum Akt activity, which was believed to be regulated in a PI3K-dependent manner (Kandel and Hay, 60 1999a). The finding that C G stimulated Akt phosphorylation after LY294002 treatment indicated that an alternated pathway may be involved in Akt activation. Many researchers believed that the mammalian target of rapamycin (mTOR) may be the same enzyme as P D K 2 , which phosphorylated Akt specifically at Ser-473 (Chan and Tsichl is, 2001). However, inhibition of m T O R with rapamycin did not lead to Akt inhibition in N S C 3 4 cel ls. Recent work demonstrated that the mTOR-r ictor complex is rapamycin insensitive. However, the mTOR-raptor complex is rapamycin sensitive and is believed to be the main stream P D K 2 kinase that mediates A k t S e r " 4 7 3 phosphorylation (Pearse et al., 2007; Proud, 2007). Therefore, in the N S C 3 4 cell system, inhibition with rapamycin did not sufficiently block A k t S e r " 4 7 3 phosphorylation because of the mTOR-r ictor complex. Whether or not C G -stimulated A k t S e r _ 4 7 3 phosphorylation depends on mTOR-raptor activity will require further studies. However, other k inases that mediate A k t S e H t 7 3 phosphorylation may include Src , cAMP-dependent protein kinase (PKA) (Datta et al., 1999), integrin linked kinase (ILK) (Chan and Tsichl is, 2001), and P K C (Duronio etal., 1998). S rc mainly phosphorylates Akt at several conserved tyrosine sites (Conus et al., 2002), which were not monitored in this experiment. The roles of these protein kinases on Akt activation were not determined and cannot be excluded as a mechanism for CG- induced Akt activation. It is possible that these protein k inases have an overlapping role with P I3K/PDK1 in activating Akt during C G exposure. 4.2.2 Erk suppression Serum stimulation of N S C 3 4 cells resulted in Erk1/2 activation. However, this effect was ablated when stimulating with serum in the presence of C G . Serum deprivation may have changed the physiology of N S C 3 4 cells maintained in a serum free condition. Therefore, these cells may have entered a quiescent state and when chal lenged with C G , they were unable to activate survival signaling pathways. Erk1/2 signaling is known to play a prominent role in cellular proliferation and survival (Widmann et al. , 1999). However, there is increasing evidence showing that Erk1/2 phosphorylation is also upregulated during excitotoxic or oxidative stress conditions (Guyton ef al., 1996; Ruffels ef al., 2004; Zhuang ef al., 2007). Activation of Erk1/2 during oxidative stress was described to occur in two phases: an early protective phase and a late pro-apoptotic phase (Luo and DeFranco, 2006). N S C 3 4 cells exposed to C G showed a selective suppression of Erk1 phosphorylation but did not alter Erk2 phosphorylation. Moreover, Erk1 inhibition lasted 30 min, after which Erk1 phosphorylation returned to its basal level (Figure 15A,B). These two isoforms were 61 traditionally believed to have functional redundancy (Guyton ef al., 1996). Therefore, the selectivity of CG- induced Erk1 suppression over Erk2 is unclear. A recent finding by Vantaggiato and col leagues (Vantaggiato ef al., 2006) suggested an alternate relationship between Erk1 and Erk2. The authors claimed that Erk2 is the primary survival k inase, whereas Erk1 affects the overall signaling output by antagonizing Erk2 activity (Vantaggiato ef al., 2006). In this case , Erk1 inhibition via an undefined mechanism provided an enhanced survival signaling mediated by Erk2 to counteract C G toxicity. Mek1/2 inhibition occurred in parallel with Erk1 inhibition (Figure 16B). Raf1 , an upstream activating kinase of Mek1/2, was not inhibited as determined by the level of phosphorylation at the inhibitory Ser-259 site (Figure 16C). This indicates that CG-med ia ted Erk1 inhibition occurred via inhibition of Mek activity. Inhibition of both Mek and Erk1 may have involved various mitogen-activated protein kinase phosphatases (MKP) , which dephosphorylated critical residues, rendering Mek and Erk1 inactive (Bhalla ef al., 2002). In an animal model of epi lepsy, M K P levels were found to be upregulated, which suppressed Erk1/2 phosphorylation during kainic acid-induced seizures (Gass ef al., 1996). Furthermore, M K P expression levels were upregulated as a defense mechan ism in rat mesangial cel ls exposed to hydrogen peroxide (Xu ef al., 2004). M K P activities were not monitored in these C G toxicity studies. Therefore, CG- induced inhibition of Mek and Erk1 activity via M K P activity can not be ruled out. Ruffels and col leagues have previously demonstrated that increased Akt activity by hydrogen peroxide suppressed Erk1/2 signaling (Ruffels ef al., 2004). In this study, the time course of CG-media ted Akt activation conincided with CG-media ted Erk1 suppression. However, in contrast to the findings of Ruffels and col leagues, CG-med ia ted Akt activation did not inhibit Erk1 (Figure 19A). Although inhibition of the PI3K/Akt pathway with LY294002 caused a compensatory activation in Erk1/2 as compared to control, C G treatment suppressed this compensatory effect for Erk1 only. This further supported the claim that C G selectively suppressed Erk1, but in an Akt- independent manner. In previous studies, treatment of the Mek inhibitor, U0126 suppressed glutamate-and hydrogen peroxide-induced Erk1/2 activity and protected against hippocampal cell death (Satoh ef al., 2000; Ruffels ef al., 2004). However inhibition of the Mek/Erk pathway with U0126 neither protected nor exacerbated CG-media ted cell death (Figure 21A). This indicated that Erk1/2 did not play a role in CG-media ted toxicity in N S C 3 4 cel ls. U0126 (10 uM for 20 h) was used for inhibition of Mek1/2/Erk1/2 pathway. At this concentration and duration of exposure, U0126 alone did not have any effect on N S C 3 4 cell viability. However, 62 similar to an observation by Satoh et a l . ( 2000), U0126 was toxic when the cells were treated at 10 uM for more than 24 h. Khabaz ian et al. (2002) did not observe any changes in Akt and Erk1/2 phosphorylation in CG-treated rat cortical cultures, even when C G was administered at the same concentration used in our experiments. There are two main reasons that could explain the discrepancies. First, the duration of C G treatment was different. Khabaz ian and col leagues examined the activities of Akt and Erk1 after 18-24 h of treatment. In the present study, the phosphorylations of these kinases were changed within min of C G exposure. Our results demonstrated that these kinases responded rapidly to C G exposure, but returned to basal phosphorylation levels after four h of treatment. Secondly, the cell culture system used in this study was different from Khabaz ian et al. Due to the genetic differences, the environment where these cells were obtained, and the age of the cells, it is likely that these cells will differentially respond to the same stimulus. 4.2.3 J N K and p38 activation J N K and p38 M A P K phosphorylation were upregulated in various animal models of neurodegeneration. For example, in a mouse model of familial A L S , p38 M A P K was activated in the cortical and spinal motor neurons during the presymptomatic stage (Holasek ef al., 2005; Wengenack ef al., 2004). Administration of minocycl ine, which suppressed p38 M A P K phosphorylation, delayed the onset of motor deficits (Kriz ef al., 2002; Zhu ef al., 2002), indicating that p38 M A P K played a vital role in motor neuron apoptosis. In mice exposed to the neurotoxin 1-methyl-4-pheny-1, 2, 4, 6- tetrahydropyridine (MPTP) , the phosphorylation of J N K was upregulated. Inhibition of J N K with pharmacological inhibitors protected against MPTP- induced dopaminergic neuron death, implicating a role of J N K in MPTP-med ia ted cell death in mice (Hunot et al., 2004). J N K and p38 M A P K pathways could be activated by a wide range of cytotoxic stimuli, including cytokines, radiation, osmotic dysregulation, heat shock, oxidative injury, amongst others (Martindale and Holbrook, 2002). Both J N K and p38 M A P K signaling pathways have been linked to commitment to apoptosis. C G exposure to N S C 3 4 cells resulted in J N K and p38 M A P K activation. Unlike the effect of C G on Akt and Erk1/2, which occurred within min of C G exposure, J N K and p38 M A P K upregulation required several h (Figure 20). It is unclear whether C G binds to a membrane receptor that stimulates the J N K and p38 M A P K pathways or J N K and p38 M A P K activation are simply a cellular response to prolonged C G exposure. S ince the activation of J N K and p38 M A P K were temporally 63 delayed, C G exposure may have caused other cytopathological conditions, which in turn activated these kinases. Furthermore J N K and p38 M A P K activation in N S C 3 4 cells exposed to C G was not related to CG- induced cell death. Pharmacological inhibitors that specifically inhibited J N K or p38 M A P K did not prevent CG- induced cell death (Figure 21B). These stress-activated protein kinases have been found to phosphorylate cytoskeletal components, which lead to disruption of the neuronal cytoskeleton. For example, a previous study demonstrated that J N K phosphorylated microtubule regulator proteins and prevented stabilization of microtubules, which lead to cytoskeletal dysfunction (Neidhart et al., 2001). p38 M A P K was found to phosphorylate neurofilaments leading to cytosolic accumulation of extensively phosphorylated neurofilaments observed in A L S patients (Ackerley e ra / . , 2004). Furthermore, both p38 and J N K were found to phosphorylate Tau and related proteins, but did not necessary promoted neuronal apoptosis in human tauopathy cases (Atzori et al., 2001b). Cytoskeletal rearrangements were observed in N S C 3 4 cells exposed to C G , which was possibly a stress-induced differentiation response (Chapter 5). These cytoskeletal rearrangments, including accumulation of cytoskeletal elements and Tau phosphorylation, may be required the activities of J N K and p38 M A P K . 4.3 Conclusion In this study, we examined the various kinase pathways activated by C G treatment to a motor neuron cell line, N S C 3 4 cells (Table 1). C G exposure to N S C 3 4 cells maintained in complete media activated Akt signaling in N S C 3 4 cells. CG- induced Akt activation was involved in protecting the cel ls against C G toxicity, s ince LY294002 exacerbated C G -mediated cell death. C G induced Akt activation was mTOR-independent and may have involved multiple upstream protein kinases in addition to PI3K. Erk1, but not Erk2 was suppressed by C G treatment. J N K and p38 M A P K were activated by C G treatment. The role of Erk1/2, J N K and p38 M A P K in CG-med ia ted toxicity is unclear. Pharmacological inhibitors that target activation of these protein k inases did not prevent or exacerbate CG-med ia ted cell death. 64 Time (min) , Serum + CG Serum only 0 2 4 8 16 32 2 4 8 16 32 -S2K pAkt" ^mmmm Total A k : * * ^ * * , M M / » y > tmP&^m 1 1 < JS 1.0 Q 2 0.5-j < 0.0 I I Cartral 2 il 32 T i m e of C G treatment (min) Figure 11. CG suppresses serum stimulation of Akt activity. NSC34 cells were serum deprived for 12 h and then stimulated with 10% serum in the presence or absence of 250 pM C G for the indicated times. Western blotting was performed with both anti-Aktl and anti-phospho-Aktl 5 6 ' " 4 7 3 antibodies on the same blot. A representative blot is shown. Akt phosphorylation is expressed as the phosphorylation ratio of phospho-Akt S e M 3 7 over total Akt. C G treatment suppresses serum stimulation of Akt phosphorylation (unpaired Student's t-test *p<0.05). The data are represented as the mean±S.E.M (n=6). 65 A Time (min) 0 2 ' 9 16 32 6 Time (min) 10 20 ;>C 60 '23 2*0 m 1440 0 Totai Akt *»•,.,,•„„ — — ' * Total Art — — - * — • :• C G concentration (jiM) Figure 12. C G treatment induced Akt activation in NSC34 cel ls maintained in complete media. NSC34 cells maintained in complete media were treated with C G . Western blotting was performed with both anti-Akt and anti-phospho-Akt S e M 7 3 antibodies on the same blot. Akt phosphorylation is expressed as the phosphorylation ratio of phospho-Akt S e r _ 4 7 3 over total Akt. A representative Western blot is presented for each experiment. (A) C G treatment induced rapid activation of Akt (One way ANOVA F 5 29=5.18 p<0.01). (B) CG-induced Akt activation is sustained for at least 480 min, but returns to basal level at 1440 min (One way ANOVA F 8 ii 8=3.22 p<0.05). (C) CG-induced Akt activation is also concentration-dependent. 250 pM of C G treatment for 5 min can trigger more than 2-fold Akt activation (One way ANOVA F5,3o=2.63 p<0.05). Treatment with 0.3% DMSO demonstrated that the DMSO in the highest C G concentration is not involved in activating Akt. Tukey's honest posf hoc shows significant changes compared to control cells without treatment (* p<0.05). Significant changes of percentage from controls (%CFC) are indicated on top of each bar. Combined results represent the mean±S.E.M (n=6). 6 6 Time (min) 0 2 4 8 16 32 pPD<1i;r * ^ ~ 0 2 4 8 16 32 T i m e of C G t rea tmen t (min) Figure 13. CG-induced activation of Akt independent of PDK1 activity. NSC34 cells were grown in complete media and treated with 250 uM of C G for the indicate times. PDK1 phosphorylation was assessed by Western blot analysis using an anti-phospho-PDK1 antibody. Actin was used as a loading control on the same membrane. PDK1 phosphorylation is expressed as the phosphorylation ratio of phospho-PDK1 over actin. The phosphorylation of PDK1 was not changed by 250 uM C G treatment (F 5 1 2=0.73 p>0.05). The data represent the mean±S.E.M (n=3). 6 7 1 Serum + C G Serum only Time (min) 0 2 4 8 16 32 2 4 8 16 32 pErkl pErk2 : = —mm~**m Total Erk Total Er! U J TS 0?5 *=• o .00-B a 1 00-, UJ C M n - £ : OJ CL -43K -40K 43K 4CK ] Centre-! I Treated! l X l - I 1 ! 16 32 8 Time of C G treatment (min) 1 1 J i i i Time of C G treatment (min) Figure 14. C G interferes with serum stimulation of Erk1/2 activity. NSC34 cells were serum deprived for 12 h and then stimulated with 10% serum in the presence or absence of 250 pM C G for the indicated times. Western blotting was performed with both anti-Erk1/2 and anti-phospho-Erk1/2 antibodies on the same. Erk1/2 phosphorylation is expressed as the phosphorylation ratio of phospho-Erk1/2 over total Erk1/2. A representative Western blot is presented for this experiment. Serum stimulation of serum deprived NSC34 cell induced Erk1/2 activation for 2-4 min and then returned back to basal levels. The presence of C G suppressed serum stimulation of (A) Erk1 and (B) Erk2 phosphorylation in serum deprived NSC34 cell (unpaired Student's t-test *p<0.05; ** p<0.01; ***p<0.001). Data are represented as the mean±S.E.M (n=6). 68 A Time (min) 0 pErk l — p E n \ 2 " " " M Total Erk1 ~ Total Erk2 -43K •-40K I-43K I-40K F=s«»-102 -36% jp«0 001 I I I ! . . 0 2 4 8 16 32 Time of C G treatment (min) 8 16 32 Time of C G treatment (min) E CG (uM) DMSO 0 5 25 100 250 , T202+Y2G4 -iigTnTHrniiiriiritmiTiiiiWTwrrMi pErkl pErk f* -43K -40K Total Erk1 * JW m Total Erk2 w L i i a 1 o n o g 1 _ o 10 lllll. DMSO 0 5 25 100 250 C G concentration (uM) p>0 05 min DMSO 0 5 25 100 250 C G concentration (uM) p E r k f pErk2 Total Erk1 Total Erk2 ) 10 20 30 50 120 240 480 14 40 0 • M 4 3 K D 3 B L U 0 10 20 30 60 120 240 480 1440 Time of C G treatment (min) l l l l l l l l l 0 10 20 30 60 120 240 480 1440 T ime of C G treatment (min) 6 9 Figure 15. CG treatment induced E r k 1 suppression, but not E r k 2 in N S C 3 4 cells grown in the presence of serum. NSC34 cells maintained in complete media were treated with 250 pM of C G . Western blotting was performed with both anti-Erk1/2 and anti-phospho-Erk1/2 antibodies on the same blot. Erk1/2 phosphorylation is expressed as the phosphorylation ratio of phospho-Erk1/2 over total Erk1/2. A representative Western blot is presented for each experiment. (A) C G treatment induced suppression of Erk1 phosphorylation from 2 to 32 min (One way ANOVA F 5 i 3o=10.2 p<0.0001) but (B) Erk2 phosphorylation was not significantly affected. (C) A longer time course experiment shows that Erk1 was suppressed very early (min) during C G treatment, but returns to basal level during longer treatments (h) (One way ANOVA F8,18=4.06 p<0.0001). (D) Erk2 phosphorylation was not affected by longer C G treatment time. (E) CG-induced suppression of Erk1 phosphorylation is concentration-dependent (5 min) (One way ANOVA F 5 3 0=3.77 p<0.001). Treatment of 0.3% DMSO served as a control to show that DMSO in the highest C G concentration did not have any effect on Erk1 suppression. In all experiments, Tukey's honest post hoc shows significant changes compared to control cells without treatment (* p<0.05). The percentage from controls (%CFC) are indicated on top of each bar. Combined results represent the mean±S.E.M (n=6). 70 Figure 16. Role of Raf1 and Mek1/2 in C G suppression of Erk1. NSC34 cells were grown in complete media and treated with 250 pM of C G for the indicate times. (A) Western blotting analysis was performed using anti-phospho-MEK1 S e r - 2 1 7 + S e r " 2 2 1 a n c | anti-phospho-Raf1 S r t " 2 5 9 antibodies on the same blot. The blots were stripped and reprobed with anti-actin antibody on the same membrane to confirm equal protein loading. (B) Mek1/2 and Raf phosphorylation were expressed as the phosphorylation ratio of phospho-Mekl e r ' 2 1 7 + S e r " 2 2 1 o r phospho-Raf1 S e r* 2 5 9 over actin. Mek1/2 phosphorylation was reduced within min of C G treatment (One way ANOVA F5 > 1 2=4.14 p<0.05; Tukey's honest posf hoc * p<0.05). The observed suppression of Erk1 phosphorylation parallels the reduction in Mek1/2 phosphorylation. (C) No significant changes were observed in the inhibitory phosphorylation site R a f 1 S e r " 5 9 , the direct upstream modulator of Mek1/2. The percentage from controls (%CFC) is indicated on top of each bar where there is a significant change. The data represent the mean±S.E.M (n=3). 7 1 Control LY294002 U0126 C G - + - + - + C G - + - + - + Control LY294002 U0126 Figure 17. Role of Mek1/2 and PI3K in CG-induced Akt phosphorylation. NSC34 cells maintained in complete media were treated with either 10 pM of the Mek1/2 inhibitor U0126 or 30 pM of the PI3K inhibitor LY294002 for 1 h. The inhibitors were then removed and the cells were incubated with complete media in the presence or absence of 250 pM of C G for 10 min. Western blotting was performed with both anti-Akt and anti-phospho-Akt S e r _ 4 7 3 antibodies on the same blot. Akt phosphorylation is expressed as the phosphorylation ratio of phospho-Akt S e M 7 3 over total Akt. A representative Western blot of PI3K or Mek1/2 inhibition on CG-induced Akt activation is shown. C G treatment without inhibitor pretreatment promoted Akt phosphorylation (unpaired Student's t-test *p<0.05). C G treatment was able to induce Akt phosphorylation followed by treatment with LY294002 (unpaired Student's t-test **p<0.01). C G treatment followed by pretreatment with U0126 did not have any effect on Akt phosphorylation. Combined results represent the mean+S.E.M (n=6). 72 Control LY294002 Rapamycin - + - + - + B Control LY2940Q2 Rapamycin p A k t 3 4 " Total Ak t jam i-62K p A k t T *-62K Total Ak t + - + -* , • • -62K i l i i in I i h i I^B H i I H , J » . iH fflS MM... MM — MM Mt MM CG - + + - + CG - + - f - + Control LY294002 Rapamycin Control LY294002 Rapamycin Figure 18. CG-induced Akt phosphorylation independent of mammalian target of rapamycin (mTOR). NSC34 cells maintained in complete media were treated with either 30 pM of LY294002 (PI3K inhibitor) or 200 nM of rapamycin (mTOR inhibitor) for 1 h. The inhibitors were then removed and the cells were incubated with complete media in the presence of absence of 250 uM of C G for 10 min. Western blotting was performed with anti-Akt and either anti-phospho-Akt S e M 3 or anti-phospho-AktThr" 3 0 8 antibodies on the same blot. Akt phosphorylation is expressed as the phosphorylation ratio of phospho-Akt over total Akt. A representative Western blot for each experiment is presented in the figure. (A) As determined before, C G treatment can induce phosphorylation of Akt S e r"* 7 3 phosphorylation under basal condition or following inhibition of the PI3K pathway with LY294002 (unpaired Student's t-test *p<0.05). mTOR is a downstream target of Akt that was previously found to phosphorylate Akt at Ser-473. However, inhibition of mTOR with rapamycin did not have any effect of Ak t S e r _ 4 7 3 phosphorylation. (B) Akt T h r " 3 0 8 phosphorylation is also increased with C G treatment or following inhibition of the PI3K with LY294002 (unpaired Student's t-test *p<0.05, ***p<0.001). Phosphorylation of Akt T h r the meamS.E.M (n=3) is not affected by inhibition with rapamycin. Combined results represent 73 Control LY294002 U0126 - + - + - + Control L Y 2 9 4 0 0 2 U 0 1 2 6 C G - + - ± _ - + Control L Y 2 9 4 0 0 2 U 0 1 2 6 Figure 19. Role of Mek1/2 and PI3K in CG-induced Erk1/2 phosphorylation. NSC34 cells maintained in complete media were treated with 10 uM of U0126 or 30 uM of LY294002 for 1 h. The inhibitors were then removed and the cells were incubated in complete media with or without 250 pM of C G for 10 min. Western blotting was performed with both anti-Erk1/2 and anti-phospho-Erk1/2 antibodies on the same blot. Erk1/2 phosphorylation is expressed as the ratio of phospho-Erk1/2 over total Erk1/2. A representative Western blot is presented in this figure. (A) C G treatment without U0126 suppressed Erk1 phosphorylation as previously demonstrated (unpaired student's t-test *p<0.05). LY294002 pretreatment caused a compensatory increased in Erk1 phosphorylation, but this effect was ablated by C G treatment (unpaired Student's t-test *p<0.05). Inhibition with U0126 followed by C G treatment did not have any effect on Erk1/2 phosphorylation. (B) Erk2 phosphorylation following inhibitor and C G treatment did not have any significant effects. Combined results represent the mean±S.E.M (n=6). 74 A n Time (min) 10 20 30 60 120 240 480 1440 0 _ . -46K PJNK mm -41K -42K p-p38 MAPK Ac in i ° C G cone. (u.M) DMSO 0 5 25 100 250 'WLWWM' z p-p38 MAPK * — .^m^mmm,,^ Figure 20. CG treatment induced late activation of stress-activated protein kinases. NSC34 cells were grown in complete media and treated with 250 pM of C G for the indicate times. Cell lysates were analyzed for stress-activated protein kinases (JNK1 (46) and JNK2 (41)) phosphorylation using a phospho-specific JNK1/2 antibody. Another blot under the same experimental paradigm was used for assessing p38 MAPK phosphorylation by using a phospho-specific p38 MAPK antibody. The blots were stripped and probed with anti-actin to confirm equal protein loading in each lane. Representative blots are shown. (A) Time course profile for C G induce JNK1/2 and p38 MAPK phosphorylation in NSC34 cells treated with 250 pM of C G for the indicated times. (B) Concentration-response analysis of JNK1/2 and p38 MAPK activation in NSC34 cells treated with increasing doses of C G for 18 h. The data represent the mean±S.E.M (n=3). 75 Control U0126 LY294002 Control SB230580 SP600125 Figure 21. Effect of Mek1/2, PI3K, JNK1/2, and p38 MAPK inhibition on CG-treated NSC34 cell viability. NSC34 cells maintained in complete media were pretreated with 10 pM of the Mek1/2 inhibitor U0126, 30 pM of the PI3K inhibitor LY294002, 10 pM of the p38 MAPK inhibitor SB230580, or 10 pM of the JNK1/2 inhibitor SP600125 for 1 h followed by 250 pM C G treatment with or without these inhibitors for another 20 h. The inhibitors were then removed and complete media with or without C G were added for a total of 48 h. Cell viability was assessed using the MTT reduction assay. (A) 250 pM of C G treatment resulted in approximately 40% decreased in cell viability (unpaired Student's t-test **p<0.01). C G treatment in the presence of U0126 or LY294002 also resulted in a significant decreased in cell viability when compared to inhibitor treatment alone (unpaired Student's t-test **p<0.01). Inhibition of PI3K with LY294002 exacerbated C G induced decreased in cell viability (CG alone vs. C G with LY294002; unpaired Student's t-test *p<0.05). (B) C G treatment in the presence of SB230580 or SP600125 did not promote cell survival (inhibitor alone vs. inhibitor with C G , unpaired Student's t-test *p<0.05, **p<0.01). Inhibitor treatment alone did not have any significant effect on cell viability as compared to control cells (no treatment). Data are represented as mean±S.E.M (n=12). 76 Table 1. Summary of C G ' s modulatory role on survival and stress kinases Kinases Investigated Duration of exposure for maximal effect Increased/ decreased Phosphorylation (%CFC) Role in CG-toxicity A k t s 4 / a 32 min Increased 229% * Cel l survival r£ r k^T202+Y204 16 min Decreased -76% * Unknown E r k 2 T 1 8 5 + Y 1 8 7 - No change - Unknown J N K T 1 8 3 + y 1 8 5 8 h Increased > 10000% Unknown p38 M A p K T 1 8 0 + Y 1 8 2 18 h Increased > 10000% Stress- induced differentiation? Comparison of the maximal effect of 250 p M C G treatment on survival and stress-activated kinase activities as compared to controls. * p<0.05 % C F C (percentage change from control) 77 CHAPTER 5. NSC34 cells differentiate to resist cholesteryl glucoside toxicity Hypothesis: C G toxicity mediates N S C 3 4 cells to undergo pathology-associated morphological changes prior to cell death. Specific aims: 1. To characterize the morphological changes in CG-treated N S C 3 4 cel ls 2. To determine if morphological changes confers resistance against CG-med ia ted cell death 5.1 Results 5.1.1 Quantitative assessment of cell morphology In the previous studies, cholesteryl glucoside (CG) was found to mediate reduction in N S C 3 4 cell viability in a dose- and time- dependent manner. C G treatment in the range of 50-250 pM for up to seven days resulted in over 50% reduction in cell viability (Figure 2, Chapter 3). Qualitative observations at this point showed mostly dying cel ls that were rounded and lack neurites (Figure 22B). However, some of the CG-treated cells exhibited morphological changes that were not observed in the control cells. For further analysis, N S C 3 4 cells were treated with 100 pM of C G for four days to induce these abnormal morphologies. These cells were then fixed and stained with p-Tubulin III, a neuronal specif ic cytoskeletal protein. A representative image of p-Tubulin III stained N S C 3 4 cells without treatment is presented in Figure 22A. A small population (estimated to be ~20%) of cel ls appeared larger under the microscope with extended neurites that were not observed in the control cells. Furthermore, some of the extended neurites developed focal swell ings that resembled neurite headings or blebs (Figure 22C, white arrows). Neurite headings are swell ings in the neuronal processes and have been found to contain aggregation of various cytoskeletal elements (Roediger and Armati, 2003). To quantitatively assess the morphological changes, N S C 3 4 cel ls were treated with 0, 10, or 100 p M of C G for 4 days. These cells were fixed, stained with p-Tubulin III, and observed under a fluorescent microscope (20X objective lens). The images of abnormal cell morphologies were captured using a C C D camera and quantified using an automated Axiovert 4.5 imaging software. The s o m a s ize was measured by the distance around the periphery of the cell body in micrometers and expressed as the soma perimeter. There were 78 significantly larger soma in N S C 3 4 cells treated with 100 uM of C G for 4 days as compared to control cells (Krustal-Wallis non parametric test, p<0.001). N S C 3 4 cel ls treated with 10 p M of C G did not show any significant changes in the soma size from the control cells. However, a ten-time higher exposure for the same duration significantly induced larger soma s izes (Krustal-Wallis non parametric test, p<0.01) (Figure 22D). The total processes lengths were the summed lengths of all processes that extended 25 pm or longer from an artificially marked boundary of the cell periphery. N S C 3 4 cells treated with 100 p M of C G exhibited neurites that were nearly two-fold longer than control cells and 10 p M CG-treated cells (Krustal-Wallis non parametric test, p<0.001). The total processes lengths were not different between the 10 pM treatment and control (Figure 22E) . To determine if the increased summed neurite length resulted from more observable neurites, the number of neurites was manually determined. A neurite was considered as 25 pm or longer initiating from an artificially marked boundary of soma periphery. Although there was a trend that 100 pM of C G treatment have more neurites, there were no statistical differences between the groups (Figure 22F). 5.1.2 Qualitative assessment of cell morphology To assess if the cytoskeleton was involved in CG- induced morphological changes, N S C 3 4 cells treated with or without 100 pM of C G for 4 days were fluorescently double stained with two cytoskeletal proteins: (3-Tubulin III and the heavy subunit of neurofilament (NFH). The antibodies that recognized these proteins independently labeled the extended processes and the neurite blebs (Figure 23). This finding indicated that CG- induced morphological changes cause cytoskeletal rearrangements. Futher, some of these proteins aggregated and formed swellings in processes, which was indicative of cytoskeletal disruptions seen in neurite blebs. Another common aggregated protein found in these swell ings was the phosphorylated form of the microtubule-associated Tau . Previous studies have found increased Tau phosphorylation and phospho Tau aggregates in neurons of Alzheimer 's d isease and some cases of motor neuron d iseases with dementia (Vega e ra / . , 2005; Strong et al., 2006). Moreover, some of these phosphorylation events were considered abnormal, since only patients that have neurodegenerative d iseases expressed these site-specific Tau phosphorylations (Hoffmann et al., 1997). Immunoreactivity to AT8 antibody, which recognized specifically phospho-Ser-202 and phospho-Ser-205 of Tau, had been associated with various neurological conditions including A D . To assess if abnormal Tau 79 phosphorylation was present in CG-treated N S C 3 4 cel ls, these cells were treated with 100 pM of C G for 4 days and immunostained with N F H and phospho-Tau (AT8 clone). C G treated N S C 3 4 cells were found to have immunoreactivity with the A T 8 phospho-Tau antibody in the neurite blebs and soma (Figure 24). Control cells have minimal AT8 phospho-Tau immunoreactivity in their soma. The TAR-DNA-b ind ing protein 43 (TDP43) was recently identified as a majory pathological protein in patients that suffered from sporadic A L S and frontotemperol dementia (Neumann ef al., 2006; Mackenzie ef al., 2007). This protein is usually located in the nucleus. However, during pathological conditions such as sporadic A L S , T D P 4 3 abnormally accumulates in the cytoplasm. To determine if C G treatment triggered cytoplasmic accumulation of T D P 4 3 , N S C 3 4 cells treated with 100 pM of C G for 4 days were double stained with anti-p-Tubulin III and ant i -TDP43 antibodies. The p-Tubulin III stain marked the boundary of the cell . Similar to other experiments, these cells were mounted in a DAPI mounting medium, which marked the nucleus. T D P 4 3 staining showed that T D P 4 3 co-local ized with DAPI , which indicated no T D P 4 3 accumulation in the cytoplasm. Rather, T D P 4 3 was observed to remain in the nucleus (Figure 25). However, some of these C G -treated cells still exhibited enlarged soma s izes and extended neurites, without any T D P 4 3 pathology. The previous experiments demonstrated that 100 pM of C G treatment to N S C 3 4 cells were associated with morphological changes that included enlarged soma, extended neurites, cytoskeletal protein aggregation, and phospho Tau immunoreactivity without T D P 4 3 pathology. To determine whether the cytoskeletal changes was a cause of cell death, N S C 3 4 cells treated with 100 pM of C G for 5 days were stained with trypan blue dye. The cells were then observed with phase contrast microscropy under a 40X objective lens. Figure 26A showed an image of N S C 3 4 cells without C G treatment, which were capable of excluding the trypan blue dye. N S C 3 4 cells treated with C G either exhibited differentiated morphologies or were rounded (Figure 26B). The rounded cells were non-viable s ince they could not exclude the trypan blue dye (Figure 26B, black arrows). On the contrary, the enlarged N S C 3 4 cells with long processes were viable due to the absence of trypan blue dye staining. 5.2 Discussion In this study, we found that a subpopulation of CG-treated N S C 3 4 cells displayed cellular morphologies that were not observed in control cells. Immunostaining these cel ls 80 with p-Tubulin III antibody and quantification of the cell morphology after C G treatment for 4 days demonstrated increased soma s izes (Figure 22D) and total neurite lengths (Figure 22E), without any significant changes in the number of processes (Figure 22F) . Furthermore, the abnormal processes appearred to contain focal swell ings (Figure 22F, white arrows). Both the abnormal processes and swell ings were immunoreactive to cytoskeletal proteins such as p-Tubulin III, N F H , and phospho Tau . Immunoreactivity with the latter is typically associated with certain neurological d iseases. C G treatment did not trigger abnormal cytoplasmic accumulation of T D P 4 3 , which has been observed in some neurological disorders, including sporadic cases of amyotrophic lateral sclerosis (ALS) . The N S C 3 4 cel ls that have differentiated and developed morphological changes induced by C G are viable, s ince they were capable of excluding the trypan blue dye (Figure 26). These findings indicate that C G triggered N S C 3 4 cell to undergo differentiation as a morphological response to CG- induced toxicity. 5.2.1 Morphological changes in CG-treated N S C 3 4 cells The subpopulation of CG-treated N S C 3 4 cells had enlarged nuclei and soma, similar to chromatolysis observed in axotomized spinal motor neurons (Mcllwain and Hoke, 2005). The reason for this morphological response has not been clearly explained. On the one hand, previous researchers have proposed that cell enlargement may be a consequence of osmotic dysregulation which resulted in excess water accumulation in the cell (Mcllwain and Hoke, 2005). On the other hand, gradual build-up of proteins and other macrmolecules as seen in various cytoskeletal disruption models may play a role in cell enlargement (Brattgard e ra / . , 1957; Edstrom, 1959). The proportional increased in nucleus s ize with the cell body was a result of coordinated growth, s ince a similar scal ing was observed in the control cells. Enlarged cell bodies and nuclei were also characteristics of cellular aging or replicative senescence , where the cells do not respond to mitogens and stop proliferation (Cristofalo ef al., a l . , 2004). It is more likely that C G mediated neurite disruption which caused enlarged nuclei and cell bodies rather than promoting cellular senescence . The reason is two-fold. The formation of neurite blebs was observed in C G -treated N S C 3 4 cell , which indicated neurite disruption. Secondly, an MTT reduction assay used for assess ing cell viability constantly demonstrated a reduction in cell viability from C G treatment. To reach replicative senescence , cells maintained in vitro need to undergo rapid proliferation followed by a gradual loss of replicative potentials (Cristofalo ef al., 2004). No evidence of increased cell proliferation due to C G treatment was observed. Futhermore, 81 reaching replicative senescence requires several subcultivations (ie. weeks) (Hayflick, 2007), yet CG- induced cell enlargement requires at most a few days. Increased neurite length in some N S C 3 4 cel ls treated with 100 u M was not due to increased numbers of neurite, but due to neurite extension. Neurite outgrowths in cells were associated with growth factor induced-neuronal differentiation. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) have been found to mediate differentiation in the rat pheochromocytoma cell line (PC12 cells), which was characterized by neurite outgrowth (Morooka and Nishida, 1998). Moreover, testosterone treatment to N S C 3 4 cel ls stably transfected with androgen receptors a lso induced neurite outgrowth (Marron et al., 2005) . Recently, various environmental stress factors were found to induce neuronal differentiation and increase neurite extension. For example, heat shock (44°C for 10 min) was found to induce neurite outgrowth in the rat P C 1 2 cells via the p38 M A P K signaling pathway (Kano ef al., 2004). Furthermore, hyperosmotic shock (240 m M NaCI for 7 days) was also found to trigger stress- induced differentiation neurite extension in P C 1 2 cel ls (Kano et al., 2007). Stress- induced neurite outgrowth was believed to be a distinct process from growth factor-mediated neuritogenesis. Stress- induced neurite extension may be one of the many cellular defense mechanisms that conferred cytoprotection or molecular adaptation to the stress stimuli (Kishi ef al., 2001; Kano ef al., 2007). The signaling cascade underlying growth factor-induced differentiation involved the Raf1/Mek1/2/Erk1/2 signaling cascade, whereas stress-induced differentiation usually required p38 M A P K signaling. C G -induced neurite outgrowth resembled the conditions of a stress-induced differentiation. Late activation of p38 M A P K was found to be not involved in mediating cell death (Chapter 4). Therefore, it would be tempting to speculate that the late activation of p38 M A P K in C G -treated N S C 3 4 cells was involved in differentiation and promotion of neurite outgrowth. It is unclear why a homogenous cell culture of N S C 3 4 cells would respond heterogenously to the same stimuli. Given that N S C 3 4 cells are a purely motor neuron-derived cell line, it was expected that all of these cells responded to C G toxicity in the same manner, by undergoing cell death. However, some CG-treated cells were resistant to prolonged C G exposure. Select ive cell death had been previously documented. For example, in A L S pathology, not all spinal cord motor neurons were affected (Boillee ef al., 2006) . Furthermore hydrogen peroxide treatment only induced cellular disruption in 6 0 % of a homogenous fetal human cortical neuron culture, leaving the other 4 0 % unaffected (Roediger and Armati, 2003). The reason for this selectivity in cell death remained unknown, especial ly when all the cells were maintained under the same condition. It was previously 82 proposed that cells in vitro grow asynchrously, each reaching a mature or senescent state at different rates (Cristofalo e ra / . , 2004). If this model correctly described the different status of each cell maintained in a dish, then it would be logical to observe differential responses to the same stimulation, where older cells die first and younger cell differentiated for adaptation to the stress. Older cells were believed to have decreased potentials of replicating and mediating responses to stress (Cristofalo et al., 2004). A lso , the possibility that massive cell death occurred due to C G treatment provided space and nutrient for resistant cells to differentiate and grow cannot be ruled out. 5.2.2 Neurite swell ings and cytoskeletal disruption Focal neurite swelling in dendrites and axons (neurite beading) was thought to be a neuropathological sign in ischemia (Hori and Carpenter, 1994), epi lepsy (Swann ef al., 2000), aging (Saito ef al., 2003), and neurodegenerative d iseases such as Alzheimer d isease (Dickson et al., 1999), Parkinson's d isease (Mattila ef al., 1999), and amyotrophic lateral sclerosis (Delisle and Carpenter, 1984; Takahashi et al., 1997). Other toxic stressors that induced neuritic headings included glutamate (Park ef al., 1996), nitric oxide (NO) (Faddis etal., 1997), hypoxia (Hasbani etal., 1998), and hydrogen peroxide (Ikegaya etal., 2001; Roediger and Armati, 2003). Neurite beading was found in an experimental autoimmune encephalomyelit is model and shown to correlate with d isease severity. This finding indicated that neurite bleb formation was an irreversible process and parallelled neuronal damage (Zhu ef al., 2003). Neurite beading was usually implicated as a cause of neuronal dysfunction due to disruption of the cytoskeleton and cellular transport system (Takeuchi ef al., 2005). Therefore immunofluorescent staining of neurite headings commonly showed aggregation of vesicular cargo, transport proteins, and cytoskeletal proteins (Menzies ef al., 2002; Takeuchi ef al., 2005). The neurite headings were first observed in CG-treated N S C 3 4 cells with a fluorescent neuronal specif ic pMubulin III stain, which was later found to colocal ize with the heavy subunit of neurofilament, another neuronal cytoskeletal protein. This finding indicated that C G toxicity paralleled cytoskeletal disruption in some N S C 3 4 cel ls. The neurite headings were immunoposit ive for a disease-associated phospho Tau . There was also some phospho Tau immunoreactivity observed in the cell body. Tau is a microtubule associated cytoskeletal protein and has many phosphorylation sites. The antibody (AT8) that specifically recognized a disease-associated of Tau phospho-site was used. This antibody recognized phosphorylation sites found in certain neuropathological 83 conditions (Neumann ef al., 2006). The control cells without C G treatment also have a small amount of phospho-Tau immunoreactivity. Arguably, cells maintained in vitro to some extent are under stress, which may explain the phospho-Tau immunoreactivity (Brown, 2005). Tau pathology has been implicated in the pathogenesis of various age-related neurodegenerative d iseases such as Alzheimer 's d isease (AD), frontotemporal dementia, amongst others (Spillantini and Goedert, 1998; Lee et al., 2001). Although some transgenic animals overexpressing Tau develop deleterious neuropathological conditions, it is unlikely that Tau pathology is the causal factor in most neurological d iseases . For example, c lassical amyotrophic lateral sclerosis patients are free of Tau pathologies (Strong ef al., 2005) and there is no direct evidence to show that Tau lesions induce neuronal death in A D cases (Mori, 2000). 5.2.3 Stress- induced differentiation for protection C G treatment induced morphological changes that included stress-induced neurite extension, neurite swell ings, cell enlargement, and immunoreactivity with d isease-associated phospho Tau . Trypan blue staining of these cells showed that they were capable of excluding the trypan blue dye, which indicated that these were viable cel ls. Although these morphological changes were observed only in CG-treated cells, we do not know whether or not C G directly induces these changes. Several possibilities of how and why the observed morphological changes occurred will be d iscussed below. First, it is possible that C G treatment eliminated most cells, which provided space for the remaining cells to grow. Due to the properties of an immortalized cell line, we would expect that increased nutrients and growing space will trigger these cells to continue to proliferate rather than differentiate. Secondly, we do not know if C G treatment selectively eliminated smaller cells or cells with shorter processes. However, prior to C G treatment, the N S C 3 4 cells have a homogeneous appearance, that show an irregular, flatten shape and occasional ly extended short processes. W e bel ieved that the appearance of enlarged cel ls with extended processes is an artifact due to C G treatment. To further investigate whether cell s ize influenced C G -mediated cell death, we could label treated cells with a cell death marker (eg. active caspase-3) and assess the proportions of cell death in smaller cells and bigger cells. Thirdly, the observed morphological changes may be events that preceded cell death in CG-treated N S C 3 4 cells, albeit each cell undergoes the changes at different rates. However, when stained with trypan blue, treated cells with abnormal morphologies are viable. Finally, C G -treatment may induce some N S C 3 4 cells to undergo stress-induced differentiation which 84 could mediate an adaptive response to C G . After differentiation, these cells may have loss their potential to respond to C G , thus they are resistant to C G toxicity. The selectivity for certain treated cells and not others are unclear. Taken together the different possibilities d iscussed above, we proposed a potential mechanism for the morphological changes observed in CG-treated N S C 3 4 cells. Prolonged C G treatment triggers a subpopulation of N S C 3 4 cells to undergo stress-induced differentiation and extend neurites via the p38 M A P K signaling pathway. The extended neurites will be susceptible to cytoskeletal disruption, which forms neurite headings. Cytoskeletal disruption in the treated N S C 3 4 cells may result in buildup of macromolecules which lead to enlarged cell s izes . At this stage, the differentiated N S C 3 4 cells reached a new state that rendered them unresponsive to C G , which prevented CG-media ted cell death. 5.2.4 T D P 4 3 proteinopathy and neurodegeneration The T A R DNA-binding protein (TDP) 43 is a ubiquitously expressed DNA-binding protein with a molecular mass of 43 kDa (Buratti and Baral le, 2001). It was originally identified for its nucleic acid binding capacity and its role in suppression of HIV-1 gene expression by blocking the assembly of transcription complexes (Ou et al., 1995). Subsequent reports have demonstrated that T D P 4 3 is a splicing regulator of the cystic fibrosis t ransmembrane conductance regulator ( C F T R ) and inhibits alternative splicing (Buratti ef al., 2001; Buratti ef al., 2004; Aya la ef al., 2006). This mechanism has been associated with d isease penetrance of cystic fibrosis (Ayala ef al., 2006). The physiological function of T D P 4 3 in brain cells has not yet been determined. Recent studies have identified T D P 4 3 as a major component of neuronal inclusions found in several neurodegenerative d iseases such as frontotemporal dementia (Neumann ef al., 2006), Park insonism dementia complex (PDC) (Hasegawa etal., 2007), and sporadic cases of A L S (Mackenzie ef al., 2007; Robertson ef al., 2007). It should be noted that familial cases of A L S with superoxide dismutase mutations do not have T D P 4 3 accumulat ion, which indicates a distinct pathomechanism between sporadic and familial cases of A L S (Mackenzie ef al., 2007). T D P 4 3 is normally localized in the nucleus. Under pathological conditions, T D P 4 3 is eliminated from nuclei of inclusion-bearing neurons, which may alter the normal T D P 4 3 activity. Furthermore, T D P 4 3 in these neurodegenerative cases were found to be highly phosphorylated and ubiquitinated (Neumann et al., 2006; Arai ef al., 2006; Mackenz ie ef al., 2007). The modified T D P 4 3 then pathologically accumulated in the 85 cytoplasm and contributed to inclusion formations or may assume a fi lamentous, skein-l ike appearance (Saito ef al., 2003; Neumann ef al., 2006; Hasegawa ef al., 2007). It is unclear whether small amounts of T D P 4 3 may be present in the cytoplasm under physiological conditions and if T D P 4 3 pathology entails a problem of shuttling into the nucleus or a problem of nuclear retention (Arai ef al., 2006; Mackenzie ef al., 2007). Whether or not abnormal accumulation of T D P 4 3 pathology is the cause of motor neuron death has not been demonstrated. Sporadic cases of classical A L S , Guamanian A L S , and Guamanian P D C have shown abnormal cytoplasmic accumulat ions of T D P 4 3 . Given that these d iseases are likely to have an environmental etiology linked to steryl g lycosides, challenging neurons in culture with the putative neurotoxin, C G may also induce T D P 4 3 pathology. However, C G treatment to N S C 3 4 cel ls did not induce cytoplasmic accumulation of T D P 4 3 . Furthermore, T D P 4 3 did not display any filamentous appearance as observed in the human neurodegenerative cases . Poss ib le interpretations of this are that CG- induced cell death is not linked to T D P 4 3 pathology in A L S cases , or the mode of neuronal degeneration induced by C G is different for N S C 3 4 cells maintained in vitro and motor neurons in vivo, which may require other factors. 5.3 Conclusions Prolonged C G treatment resulted in substantial cell death and induced morphological changes in a population of resistant cells. These cells displayed enlarged cell bodies and nuclei, with increased neurite extension. The reason for the heterogenous cellular response within a homogenous cell line is unclear. Futhermore, the neurite develop focal neuronal blebs characterized by aggregration of cytoskeletal protein p-tubulin III and N F H . C G -treatment a lso induced pathological Tau phosphorylation which showed immunoreactivity not only in the cell bodies but also the neuronal blebs. CG- induced morphological changes did not appear to be directly linked to T D P 4 3 pathology. Finally, the morphological changes did not correlate with cell death, indicating that N S C 3 4 cel ls may undergo differentiation as an adaptive mechanism for C G toxcity. 86 A t C o n t r o l % H m $§*. ^ * B 100 pM C G c * I *> .... r * •» 100 | i M C G D 200-100-1 I CG concentration [yMi) > I I ~ 35 cs to 0 10 100 0 10 100 C G concentration (JJM) Figure 22. Morphological quantification of CG-induced cytopathology in NSC34 cells. Four days of 100 pM C G treatment resulted in over 50% reduction in cell viability as compared to control cells. However, a subpopulation of treated cells appeared bigger under the microscope and had long processes. (A) p-tubulin III staining of control cells. (B) p-tubulin III staining of C G treated cells for 3 days, which shows rounded, dying morphology. (C) However, a sub-population of treated cells appeared bigger and laid out long processes. Furthermore, neuronal headings were observed on some of the long processes (indicated by white arrows). NSC34 cells treated with 100 pM of C G for 3 days were fixed and stained with p-tubulin III. Abnormal processes and enlarged cell size were quantified. Kruskal-Wallis non-parametric test (** p<0.01; ***p<0.001) showed that treatment induced NSC34 cells to exhibit longer processes (D) and larger soma perimeters (E), with no changes in the number of processes per cell (F). Images were taken from 6 random fields per coverslip and viewed under a 20X objective lens. Scale bar=100 pM. Morphological quantifications were performed using Axiovert 4.5 imaging software and data were represented as mean±S.E.M (n=6 per C G concentration). 87 88 Figure 23. Abnormal processes and axonal blebs in CG-treated NSC34 cells are immunoreactive with p-tubulin III and NFH. Labeling of the heavy subunit of Neurofilament (NFH) and p-Tubulin III in NSC34 cells showed co-localization of these two cytoskeletal elements in the processes and axonal blebs. NSC34 cells were either not treated or treated with 100 pM of C G for 6 days. Panels A and E show DAPI staining of the nuclei; B and F show staining of p-Tubulin III; C and G staining of NFH; D and H are merged images. Images were captured using a fluorescence microscope under a 40X objective lens. Scale bar=100 pM. 8 9 Control 100 \iM CG m E D API phospho Tau F % phospho Tau C NFH G D Mer ged H Merged 9 0 Figure 24. CG-treated N S C 3 4 cells are immunopositive for disease-associated phospho-Tau. C G treatment (100 uM C G for 6 days) induces extended processes with axonal blebs in NSC34 cells. Labeling of the heavy subunit of neurofilament (NFH) and pair-helical filament (PHF) Tau phosphorylated at Ser-202 (AT8 clone) showed co-labeling at the axonal blebs. The cell bodies of treated cells have traces of phospho-Tau immunoreactivity. NSC34 cells were either not treated or treated with 100 pM of C G for 6 days. Panels A and E show DAPI staining of the nuclei; B and F show staining of NFH; C and G staining of PHF Tau; D and H are merged images. Images were captured using a florescence microscope under a 20X objective lens. Scale bar=100 pM. 91 Control 100 u M CG 92 Figure 25. TDP43 pathology is not present in CG-treated NSC34 cells. NSC34 cells treated with 100 uM C G for 6 days did not reveal any TAR DNA binding protein (TDP) 43 pathology. TDP43 is normally found in the nucleus but accumulates abnormally in the cytosol during pathological conditions as documented in motor neurons of patients with sporadic ALS. No abnormal accumulation of TDP43 was observed in C G treated NSC34 cells. Panels A and E show DAPI staining of the nuclei; B and F show staining of p-Tubulin III; C and G staining of TDP43; D and H are merged images. Images were captured using a fluorescence microscope under a 40X objective lens. Scale bar=100 pM. 93 No treatment 100 M M C G for 5 days Figure 26. Viability and morphology of CG-treated NSC34 cells. NSC34 cells were treated with 100 uM of C G for 6 days to induce abnormal processes extension. The cells were stained with trypan blue and analyzed by phase contrast microscopy. (A) Control cells maintained in complete with no C G treatment. (B) CG-treated NSC34 cells with extended processes and increased soma size. These cells were viable cells because they could exclude the trypan blue dye. Dead cells were smaller, rounder, and were stained blue with the dye (marked by black arrows). Images were taken with phase contrast microscopy under a 40X objective lens. 94 CHAPTER 6 General discussion and future studies 6.1 General discussion Amyotrophic lateral sc lerosis-Parkinsonism dementia complex ( A L S - P D C ) is a spectrum of neurological disorders characterized a varying combination of A L S , Park inson's, Alzheimer 's d isease features. Epidemiology and experimental studies have pointed to a dietary link between A L S - P D C and consumption of cycad seeds (Hirano et al., 1961). Biochemical studies have identified the putative neurotoxins in washed cycad flour as variant forms of steryl g lycosides molecules (Khabazian ef al., 2002). Steryl g lycosides are commonly found in the environment, but their biological roles remain elusive. Cholesteryl glucosides (CG) , a variant form of the cycad-derived steryl g lycosides, have both toxic and protective properties in mammalian cells. In one study, C G treatment in rat cortical neurons triggered cell death via apoptosis (Khabazian ef al., 2002). However, other authors found that briefly exposing C G to fibroblast cells protected against heat shock (Kunimoto ef al., 2002). In the current study, we investigated the role of C G in a motor neuron hybrid cell line, N S C 3 4 cel ls. Specif ical ly, we determined whether C G protected N S C 3 4 cells, from serum deprivation stress and examined the biochemical mechanisms triggered by C G with respect to regulation of cell survival and cell death. 6.1.1 Cytotoxicity triggers cholesteryl glucoside (CG) synthesis A transient increase in endogenous C G content may be a defense mechanism for protecting against cytotoxic stresses. However, prolonged C G exposure is toxic and leads to cell death. The outcome likely depends on the duration of C G exposure (Figure 27). Typically, a brief episode of stress promoted cell survival, while a prolonged exposure caused cell death. An intermediate exposure time may temporarily arrest growth until C G is removed from the system. Heat shock in fibroblast cells and slime molds were found to increase endogenous synthesis of C G . In the current study, N S C 3 4 cells were found to possess the capability of endogenous C G synthesis during serum deprivaion. This finding indicated the possibility that steryl glycosylation in mammalian cells may be involved stress signal transduction (Kunimoto ef al., 2002). It is tempting to speculate that various neurotoxins or genetic acidents trigger mammalian synthesis of C G . The transient increase in C G may confer cytoprotection against the stresses. However, prolonged C G exposure due to the continuous stress stimulus may trigger cell death. A previous report by Kunimoto ef al., (2002) demonstrated that small amounts (2 pM) of C G can induce at least a five-fold increase in endogenous C G . S ince A L S - P D C patients were continuously subjected steryl 95 glycosides over time from cycad seed consumption, we suggest the possibility that the cycad-derived steryl g lycosides triggered endogenous synthesis of C G , which is neurotoxic. 6.1.2 Activating the PI3K/Akt pathway for neuroprotection Activation of the PI3K/Akt signaling pathway during cell stress was known to be important in conferring cytoprotection (Martindale and Holbrook, 2002). For example, inhibition of this pathway using the PI3K inhibitors, wortmannin and LY294002, blocked activation of Akt by hydrogen peroxide and increased cell death (Sonoda et al., 1999; Ruffels et al., 2004). Growth factor-mediated neuroprotection from cytotoxic insults primarily activated the PI3K/Akt pathway through specif ic receptor tyrosine kinases (Ding et al., 2000; Li et al., 2003). The survival signals then lead to phosphorylation-dependent suppression of intracellular apoptotic factors such as B A D , caspase9, and G S K 3 a/p (reviewed in Datta et al., 1991; Vukosav ic ef al., 1999). Therefore, neurotrophic growth factors were suggested as canadidates for therapeutic interventions for motor neuron d isease. Viral-delivery of the insulin-like growth factor increased the viability of motor neurons in the mouse mutant superoxide dismutase (mSOD) model of A L S . An increased Akt activity correlated with the delayed onset of the d isease in these mice, suggesting that Akt protected against the toxicity generated by the m S O D gene. This recent study found that N S C 3 4 cells exposed to C G stimulated Akt phosphorylation, which paralleled preconditioning for serum deprivation (Table 1). C G stimulation of Akt phosphorylation is dependent on the PI3K and independent of the mammal ian target of rapamycin. Whether or not C G binds to a receptor that lead to Akt activation is not known. However, this is not the first study to show that a pro-apoptotic stimulus can induce an anti-apoptotic response. Hypoxia and hydrogen peroxide, which are known to be toxic, could precondition neurons to various stresses. Furthermore, these stimuli and C G lead to activation of the PI3K/Akt pathway. Hence, activation of the PI3K/Akt pathway could be part of a general adaptive response to cytotoxic stresses. Given the importance of the PI3K/Akt signaling pathway in protection against various cytotoxic stresses in vitro, we suggest that specifically enhancing this pathway in neurons should be considered in the development of neuroprotective strategies. 6.1.3 Stress differentiation: an adaptive process N S C 3 4 cells treated with C G least three days resulted in over 5 0 % cell death. However a small percentage of the viable CG-treated cells exhibited enlarged cell bodies and extended neurites. Some neurites contained focal swell ings that were immunoreactive 96 with cytoskeletal elements and phospho Tau. W e do not know if C G treatment directly triggered the morphological changes or these were simply secondary changes due to other downstream effects. W e speculated that these cells may be undergoing a form of stress-induced differentiation, which conferred resistance against C G toxicity. Stress- induced differentiation in neuronal cells has been previously reported. For example, P C 1 2 cel ls exposed to heat shock or osmotic shock triggered neurite extension as a form of differentiation (Kano ef al., 2004; Kano ef al., 2007). Dinitrophenol (DNP) is commonly known as a toxic, mitochondria uncoupling agent and was found to trigger differentiation and promoted neurite outgrowth in the neuronal N2a cell line (De Fel ice and Ferreira, 2006). Low dose D N P treatments promoted neurite extension in primary cortical and hippocampal cultures (De Fel ice and Ferreira, 2006). The stress-induced differentiation was believed to confer protection against the stress insults; however, the mechanism underlying this phenomenon remained elusive (De Fel ice and Ferreira, 2006; Kano ef al., 2007). Conceivably, different forms of stress-induced differentiation might be a common neuronal reponse to various toxic chal lenges. For example, we speculate that axonal sprouting observed in A L S patients may be a form of stress-induced differentiation. In the mouse m S O D model of A L S , researchers have reported axonal sprouting during presymptomatic animals, where axons from affected motor neurons attempt to reinnervate the target muscle (Shefner ef al., 1999; Tarn ef al., 2001). Interfering with axonal outgrowth by various toxins was found to increase the rate of d isease progression (Gordon ef al., 2004). 6.2 Future studies The signaling pathways leading to Akt activation have not been thoroughly elucidated in relation to the toxic effect of C G exposure in N S C 3 4 cells. C G stimulation after PI3K inhibition was able to induce Akt phosphorylation on two critical phosphorylation sites. This finding indicated that other pathways may overlap with P I3K/PDK1 in activating Akt. Determining this kinase may offer a novel mechanism for activating Akt and possibly identify a new P D K 2 enzyme (Chan and Tsichl is, 2001). Several candidate kinases were previously identified as potential activators of Akt, which included P K A , ILK, and Src . Future studies will assess the expression and activation pattern of these kinases in N S C 3 4 cells exposed to C G . Further experiments using pharmacological inhibitors, which specifically inhibit these k inases, could be done to determine the role of these kinases in CG- induced Akt activation. Although brief C G stress was protective, but prolonged C G exposure was toxic. The mechanism by which prolonged C G treatment mediated toxicity in N S C 3 4 cells has not 97 been clearly defined. Previously, it has been shown that exposure of rat cortical culture to the steryl glycoside-containing fraction of cycad seeds resulted in depolarizing field potential that paralleled an increase in P K C B activity (Khabazian ef al., 2002). These data indicated that steryl g lycosides may induce excitotoxicity that lead to neuronal death. Furthermore, abnormally upregulated activity of P K C p was reported in the spinal cords of patients who died from sporadic A L S (Lanius ef al., 1995; Krieger ef al., 1996). Therefore, C G could mediate cell death via the actions of various P K C isoforms. Future experiments will focus on studying the activities and expression of P K C . Furthermore, experiments using pharmacological inhibitors for P K C such as RO320432 or co-treatment of C G with phorbol-esterase could determine the role of P K C in CG- induced cell death. W e believe that the neurodegenerative d iseases are not likely to be caused by a single factor. Rather, a complex interplay between multiple factors is likely to trigger d isease manifestation. Although exposure to steryl glycosides can be a contributing factor in neurodegeneration, the possibility that age and susceptibility genes may be participating in neurodegenerative d isease processes cannot be excluded. Susceptibil ity genes, including the amyloid precursor protein, mutant superoxide dismutase, parkin, amongst others, may be the underlying factor explaining how a common environmental toxin can induce specif ic neurodegenerative phenotypes. Moreover, steryl glycosides, either endogenously synthesized or exogenously acquired, can mediate neurotoxicity through a complicated cascade involving the interaction of susceptible genes, and old age. This issue can be approached by exposing transgenic animals to the putative neurotoxins. For example, animals with the mutant superoxide dismutase may be challenged with flour derived from cycad seeds or steryl g lycosides. On the one hand, it is predicted that transgenic animals exposed to the putative neurotoxins will manifest behavioral deficits earlier and show more severe destructions to their spinal motor neurons. On the other hand, neurotoxin exposure or inheriting a susceptible gene alone will eventually manifest the d isease phenotype, but at significantly slower rates. On-going studies in the laboratory have been studying the steryl glycoside contents in humans, particularly patients suffering from A L S . Specifically, we are trying to understand how steryl g lycosides are acquired and if this c lass of lipids accumulates in the central nervous to cause damage. Moreover, a batch of mice has been fed cycad over a period of 6 months. Blood samples and various parts of their central nervous systems will be subjected to lipid analysis using high performance liquid chromatography and mass spectrometry to see whether these mice accumulate steryl g lycosides in their body after eating cycad for an 98 extensive period of time. A n in depth understanding on the distribution and metabolism of steryl glycosides in the human body may render this family of compounds a novel lipid biomarker for A L S and related neurodegenerative d iseases. 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