CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC MOUSE MODEL FOR FRAGILE X-ASSOCIATED

TREMOR/ATAXIA SYNDROME (FXTAS)

THESIS

A thesis submitted for the degree of Master of Biomedical Science majoring on Genetic Counseling

by FATWA ADIKUSUMA

G4A008039

POST GRADUATE PROGRAM DIPONEGORO UNIVERSITY

SEMARANG 2011

CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC MOUSE MODEL FOR FRAGILE X-ASSOCIATED

TREMOR/ATAXIA SYNDROME (FXTAS)

THESIS

A thesis submitted for the degree of Master of Biomedical Science majoring on Genetic Counseling

by FATWA ADIKUSUMA

G4A008039

POST GRADUATE PROGRAM DIPONEGORO UNIVERSITY

SEMARANG 2011

ii

THESIS

CHARACTERIZATION OF AN INDUCIBLE TRANSGENIC MOUSE MODEL FOR FRAGILE X-ASSOCIATED

TREMOR/ATAXIA SYNDROME (FXTAS)

By FATWA ADIKUSUMA

G4A008039

Has been defended in front of the defense committee On April 13th, 2011

and has been approved by:

The Netherland Indonesia Principal Supervisor, Principal Supervisor, Renate Hukema, PhD Prof. Sultana MH Faradz, PhD NIP. 130 701 415 Supervisor, Supervisor, Rob Willemsen, PhD Prof. DR. dr. Sarjadi, Sp.PA (K) NIP. 130 352 547

Recognition, Science

Post Graduate Program Diponegoro University

DR. dr. Winarto Sp.MK, SpM(K) NIP. 130 675 157

iv

DECLARATION

I hereby declare that this submission is my own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by

another person nor material which to a substantial extent has been accepted for the

award of any other degree or diploma of the university or other institute of higher

learning, except where due acknowledgement is made in the text.

Semarang, April 2011

Fatwa Adikusuma

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ACKNOWLEDGMENT

Thanks God for Your destiny to give me an amazing study experience in

this program, Genetic Counseling Biomedical Science Diponegoro University. In

this program many people and parties have contributed a lot for my study. I would

like to thank to them.

I would like to express my gratitude to my supervisors: Renate Hukema,

PhD, Rob Willemsen, PhD, Professor Sultana MH Faradz MD, PhD, and

Professor Dr. dr. Sarjadi, SpPA (K) for the supervision, advices and support.

My special thanks to Professor dr. Sultana M.H. Faradz, PhD. Head of

Master Biomedical Science Majoring on Genetic Counseling Diponegoro

University Semarang for making this study happen and possible. Every single step

of this study has your contribution. Thank you for opening this program,

encouraging us to have better knowledge and experience, for your care to us, for

finding the scholarship, for supervising me, for everything tha

entirely. dr. Tri Indah Winarni, MSi.Med for

her support to this program as well.

I also thank to Dr. Abe Susanto, MSc from from Bureau of Planning and

International Cooperation, Ministry of National Education The republic Indonesia,

who

I would like to thanks to Professor dr. Ben Oostra PhD and Rob

Willemsen PhD, from Department of Clinical Genetics, Erasmus MC Rotterdam,

The Netherlands, for making it possible to study and experience an advanced

research at their laboratory.

vi

I would like to thank to colleagues in Fragile X group Erasmus MC who

gave many contributions whilst I was in the laboratory. Thanks to Edwin Mientjes

PhD, Liesanne and Ronald Buijsen.

I would like to extend my acknowledgement to dr. Endang

Ambarwati,Sp.RM, dean of the Faculty of Medicine Diponegoro University

Semarang, and also to

Program in Biomedical Science Post Graduate Program Diponegoro University

Semarang, Faculty of Medicine, Diponegoro University Semarang.

Last but not least, I would like to thank to my family and many people

who contribute in supporting me to finish this study.

Semarang, April 2011

Fatwa Adikusuma

vii

LIST OF ABBREVIATIONS

ASO : antisense oligonucleotides

(CGG)n : CGG with (n) number of repeat

C57BL/6 : a type of mouse strain which is widely used for

laboratory mouse

COS-7 : CV-1 (simian) in Origin, and carrying the SV40

genetic material line 7

DM : myotonic dystrophy

DMPK : myotonic dystrophiy protein kinase

dox : doxycyline

eGFP : enhanced green fluorescent protein

FISH : fluorescence in situ hybridization

Fmr1 : fragile x mental retardation 1 (for animal)

FMR1 : fragile x mental retardation 1 (for human)

Fmrp : fragile x mental retardation protein (for animal)

FMRP : fragile x mental retardation protein (for human)

FXTAS : fragile x-associated tremor/ataxia syndrome

Gapdh : glyceraldehyde 3-phosphate dehydrogenase

GFA : glial fibrillary acidic

GFP : green fluorescent protein

GLAST : glutamate aspartate transporters

hnRNP : Heterogeneous nuclear ribonucleoproteins

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Hsp : heat shock protein

IP : intraperitoneal

KI : knock-in

MBNL1 : muscleblind-like 1

MCP : middle cerebellar peduncle

MRI : magnetic resonance imaging

Pcp : Purkinje cell protein

POF : premature ovarian failure

PrP : prion protein

RT-Q-PCR : reverse transcriptase-quantitative-polymerase

chain reaction

rtTA : reverse-tetracycline transactivator

SCA : spinocerebellar ataxia

Tet : tetracycline

UTR : untranslated region

ZNF9 : zinc finger protein 9

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TABLE OF CONTENTS

DECLARATION ................................................................................................................iv

ACKNOWLEDGMENT ..................................................................................................... v

LIST OF ABBREVIATIONS ............................................................................................ vii

TABLE OF CONTENTS .................................................................................................... ix

LIST OF TABLE, FIGURES, AND APPENDICES .......................................................... xi

ABSTRACT ...................................................................................................................... xiii

ABSTRAK ........................................................................................................................ xiv

CHAPTER 1

INTRODUCTION .............................................................................................................. 1

1.1. BACKGROUND ..................................................................................................... 1

1.2. RESEARCH QUESTIONS...................................................................................... 5

1.3. RESEARCH PURPOSES ........................................................................................ 6

1.4. RESEARCH BENEFITS ......................................................................................... 7

1.5. RESEARCH ORIGINALITY .................................................................................. 7

CHAPTER 2

LITERATURE REVIEW ................................................................................................... 8

2.1. FXTAS: THE FMR1 GENE AND CLINICAL ASPECTS ..................................... 8

2.2. NEUROPATHOLOGICAL OF FXTAS ............................................................... 11

2.3. MOLECULAR PATHOGENESIS OF FXTAS .................................................... 14

2.4. MOUSE MODEL OF FXTAS ............................................................................... 17

CHAPTER 3

THEORITICAL FRAMEWORK, CONCEPTUAL FRAMEWORK, AND HYPOTHESES ................................................................................................................. 20

3.1. THEORITICAL FRAMEWORK .......................................................................... 20

3.2. CONCEPTUAL FRAMEWORK .......................................................................... 21

3.3. HYPOTHESES ...................................................................................................... 22

CHAPTER 4

RESEARCH METHODS ................................................................................................. 23

4.1. RESEARCH FIELD .............................................................................................. 23

4.2. SETTING/LOCATION, RESEARCH PERIOD ................................................... 23

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4.3. RESEARCH DESIGN ........................................................................................... 23

4.4. POPULATIONS AND SAMPLES ........................................................................ 23

4.5. RESEARCH VARIABLES ................................................................................... 24

4.6. OPERATIONAL DEFINITIONS .......................................................................... 25

4.7. RESEARCH PROTOCOLS .................................................................................. 27

4.8. FRAMEWORK OF RESEARCH .......................................................................... 36

4.9. DATA ANALYSIS ................................................................................................ 44

4.10. ETHICAL IMPLICATION.................................................................................. 44

CHAPTER 5

RESULTS ......................................................................................................................... 45

5.1. Previous study: cell culture and organotypical slices ............................................ 45

5.2. The workable of Tet-on-nCGG-eGFP: in vivo study using Tet-on-nCGG-eGFP/hnRNP-rtTA ....................................................................................................... 45

5.3. The workable of Tet-on-99CGG-eGFP/GFA2-rtTA in vivo ................................. 48

5.4. The workable of Tet-on-99CGG-eGFP/PrP-rtTA in vivo ...................................... 48

5.5. Transgene expression outside the brain in Tet-on-99CGG-eGFP/PrP-rtTA bigenic mice ............................................................................................................................... 50

5.6. Best founder of PrP-rtTA ....................................................................................... 53

5.7. Best founder of Tet-on-99CGG-eGFP ................................................................... 54

5.8. Formation of ubiquitin intranuclear inclusion in the Tet-on-99CGG-eGFP/PrP-rtTA bigenic mice ......................................................................................................... 57

CHAPTER 6

DISCUSSION ................................................................................................................... 62

6.1. Leakage of Tet-on-99CGG-eGFP/PrP-rtTA .......................................................... 63

6.2. Toxic RNA and inclusions ..................................................................................... 64

6.3. Reversibility study ................................................................................................. 66

6.4. Astrocytes and glutamate excitotoxicity involvement for disease progression ..... 67

CHAPTER 7

CONCLUSION AND SUGGESTION ............................................................................. 72

REFERENCES ................................................................................................................. 73

CURRICULUM VITAE ................................................................................................... 81

APPENDICES .................................................................................................................. 85

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LIST OF TABLE, FIGURES, AND APPENDICES

Tables

Table 1. Proteins found in the human brain inclusions

Figures

Figure 1. CGG length, FMR1 expression and the clinical outcomes

Figure 2. Ubiquitin-positive intranuclear inclusions in human neurons, human astrocytes, murine neurons

Figure 3. Immunohistochemistry for GFP in liver, kidney, and intestine of bigenic mice Tet-on-nCGG-eGFP/hnRNP-rtTA with dox treatment

Figure 4. Immunohistochemistry for GFP in brain slices of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA after 2 weeks of dox in drinking treatment

Figure 5. Immunohistochemistry for GFP in tissues outside the brain of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA after dox intraperitoneal injection

Figure 6. Western blot for GFP on kidney of tet-on-99CGG-eGFP/PrP-rtTA mouse after dox IP treatment

Figure 7. RT-Q-PCR for GFP RNA from kidney of tet-on-99CGG-eGFP/PrP-rtTA mouse after dox IP treatment

Figure 8. rtTA mRNA levels from the brains of different PrP-rtTA founder

Figure 9. Immunohistochemistry for GFP in the liver slices comparing founder 31 and 39 of Tet-on-99CGG-eGFP

Figure 10. GFP RNA levels in the liver comparing founder 31 and 39 of Tet-on-99CGG-eGFP

Figure 11. Western blot analysis for GFP in the liver comparing founder 31 and 39 of Tet-on-99CGG-eGFP

Figure 12. Immunohistochemistry for GFP in the brain slices of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA after dox treatment

xii

Figure 13. GFP RNA levels in the brain of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA after dox treatments

Figure 14. Western blot for GFP of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA after different time points of dox treatment

Figure 15. Immunohistochemistry of ubiquitin in the brain slices of bigenic mice Tet-on-99CGG-eGFP/PrP-rtTA with 12 and 16 weeks of dox treatment

Appendices

Appendix 1. rtTA RT-Q-PCR from brain result for choosing the best PrP-rtTA founder

Appendix 2. Western blot for GFP in liver of mice from founder 39 vs 31 of Tet-

on-99CGG-eGFP

Appendix 3. RT-Q-PCR for GFP in liver of mice from founder 39 vs 31 of Tet-on-99CGG-eGFPA

Appendix 4. RT-Q-PCR for GFP after different time points of dox treatment

Appendix 5. Western blot for GFP after different time points of dox treatment

Appendix 6. Ethical clearance document

xiii

ABSTRACT

Background: FXTAS (fragile X-associated tremor/ataxia syndrome) is a neurodegenerative disease with tremor and ataxia as the primary symptoms and ubiquitin-positive intranuclear inclusions as the major neuropathological hallmark. FXTAS affects premutation males with an expanded premutation CGG repeat in the promoter of the FMR1 gene. The expanded premutation CGG RNA is considered toxic. Inducible transgenic mice were generated, using the Tet-on system to express transgene expanded premutation 99CGG along with an eGFP reporter under control of dox (doxycycline) induction, as well as specific driver promoter, the GFA2-rtTA and PrP-rtTA. This study aims to characterize those mouse models so they would be ready to be used for further studies.

Methods: Immunohistochemistry, Western blot, and RT-Q-PCR techniques were used to characterize the transgene expression of the transgenic mice. Characterization of ubiquitin inclusions formation used immunohistochemistry technique.

Results: The Tet-on-99CGG-eGFP transgene worked in vivo, while Tet-on-11CGG-eGFP lost its expression. Tet-on-99CGG-eGFP/GFA2-rtTA bigenic mice did not work in vivo, while Tet-on-99CGG-eGFP/PrP-rtTA bigenic mice did work. As expected, the Tet-on-99CGG-eGFP/PrP-rtTA mice formed the FXTAS hallmark, the ubiquitin-positive intranuclear inclusions after 12 weeks of dox induction.

Conclusions: Mouse model Tet-on-99CGG-eGFP/PrP-rtTA worked well, and were ready to be used for main further studies.

Keywords: FXTAS, FMR1 gene, transgenic mice, Tet-on, PrP, GFA2, reversibility, ubiquitin inclusions

xiv

ABSTRAK

Latar belakang: FXTAS (fragile X-associated tremor/ataxia syndrome) merupakan penyakit neurodegeneratif dengan gejala utama tremor dan ataxia serta inklusi intranuklear ubiquitin yang muncul sebagai ciri-ciri utama secara neuropatologikal. FXTAS menyerang pria premutasi yang mengalami ekspansi repetasi CGG di promotor gen FMR1. RNA CGG premutasi dipercaya toksik. Tikus transgenik yang dapat diinduksi sudah dibuat, dengan menggunakan sistem Tet-on untuk mengekspresikan transgen 99CGG terekspansi bersama-sama dengan reporter eGFP, serta dapat dikontrol melalui induksi dox (doxycycline), juga menggunakan promotor pengendali yang spesifik yaitu GFA2-rtTA dan PrP-rtTA. Studi ini bertujuan untuk mengkarakterisasi tikus tersebut sehingga model tikus tersebut siap untuk digunakan untuk studi-studi berikutnya.

Metode: Teknik imunohistokimia, Western blot, dan RT-Q-PCR digunakan untuk mengkarakterisasi ekspresi transgene pada tikus transgenik. Karakterisasi inklusi ubiquitin menggunakan teknik imunohistokimia.

Hasil: Transgen Tet-on-99CGG-eGFP dapat bekerja secara in vivo, tetapi Tet-on-11CGG-eGFP kehilangan ekspresi transgennya. Tikus bigenik Tet-on-99CGG-eGFP/GFA2-rtTA tidak bekerja, sedangkan tikus bigenik Tet-on-99CGG-eGFP/PrP-rtTA dapat bekerja dengan baik. Sesuai harapan, tikus bigenik Tet-on-99CGG-eGFP/PrP-rtTA dapat menghasilkan inklusi intranuklear ubiquitin setelah induksi dox selama 12 minggu.

Kesimpulan: Model tikus Tet-on-99CGG-eGFP/PrP-rtTA dapat bekerja dengan baik dan siap untuk digunakan untuk studi penting berikutnya

Kata kunci: FXTAS, gen FMR1, tikus transgenik, Tet-on, PrP, GFA2, reversibilitas, inklusi ubiquitin