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Targeting Ryk-signaling to attenuate multiple sclerosis by promoting oligodendrocyte differentiation and myelination

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Targeting Ryk-signaling to attenuate multiple sclerosis by promoting oligodendrocyte differentiation and myelination

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Content Targeting Ryk-signaling to attenuate multiple sclerosis by promoting oligodendrocyte differentiation and myelination Supervisor: Wange Lu, Ph.D Naibo Zhang Master of Science (BIOCHEMISTRY & MOLECULAR BIOLOGY) Keck School of Medicine, University of Southern California Degree Conferral Date: August 2015 Copyright ©August 2015 Contents Acknowledgements ........................................................................................................ 3 Background ................................................................................................................ 4 Multiple Sclerosis: Symptom, Epidemiology, Pathology and Therapies ............... 4 Myelination and Remyelination ............................................................................. 8 Wnt-Ryk Signaling ................................................................................................. 9 Drugs Profile ........................................................................................................ 12 Materials and Methods ............................................................................................. 13 NS Cs Culture ........................................................................................................ 13 Oligodendrocytes Culture ..................................................................................... 14 Immunocytochemistry .......................................................................................... 15 Imaging ................................................................................................................. 17 Cell Qualification ................................................................................................. 17 Drugs preparation ................................................................................................. 17 Results ...................................................................................................................... 18 Cell Culture Medium Condition Modification ..................................................... 18 OPC Cell Culture System Development .............................................................. 19 Myelination of Oligodendrocytes ......................................................................... 24 Ryk Inhibitor Candidates Test .............................................................................. 30 Conclusion ............................................................................................................... 33 Discussion ................................................................................................................ 3 5 References .................................................................................................................... 39 2 Acknowledgements First of all I am thankful to Dr. Wange Lu for his guidance on my research. Dr. Lu not only trained me on experimental techniques, but also teach me how to be a researcher. He broadened my view scope on science, and offered me new ideas. I also appreciate his generous financial and logistical support. I am thankful to my defense committees: Dr. Wange Lu, Dr. Zoltan Tokes and Dr. Qilong Ying, thank you for your attendance and patience. I am grateful to my mentor Dr. Hongzhen Yang, who taught me experimental skills. I also want to say thank you to all my lab members: Byoung San Moon, Bradford Steele, David Huang, Mingyang Cai, Min Gao, Qiong Wu, Wen-Hsuan Chang, Yanli Xu. For their generous help and useful opinion towards my research. 3 Background Multiple Sclerosis Multiple Sclerosis (MS) is a CNS demyelinating disease. It is one of the most common chronic disabling CNS disease in young adults[ll_ As a main symptom of MS, demyelination usually result in severe neurodegenerative lesions. MS patients show different symptoms, while specific symptom directly relates to affected locations within central neural system (CNS). Lesions occur mostly in white matter, and was reported that even cortex region and gray matter can also be severely affected[ 2 l. It is difficult to view cortex lesions directly by MRI, measuring the thickness of cortex is always a useful method for diagnosis because affected cortex is thinner than normal. Presently there is no drug that could cure CNS lesions via lesion repair. Also, there is no treatment on gray matter lesions, because T-cells can not reach gray matters. MS is also recognized as an autoimmune disease. Myelin antigen specific T-cells are activated in periphery tissue, and then migrate back to CNS. A inflammation response is triggered by those already-activated immune cells(included activated Th-cells, even B cells). MS is a progressive disease that can be separated into stages. In early 4 stage of MS, lesions and inflammation exhibit a dynamic situation, due to remyelination process. Lesions can be restored while new lesions keeps forming. RRMS (relapsing - remitting MS), which is treated as an early stage of MS, degeneration is followed by a partial or complete recovery. Irreversible lesion occurs in progressive process (RPMS: relapsing progressive MS; SPMS: secondary progressive MS; PPMS: primary progressive MS)[ 3 l. Though inflammation decreases in progressive stage, lesions may cause severe neurodegenerative symptoms, finally develop disabilities of patients. Symptom Patients exhibit wide range of physical disabilities and mental problems. Different symptoms are correlated with their specific locations of CNS lesions. Visual loss, speech failure and motor failure are usually observed in MS patients. Fatigue is one of the most common symptom, the cause of fatigue is still remain unknown. Cognitive impairment increase with the affected time, during the progressive period of the disease. People observe MS patients' respond to cognitive training, combine fMRI technology with behavioral methods, as a diagnosis standard[ 4 l. 5 Epidemiology MS affects more than 2.5 million individuals worldwide and 400,000 in the US, this number is increasing yearly. Lives of patients are shortened by 5-10 years in average. [SJ There is about 5% concordance among dizygotic twins, about 20% - 30% concordance among monozygotic twins, which indicates an inherent correlation may be included. Pathology MS has been considered as an auto-immune disease. Th-17 cells, a subset of T cells, proliferate and enter through Blood Brain Barrier, attack glia cells and axonal tissues. Glia cells and neuron debris, include myelin, glycoprotein, is delivered out via Blood Brain Barrier, causing activation of normal T cells distributed in peripheral tissues. Those activated T cells who can recognize myelin and cell debris will again directly attack myelination sheath, result in progressive CNS impairment. B cells may be included in this process, too. A strong immune reflection will happen within CNS tissue, and results into severer gliosis and axonal loss. The whole processes formed a positive feedback cycle (Figure 1. ), finally lead to a progressive, chronic CNS deficiency. 6 Hereditary predisposition (genetic) ~ Environmental Factors (Sunshine; Vitamin D; Smoking; EBV) Pathology of MS 0. 0 0~0 Tcells o ~ Activated T cells . '\ \ Myelin Sf'\d neuronal debris nfigen Autoimmune Positive Feedback Cycl~~~ 1';:::============================::::;'~ !<3 I Figurel. MS immuno-pathology. T cells are activated by myelin and axonal debris, then go through blood brain barrier, strike the myelination, result in gliosis and axonal lost again, forming a positive feedback. Yellow arrows showed the direction of cycle, gray arrows showed exogenous MS causes. Therapies There is a clinical therapy which is now widely used on MS patients, called lmmunomodulation. IFN- 13 family, is one of widely used immunomodulation therapies. The cost of the therapies increase up to about 60,000 $ per year, from the originally about lOOOOper year. The number keeps increase annually by about 6 to 7%[ 6 1. Fingolimod (also known as FTY720, first oral drug for MS) is an FDA 7 approved effective MS drug. It is a sphingosine I-phosphate receptor modulator that blocks lymphocyte egress from lymph nodes and recirculation to the CNS, and it may have a direct effect on CNS. [ 7 l But those standard therapies can only decrease immune reactions, they can not rescue MS through promote remyelination. We know that CNS lesions begin with oligodendrocytes demyelination and axonal loss. To activate remyelination is nowadays the most promising therapy. Remyelination can happen naturally. Myelination and Remyelination Myelination One of the beginning effect of MS is demyelination of oligodendrocytes. Oligodendrocytes are glia cells live in between neurons, their arms form myelin sheath on axons, protecting them from being injured by mechanic force. Oligodendrocytes secrete growth factor and nutrition factors to support neuron living. Oligodendrocytes mature in a long period of time, from months to years. Oligodendrocytes progenitor cells (OPC) can also become neurons and astrocytes. Matured OLs began to form myelin sheath, this process plays important role on electrical signal insulation(saltatory conduction), mechanical protection, and roles in learning. 8 Remyelination Remyelination is the process in which entire myelin sheaths are restored to demyelinated axons, reinstating saltatory conduction and resolving functional deficits. [8] Remyelination happens naturally in CNS. In brain and spinal cord, about 10%-15% of the cell population are microglia. Microglia, resident macrophages in CNS, are sub group of glia cells, the main, active immune defense in CNS. Microglia play significant role on attenuating inflammation, preparing better living niche for OLs. OPCs distributed throughout CNS will migrate approaching to the lesion area, differentiate and form new myelin sheath onto the naked axons. Newly formed myelin sheath may be thinner than original myelination, this occurs in some axons, due to their larger diameter. Remyelination is usually less functional than original myelination, and there are several factors contribute to the failure of remyelination. In this case, to increase remyelination becomes an important target of MS therapy. Wnt-Ryk signaling Ryk is a transmemberane Wnt receptor belongs to Receptor-like tyrosine kinase (RTK) family[ 9 l. Ryk has important functions on neurogenesis, neurite outgrowth, and axon guidance[lOJ_ When Wnt signal appears, Ryk receptor is 9 cleaved by y -secretase. Ryk intracellular domain(Ryk-ICD) entered into cytosol and is stablized by Cdc37 and Hsp90 complex, then, Ryk-ICD is translocated into nucleus[ 11 l. Interaction with Cdc37 avoid Ryk ICD undergoing ubiquitination and proteaosme degradation (Figure 2. ). The mechanism that how ICD is translocated into nucleus is still remain unknown. Ryk signal is involved in Wnt3-induced neuronal differentiation in vitro and in vivo. Wnt-Ryk signaling promotes neurogenesis and reduces oligodendrogenesis[ 12 l. 10 y -secretase - Figure 2. Schematic Wnt-Ryk signaling pathway. Wnt3 ligand binding recruits y -secretase to cleave Ryk receptor, Ryk intracellular domain(Ryk-ICD) is then translocates into nucleus, with the stabilization by Cdc37/ Hsp90 complex. The mechanism that how Ryk-ICD is translocated into the nucleus is still remain unknown, the pinkish circle with question mark represents for a factor which might mediate this process. Our hypothesis is that blocking of Ryk signaling pathway may promote remyelination. We used three small inhibitor to inhibit y -secretase cleavage step and Hsp90 complex stabilizing step, in order to avoid Ryk-ICD t:ranslocation. The three inhibitor candidates we chose are 17-AAG; DAPf and Celast:rol. 11 Drugs profile 17AAG Geldanamycin derivative l 7-allylamino-17-demethoxygeldanamycin, or tanespimycin (17-AAG), which is the first Hsp90 inhibitor entered clinical trials, binds to a conserved pocket in the N-terminal ATP-binding domain of Hsp90, as a result inhibit Hsp90 complex activity. Hsp90 has been reported over expressed in various of cancer cells, 17-AAG as an Hsp90 inhibitor that have been using in cancer treatment, for example leukemia, kidney cancer, etc. [l 3 J 17-AAG is also reported to have functions on curing Alzheimer disease, due to its inhibition on Hsp9Q[ 14 l _ We used 17-AAG as an Hsp90 inhibitor to disrupt Cdc37 and Hsp90 complex, in order to block their stabilization towards Ryk-ICD. DAPT DAFT (GSI-IX) is a y -secretase inhibitor. [lSJ y -secretase is a protease responsible for Notch I activation. Inhibit y -secretase. DAFT It has been also reported DAFT can enhance both differentiation and myelination on oligodendrocytes. 12 Celastrol Celastrol is a quinone methide triterpene present in the root extract of Tripterygium wilfordii and celasturs regelii and has been used as anti-inflam matory drugs. Studies have identified several targets of celastrol which focus ed on the inhibition ofIKK/NF-kB pathway, important target for cancer ther apy. Celastrol can inhibit hsp90 and cdc37 interaction to inhibit IKK-NF-k B pathway. As a hsp90 inhibitor, celastrol also has the possibility to inhibit w nt-Ryk pathway and promote downstream effects including oligodendrogene sis . Besides, as MS is a kind of autoimmune disease, celastrol anti-inflamma tory effect may be more than helpful when being used on MS therapy. [16][17][18] Materials and Methods NSCs culture El 1.5 mouse Neural Stem Cells (NSC) are cultured in 60mm dish. Tissue culture dish is coated by Poly-Ornithine(Sigma) in 37'C overnight, then coated with fibronectin (LifeTech) for 2 hours. Briefly rinsed with distilled water, dishes can be used to culture neural stem cells. Stored in 4 'C, plates can last for up to four weeks. (Freshly coated dish is better). NSC culture medium is defined as DMEM-Fl2 with 1 X B27, basic fibroblast growth 13 factor (bFGF, Peprotech) is freshly added at a concentration of 25nM before use. Pre-heat the medium to 37'C is recommended, in order to keep NSCs in a undifferentiated stage. To pass the NSCs, dissociate cells with AccuMax (EMD Millipore), which is a gentle enzyme reagent used for cell counting. Dissociation time is less than 5 minutes, rinse with PBS to neutralize the enzyme. Transfer cell suspension into a 15ml centrifuge tube. Pipette the cell suspension gently is necessary to avoid differentiation. Spin down cells with 1 OOOrpm for 2 minutes at room temperature. Rep late the cells at about 10,000 cells per 60mm dish. Cells can be recognized as undifferentiated up to 10 passages, if extremely care is taken. Oligodendrocytes culture Oligodendrocytes are differentiated from Oligodendrocyte progenitors, already developed culture system contains thyroid hormones. A well-defined SATO medium contains DMEM with additives: GlutaMAX (1 % ), putrescine (16 1 g/ml; Sigma-Aldrich) , thyroxine ( 400 ng/ml; Sigma-Aldrich), triiodothyronine ( 400 ng/ml; Sigma-Aldrich), progesterone (6.2 ng/ ml; Sigma-Aldrich), sodium selenite (5 ng/ml; Sigma-Aldrich), bovine serum albumin (100 1 g/ml; Sigma-Aldrich), insulin (5 1 g/ml; Sigma-Aldrich), transferrin (50 1 g/ml; Sigma-Aldrich), B27 (2%), and primocin (100 mg/ml). [l 9 J 14 A simplified medium is developed by usmg N2 supplement directly instead of putrescine-HCl, progesterone, sodium selenite, insulin and human transferrin. Since the group who use SATO medium to differentiate OPCs into OLs obtained primary OPCs from CNS, they used primocin as a gentle antibiotic(low toxicity). We did not add Primocin. According to above, we used simplified SATO medium contains DMEM, B27 (50 X, Lifetech), N2 (lOOX, Lifetech), GlutaMax (lOOX, Lifetech), Triiodothyronine (T3), Thyroxine (T4). Different groups reported that they have used different growth factors to induce oiligodendrogenesis in vitro, for example, shh, PDGF, CNTF to induce oligodendrogenesis, from epiblast cells or ES cells. Our OPCs are acquired from NSCs, by PDGF-AA (Prototech) induction. [ 20 H 21 H 22 J Immunocytochemistry 12mm diameter micro slips (VWR) are placed into 24 well plates after sterilization. After Poly-ornithine coating over night and higher cone. Fibronectin coating, we recommend to seed cells within two days. Coating on glass surface will be weakened by longer storage. Dissociate NSCs from 60mm plate gently by Accumax (EMD Millipore), seed l.5xl0 4 NSCs per well in 24 well plate. Keep the cells overnight, allow them to attach to the 15 microslip surface. Induce oligodendrogenesis with PDGF for four days, then stop proliferation by retrieve growth factors, continuously differentiate OPCs till to Dayl2, harvest the cells by fixing them with 4% paraformyldehyde (PF A) for 10 minutes , rinse with PBS once. Permeabilize the cells by 0.3% Triton X-100 (in PBS) for lOmin, blocking with 10% Normal Goat Serum (NGS, LifeTech). Dissolve primary antibodies (rat MBP/ mouse MOG/ mouse Tujl, 1:500, Abeam) in 0.1 % Triton X-100 (in PBS). 4 'C incubate overnight. Rince with PEST ( 0.1 % Tween-20 in PBS) twice, incubate with secondary antibodies for 2hrs, room temperature. Rince twice with PEST (0.5% Triton X-100 in PBS), then incubate 20min with DAPI (1: 1000 in PBS). Rince with PEST once, then nnse with distilled water once, mount (mounting solution: Floromount-G, SouthemBiotech) the microslips onto microscope slides (VWR). Allow the mounting solution to dry for 20 minutes, then smear the edges of microslips with nail polish (Electron Microscopy Sciences). Air dry the slides for about 30 minutes, store them in 4 'C. 16 Imaging Imaging is performed by Zeiss Fluorescent Microscopy and Zeiss LSM5 Confocal Microscopy. (20 X magnification) Cell quantification Each treatment includes 4 biological replications (BR) , randomly choose 8 view scopes from each biological replication (each micro slip) under confocal microscopy. Quantify MBP (myelin positive marker) positive cells, normalize by amount of DAPI positive cells. Ratio is shown in percentage, values from the 4 BRs are presented as mean+ SD. Drugs preparation Ryk signaling inhibitor candidates 17-AAG, DAFT and Celastrol are dissolved in DMSO into 1 OOmg/mL as stock solution, then dilute them into lOOuM before use (DMSO and PBS). Before being used to culture cells, diluted solution has been added into medium prior to use. 17 Results Cell Culture Medium Condition Modification Cell culture meduim contains thyroiod hormone 1s reported having function to induce oligodendrogenesis. Neural stem cells are cultured in DMEM-Fl2 medium contained 1 XB27, 25 ng/ mL basic FGF (bFGF). Under simple differentiation condition in NSC culture mudium retrieved bFGF, cells began to die from day 4 to day 6. After T3 and T4 are added in medium, cells grows better after day 4, and they can differentiate into later stages, even to day 8. All cells seeded on microslip are counted to the same. Each well with microslip that coated with Poly-Omithine and Fibronectin is seeded with 1.5 X 10 4 cells. Immunostaining with DAPI shows a better growing situation in medium contained T3 and T4. Olig2 is a oligodendroglia positive marker, more Olig2 positive cells are observed in Ryk knockout cells compared to wild type cells. Nestin, which is a early stage neuronal marker is used to show NSCs, but it recognizes OPCs as well. (Figure 3) 18 No growth factor arlded E WT :::J -0 Q) E 0 (j) z Ryk-/- ~ I- (<)- !:::.. E RykWT :::J -0 Q) E Ol c (.) :::J -0 Ryk -/- c I 0 .Ql 0 DAPI Olig2 Nestin Figure 3. Wild type (V/T) and Ryk knockout(Ryk-/-) NSCs are cultured in two different mediums. NSC medium: contains Dtv:!EM/ Fl2 with 2% B27(50X); Oligo-inducing medium: NSC medium supplied with Triiodothyronine(T3), Thyroxine(T 4). OPC Cell Culture System Development Oligodendrocyte progenitor cells (OPCs) can be obtained from post natal mice, embryonic stem cells, embryo bodies, and neural stem cells. Currently most developed protocols are: enrich and purify rat OPCs by immunomagnetic separation and gradient centrifugation; induce pluripotent mouse epiblast stem cells (EpiSCs) into functional OPCs, etc. Oligodendrogenesis is induced by different combinations of growth factors. Cells become OPCs after 2 days induction, and exhibited dying phenomenon 19 when they were suddenly changed into a new medium which doesn't contain ant growth factors. So we recommend continuing culture in induction medium with PDGF for two additional days. A schematic combinations of growth factors is shown in Figure 4. Sonic hedgehog (shh) (100 ng/ml, Recombinant human shh, Peprotech) and platelet-derived growth factor (PDGF) (10 ng/ml; Recombinant human PDGF-AA, Peprotech) and Ciliary Neurotrophic Factor (CNTF) (10 ng/ml; Human, GenScript) are reported having induction function m oligodendrogenesis. When using NSCs to generate OPCs, the fewer passages(passage 3 to 5) NSCs have been passed, the better. Day 0 1 2 3 4 NSC OPC Approach 1 shh II PDGF Approach 2 shh/PDGF II PDGF Approach 3 PDGF II PDGF Approach 4 CNTF II PDGF Figure 4. Schematic view of different combinations of growth factors. There are four groups included: Approach 1: Day 1 and Day 2: shh(lOOng/ml), Day3 and Day4: PDGF (lOng/ml); Approach 2: Day 1 and Day 2: shh (lOOng/ml)/ PDGF (I Ong/ml), Day 3 and Day 4: PDGF (lOng/ml); Approach 3: Day 1 to Day 4: PDGF (I Ong/ml); Approach 4: Day 1 and Day 2: CNTF (I Ong/ml), Day 3 and Day 4: PDGF (I Ong/ml). 20 Since Olig 2 can be used to identify oligodendrocyte cell strains, but not a specific stage of OPC, we perf 01med immunostaining with PDGFR a antibody to identify positive OPCs. (Figure 5.) (A) Approach 1: shh WTNSCs Ryk -/- (B) Approach 2: shh+PDGF WTNSCs Ryk -/- (C) Approach 3: PDGF WTNSCs Ryk -/- (D) Approach 4: PDGF WTNSCs Ryk -/- 22 Figure 5. Immunostaining ofNSCs derived OPCs with PDGFR a antibody. In (A-D), NSCs are induced with four different combinations of growth factors. (A) Approach 1: Induced with shh (lOOng/ml) for Day 1 and Day 2, then continuously cultured in PDGF (lOng/ml); (B) Approach 2: Induced with shh (lOOng/ml) and PDGF (lOng/ml) for Day 1 and Day 2, then continuously cultured in PDGF (lOng/ml); (C) Approach 3 : Induced with PDGF (lOng/ml) for Day 1 and Day 2, then continuously cultured in PDGF (lOng/ml); (D) Approach 4: Induced with CNTF (lOng/ml) for Day 1 and Day 2, then continuously cultured in PDGF (lOng/ml). Fluorescent microscopy imaging is performed, at 20 X magnification. Quantification shows percentage of PDGFR a positive cells. (Figure 6). Two of the approaches (Approach 2&3) are the best. We decided to use PDGF to induce oligodendrogenesis thereafter. Cl) (.) + 100 Ctl 0::: 80 LL *** ** - WT 0 . D K . (!) 60 c • . * ' a.. 40 . 'lo- • 0 20 . ~ 0 0 ·- . . . . Appr 1 Appr 2 Appr 3 Appr 4 Figure 6. Quantification of the ratio of PDGFR a positive cells to DAPI positive cells, 23 showed in percentage. Appr l,Appr 2, Appr 3, Appr 4 stand for Approaches 1 to 4: the four combinations of growth factors described above. WT: wild type, KO: Ryk knockout. Percentage averaged from 4 biological replications is presented as mean+ SD. Asterisks (*: p<0.05, **: p<0.01, ***: p<0.001 ). Myelination of Oligodendrocytes Allow the cells grow continuously in a differentiate condition. The new medium is simplified from a developed recipe. 12mm Cover slips in 24 well plates are boated by same concentration of poly omithine but a higher concentration of fibronectin. Also, the time coating febronectin is extended to 4 hours. In order to keep the cells alive for a longer period of time, a thicker layer of febronectin is necessary. NS Cs are seeded at a concentration of 1.5 X 10 4 cells per well. Induce the cells with PDGF for 4 days. After four days induction, we continued culturing the cells to Day 12, in simplified SATO medium. Immunostained cells with Myelin basic protein(MBP) antibodies and Tuj 1 ( J3 III- tubulin ) antibody. Cells were fixed by 4% PFA at day 12, rinsed with PBS once. Permeablize cells with 0.3% Triton X-100 for 10 minutes, rinsed with PBS again, then blocking by 10% normal goat serum for 1 hour. After rinsed with PBS, primary antibodies are added in 0.1 % Triton X-100 buffer, placed the in wet 24 chamber over night at 4 °C. Wash the wells once with PBS once, incubated the cells in secondary fluorescent conjugated antibodies (dissolved in O.lo/o Triton X-100) for two hours, at room temperature. After 20 minutes DAPI staining, mounted the slips and performed imaging by confocal microscopy (20 X ). Results showed MBP positive cells ratio is different in different inducing conditions using different growth factors, while in each group, Ryk knockout cells have larger MBP positive ratio, compared to wild type cells. (Figure 7) (A) Approach 1: shh WT . . '; Ryk-/- (B) Approach 2: shh+PDGF WT Ryk-/- (C) Approach 3: PDGF WT Ryk-/- (D) Approach 4: CNTF 26 MBP positive cell percentage quantification 6 * - WT ti) D KO - 5 - Cl> (.) 4 + a.. *** * al3 ~ ..... 2 0 ~ 1 0 Appr 1 Appr 2 Appr 3 Appr 4 Figure 7. (A-D): Irnrnunocytochernistry on Day 12 of oligodendrocytes culture, using four different corn bination of growth factors, described above. (Green: l\.1BP, Red: Tuj 1, Blue: DAPI.) (A) uses growth factors corn bination approach 1: shh. (B) uses growth factors combination approach 2: shh, PDGF. (C) uses growth factors combination approach 3: PDGF. (D) uses growth factors combination approach 4: CNTF. WT: wild type, Ryk-/-: Ryk knockout. White arrows point at the nucleus positions of l\.1BP positive cells. Magnification: 20 X , scale bars: 20 µ m. CE) Quantification of the ratio of oligodendrocytes (l\.1BP positive) among DAPI stained cells(as whole cells number), showed in percentage. Apprl,Appr2, Appr3, Appr4 stand for Approaches} to 4: the four combinations of growth factors described above. WT: wild type, KO: Ryk knockout. Percentage is showed with mean+ S.E. Asterisks(*: p<0.05, **: p<0.01, ***: p<0.001 ). 27 Quantification shows different MBP positive percentage, in each approach, there is a higher percentage in Ryk knockout cells than in wild type cells. Quantification is confirmed by Anti-Myelin Oligodendrocyte Glycoprotein (mouse MOG, 1 :500, EMD millipore) antibody staining, similar data is acquired. (Figure 8.) (A) Approach 1 : shh (B) Approach 2: shh,PDGF (C) Approach 3: PDGF (D) Approach 4: CNTF l- s 28 (E) MOG positive cell percentage quantification Figure 8. 6 -5 Cl> 0 + 4 83 ~2 0 ~ 1 0 ** * ** - WT D KO * Appr 1 Appr 2 Appr 3 Appr 4 (A-D): Immunocytochemistry on Day 12 of oligodendrocytes culture, using four different combination of growth factors, described above. (Red: MOG, Green: MAP2, Blue: DAPI.) (A) uses growth factors combination approach 1: shh. (B) uses growth factors combination approach 2: shh, PDGF. (C) uses growth factors combination approach 3: PDGF. (D) uses growth factors combination approach 4: CNTF. WT: wild type, Ryk-/-: Ryk knockout. White arrows point at the nucleus of MOG positive cells. Magnification: 20 X , scale bars: 20 µ m. (E) Quantification of the ratio of oligodendrocytes among DAPI stained cells(as whole cells nurn ber), showed in percentage of MOG positive cells. Apprl,Appr2, Appr3, Appr4 stand for Approaches I to 4: the four combinations of growth factors described above. WT: wild type, KO: Ryk knockout. Percentage is showed with mean+ S.E. Asterisks(*: p<0.05, **: p<0.01, ***: p<0.001) 29 Ryk Inhibitor Candidates Test We use this in vitro culture system to test three Ryk signaling inhibitors. DAPT is y -secretase inhibitor, while 17-AAG and Celastrol inhibits Hsp90 activity. Treatment last for 8 days on OPCs. Immunostaining with J:vffiP shows all of the three inhibitors can promote myelination in this in vitro system. Quantification is performed as the same way mentioned above. (Figure 9.) (Al) 17-AAG in WT OPC 0 <DMSO) 0.03 0.10 0.33 1.0 30 (A2) 2.5 rJ) *** - 2.0 * - ** Q) * (,) + 1.5 Q. m 1.0 :a: It- 0 .5 ~ 0 0.0 0.00 0.03 0.10 0.33 1.00 17-AAG (µM) (Bl) DAPT in WT OPC 0 ( DMSO) 0.03 0.10 0.33 1.0 31 (B2) 1.8 * ti) 1.6 * - - 1.4 <D (.) 1.2 + a.. 1.0 m .8 :E .6 ..... 0 .4 ~ .2 0 0.0 0.00 0.03 0.10 0.33 1.00 DAPT(µM) (Cl) Celastrol in WT OPC 0 <DMSO) 0.03 0.10 0.33 1.0 32 (C2) 3.0 (/) 2.5 *** Q) *** 0 2.0 + a.. 1.5 ca :::!: 1.0 ~ 0 ~ 0 .5 0.0 0.00 0.03 0.10 0.33 1.00 Celastrol (µM) Figure 9. (Al), (Bl), (Cl): Immunostaining of cells (17-AAG, DAPT, Celastrol treaments) that are thought to disrupt Ryk signaling pathway. Cells are cultured in the differentiation culture system described above, inducing step used PDGF. (A2), (B2), (C2): percentage of MBP positive cells. Percentage is showed with mean ± SD. Asterisks (*: p<0.05, **: p<0.01, ***: p<0.001) Conclusion 1. Medium containing T3 and T 4 hormone can induce oligodendrogenesis. Cells during early differentiation grow healthily in this medium. 2. According to the quantification results, we recommend to use shh/ PDGF or only PDGF to induce oligodendrogenesis. Significant increase of PDGFR 33 a positive cells can be observed in these groups. There is a significant difference between wild type and Ryk knockout cells, this result corresponded with previous data, showing that Ryk knockout can promote oligodendrogenesis. 3. CNTF can induce larger number of oligodendrocytes, but cells are not as widely spanned as other groups treated with other growth factors. This maybe because of rapid proliferation in early differentiation period. 4. A higher percentage of MBP positive cells is observed in Ryk knockout cells compared to wild type cells. Wild type cells tend to have better myelination phenotype, while Ryk knockout cells always grow into smaller shape. 5. 17-AAG, DAFT and Celastrol significantly promote myelination. Quantification results might be problematic in groups contains high concentration of inhibitors (for example, 1.0 µ M 17-AAG), due to the cell death. 34 Discussion We provided a convenient in vitro cell culture system to test myelination. Current in vitro myelination systems are mainly established by coculture methods[ 23 H 24 l_ Oligodendrocytes are differentiated from primary derived OPCs, and these OPCs are cocultured with a layer of axons generated by DRG (dorsal root ganglion cells) [ 25 lor RGC (retinal glial cells)[ 26 l. OPCs as well as DRGs or RGCs are purified by immunopanning. Cells dissociated from tissues are cultured on antibody-coated plates for weeks. The challenges are, several different antibodies are used in these protocols, and a long term purification period may lead to OPC differentiation. Some other groups also purify OPCs using gradient centrifuge, however, an antibody-associated method is still necessary after gradient centrifuge. People also use FACS sorting[ 27 l, but FACS sorting may result in mechanical injury to OPCs, this may lead to differentiation of OPCs. Recently, a new culture system substitute axons with engineered nanofibers. Oligodendrocytes wrap around those fibers, this system can be used in a high-throughput drug screening[ 28 l. This in vitro culture system supplies a high efficiency drug testing platform on myelination level. However, our protocol has following advantages. First, NSCs are easier to obtain and culture, this provides a robust source for OPC inducing. Second, to measure myelination status, we need a coculture system because myelination needs 35 presence of axons. Our differentiation system has different populations of neurons, those neurons provided axons needed by myelination. Third, our methods is cheaper and faster. Using a culture condition which is suitable for oligodendrocytes growth, we obtained MBP/ MOG positive cells from about day 10 to day 12, this period is shorter than most of in vitro culture system. According to above, we believed this system can be used as MS in vitro culture model, and is convenient for drug screening. Our results show that 17-AAG, DAFT and Celastrol can promote myelination. This indicates that inhibit Ryk signaling with small inhibitors do have function on increasing myelination. To examine whether these inhibitors can rescue demyelination via promoting remyelination, further studies in vivo need to be proved. T-cell dependent, cuprizone induced MS model or myelin proteolipid protein (PLP) induced EAE (experimental autoimmune encephalomyelitis) model are widely used as MS model. [ 29 l We plan to use cuprizone induced MS model to test drug candidates mentioned above. Symptoms might be attenuated after treat EAE mouse model with Ryk inhibitors, we will confirm it by brain section and immunohistochemistry. To make sure whether these drugs influence myelination through Wnt-Ryk signaling pathway, we are going to test the three candidates in Ryk knockout cells as well, using myelination culture 36 system described above. We also proposed that an exogenous Ryk extracellular domain (Ryk-ECD) might have a competitive function against Wnt signal. This may be another possible method to inhibit Wnt-Ryk signaling, and may results in rescue of MS. A Ryk-ECD-Fc fusion protein mammalian expression system will be established. Purified protein will be used to treat OPCs via same approaches using above. In the future, remyelination may become a therapeutic target of MS. Immunopathogenesis of MS is complex. It includes T-cells as well as B-cells, and their affected region is not restricted in white matter, but gray matter as well. The etiology of MS is remain unknown, but it is likely a combination of genetic, epigenetic and environmental factors plays the main role. Immunomodulation is now widely used in MS therapies, but it can only suppress the immune reaction. To rescue multiple sclerosis, the basic problem should be solved is how to induce remyelination in situ and restore the functions of CNS. Recently, stem cell based transplantation seems to become one of the most promising new therapies, since there is no FDA approved drugs having a specific effect on remyelination. Immunotherapy can limit the velocity of MS progression, however, it is not helpful on 37 rescuing old lesions. Therapies targeting remyelination might be a effective method to rescue MS lesions. Together with immunotherapies, remyelination-based therapies may lead to a novel direction on curing MS. References [ 1 ] Chwastiak L1, Ehde DM, Gibbons LE, Sullivan M, Bowen JD, Kraft GH. Depressive symptoms and severity of illness in multiple sclerosis: epidemiologic study of a largecommunity sample. Am J Psychiatry. 2002 Nov;l59(11):1862-8. [ 2] Ontaneda D, Hyland M, Cohen JA. Multiple sclerosis: new insights m pathogenesis and novel therapeutics.Annu Rev Med. 2012;63:389-404. [3] Hans Lassmann, Jack van Horssen and Don Mahad, Progressive multiple sclerosis: pathology and pathogenesis, Nat. Rev. Neurol. 8, 647 - 656 (2012). [ 4] Hubacher M 1 • Kappos L 2 , Weier K 2 , Stiicklin Ml, Opwis K 1 , Penner IK 1 , Case-Based fMRI Analysis after Cognitive Rehabilitation in MS: A Novel Approach. Front Neurol. 2015 Apr 8;6:78. [ 5] Ontaneda D 1 , Di Capua D 2 . Benefits versus risks of latest therapies in multiple sclerosis: a perspective review. Tuer Adv Drug Saf. 2012 Dec;3(6):291-303. [6] Hartung DM 1 , Bourdette DN 2 , Ahmed SM 2 , Whitham RH 2 , The cost of multiple sclerosis drugs in the US and the pharmaceutical industry: Too big to fail? Neurology. 2015 Apr 24. [7] Brinkmann V. 2009. FTY720 (fingolimod) in multiple sclerosis: therapeutic effects in the immune and the central nervous system. Br. J. Pharmacol. 158:1173 - 82 [8] Robin J. M. Franklin 1 & Charles ffrench-Constant2, Remyelination in the CNS: from biology to therapy, Nature Reviews Neuroscience 9, 839-855 (November 2008). [9] Lu W 1 , Yamamoto V, Ortega B, Baltimore D., Mammalian Ryk is a Wnt coreceptor required for stimulation ofneurite outgrowth. Cell. 2004 Oct 1;119(1):97-108. [ 10] Lyu Junmook 1 , Yamamoto Vicky 1 , Lu Wange. Cleavage of the Wnt receptor Ryk regulates neuronal differentiation during cortical neurogenesis. Dev Cell. 2008 Nov;l5(5):773-80. [11] Lyu J 1 , Wesselschmidt RL, Lu W. Cdc37 regulates Ryk signaling by stabilizing the cleaved Ryk intracellular domain. J Biol Chem. 2009 May 8;284(19): 12940-8. [ 12] Zhong Jiangyang 1 , Kim HT, Lyu J, Yoshikawa K, Nakafuku M, Lu Wange. The Wnt receptor Ryk controls specification of GABAergic neurons versus oligodendroc 38 ytes duringtelencephalon development. Development. 2011 Feb;l38(3):409-19. [ 13 ] Meletios-Athanassios Dimopoulosa, Constantine S. Mitsiadesb, Kenneth C. Andersonb, Paul G Richardsonb Tanespimycin as Antitumor Therapy Clinical Lymphoma Myeloma and Leukemia Volume 11, Issue 1, February 2011, Pages 17 - 22 [14] Laura Ortega a.b, Minerva Calvillo c, Felix Luna ", Francisca Perez-Severiano ct, Moises Rubio-Osornio', Jorge Guevara b, Ilhuicamina Daniel Limon", 17-AAG improves cognitive process and increases heat shock protein response in a model lesion with A J3 25 - 35. Neuropeptides 48 (2014) 221 - 232 [15] Okuhashi Y 1 , Nara N, Tohda S. Effects of gamma-secretase inhibitors on the growth ofleukemia cells. Anticancer Res. 2010 Feb;30(2):495-8. [16] Salminen A 1 , Lehtonen M, Paimela T, Kaarniranta K. Celastrol: Molecular targets of Thunder God Vine. Biochem Biophys Res Commun. 2010 Apr 9;394(3):439-42. [17] Zhang T 1 , Li Y, Yu Y, Zou P, Jiang Y, Sun D. Characterization of celastrol to inhibit hsp90 and cdc3 7 interaction. J Biol Chem. 2009 Dec 18;284(51):35381-9. [ 18 ] Allison AC 1 , Cacabelos R, Lombardi VR, Alvarez XA, Vigo C. Celastrol, a potent antioxidant and anti-inflammatory drug, as a possible treatment for Alzheimer's disease. Prog Neuropsychopharmacol Biol Psychiatry. 2001 Oct;25(7): 1341-57. [ 19] Sher F 1 , Rossler R, Brouwer N, Balasubrarnaniyan V, Boddeke E, Copray S.Differentiation of neural stem cells into oligodendrocytes: involvement of the polycomb group protein Ezh2 Stem Cells. 2008 Nov;26(11):2875-83. [ 20] Hesp ZC 1 , Goldstein EA 1 , Miranda CJ 2 , Kaspar BK 3 , McTigue DM 4 .Chronic oligodendrogenesis and remyelination after spinal cord injury in mice and rats. JNeurosci. 2015 Jan 21;35(3):1274-90. [21] Misumi S 1 , Nishigaki R, Ueda Y, Watanabe Y, Shimizu Y, Ishida A, Jung C, Hida H.Differentiation of oligodendrocytes from mouse induced pluripotent stem cells without serum. Transl Stroke Res. 2013 Apr;4(2): 149-57. [22] Tanaka T, Murakami K, Bando Y, Yoshida S.Minocycline reduces remyelination by suppressing ciliary neurotrophic factor expression after cuprizone-induced demyelination.J Neurochem. 2013 Oct; 127(2):259-70. [23] Watkins TA, Scholze AR.Myelinating cocultures of purified oligodendrocyte lineage cells and retinal ganglion cells.Cold Spring Harb Protoc. 2014 Oct 1;2014( 10):pdb.top070839. [24] Watkins TA, Scholze AR.Myelinating cocultures of rat retinal ganglion cell 39 reaggregates and optic nerve oligodendrocyte precursor cells.Cold Spring Harb Protoc. 2014 Oct 1;2014(10):pdb.prot074971. [25] Zuchero JB.Purification and culture of dorsal root ganglion neurons.Cold Spring Harb Protoc. 2014 Aug 1;2014(8):813-4. 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Abstract (if available)

Abstract Multiple Sclerosis (MS) is a CNS degenerative disease. It is one of the most common chronic disabling CNS disease in young adults. In MS, oligodendrocytes(OLs) loss is the most important character. MS is also recognized as an autoimmune disease. Recent therapies are mainly focusing on immunomodulation and remyelination. Remyelination happens naturally, however it can be avoided in CNS. We selected three small inhibitors working towards Ryk signaling pathway, in order to stimulate remyelination. However, drug screening for MS has been strongly obstructed due to limited resources of OLs as well as a proper cell culture system. So we developed a method to differentiate neural stem cells (NSCs) into matured OLs, and we also developed an in vitro OLs culture system. Using this system, we tested three drug candidates (17-AAG, DAPT and Celastrol) for gradient concentration. Results showed the amount and ratio of OLs significantly increased. Immunomodulation is a method that can only reduce the velocity of disease progression, they does not have effects on restoring existed lesions. Remyelination using oral drugs seems to be a promising future of MS therapy, while stem cell based techniques are still far away.

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Asset Metadata

Creator Zhang, Naibo (author)

Core Title Targeting Ryk-signaling to attenuate multiple sclerosis by promoting oligodendrocyte differentiation and myelination

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/30/2015

Defense Date 05/28/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag multiple sclerosis,OAI-PMH Harvest,oligodendrocytes,remyelination

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lu, Wange (committee chair), Tokes, Zoltan A. (committee member), Ying, Qi-Long (committee member)

Creator Email naibozha@usc.edu,neil.nag.zhang@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-615832

Unique identifier UC11303994

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Legacy Identifier etd-ZhangNaibo-3756.pdf

Dmrecord 615832

Document Type Thesis

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Rights Zhang, Naibo

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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