© 2009 Mukarram S El-Banna ALL RIGHTS RESERVED Mouse Models for the Neurodegenerative Lysosomal Storage Diseases Niemann- Pick Types C1 & C2 and Classical Late Infantile Neuronal Ceroid Lipofuscinosis. by Mukarram S. EL-Banna A thesis submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Masters of Science Graduate Program in Microbiology and Molecular Genetics and The Graduate School of Biomedical Sciences University of Medicine and Dentistry of New Jersey written under the direction of Dr. Peter Lobel and approved by ________________________ ________________________ ________________________ New Brunswick, New Jersey May, 2009 ABSTRACT OF THE THESIS Mouse Models for the Neurodegenerative Lysosomal Storage Diseases Niemann- Pick Types C1 & C2 and Classical Late Infantile Neuronal Ceroid Lipofuscinosis. By MUKARRAM S. EL-BANNA Dissertation Director: Dr. Peter Lobel Use of mutant mice provides us with an excellent tool for investigation of lysosomal diseases such as classical late infantile neuronal ceroid lipofuscinosis (cLINCL) and Niemann-Pick types C1 and C2 (NPC1 and NPC2). These are recessive devastating disorders in children that predominantly affect the central nervous system and result in progressive neurodegeneration and early death. Suitable mouse models enable us to assess behavioral, pathological, cellular, and molecular abnormalities associated with disease and to determine the efficacy of different therapeutic approaches. In addition, these models can be used to study the underlying function of the normal genes This thesis addresses the generation and characterization of mouse models for these diseases. These include creation of gene-targeted models for cLINCL and NPC2 diseases. Disease progression was monitored by assessing gross phenotype (weight), behavioral parameters such as trembling, gait analysis and survival. This and other analyses indicate that the gene targeted mice are good models for the human disease. We also created a mouse model with mutations in both Npc1 and Npc2. Comparison of the single and ii double deficient NPC mutants indicate that the NPC1 and NPC2 proteins function in a common pathway. We also created mutant mice that expressed different levels of tripeptidyl peptidase 1 (TPP1), the protein deficient in cLINCL. This allowed estimation of how much protein is necessary to achieve therapeutic benefits. Immunohistochemistry serves an important role in studying these mouse models and determining the distribution of the normal proteins. This is especially important when looking at protein distributions within the brain following gene or enzyme replacement therapy. We describe development of procedures to visualize TPP1, NPC1, and NPC2 in mouse brain. iii Dedication This work is dedicated to my family. iv Acknowledgements I would like to thank Dr. Peter Lobel for his guidance and for serving as my mentor. He embodies all the characteristics of a true scientist who conducts his research meticulously and with honesty and integrity. I would also like to thank Dr. David Sleat for being a great role model and teaching me about his “theory, what is his”. Without his guidance, my work would not be possible. I would like to thank Dr. Jim Millonig and Dr. Ann Stock for serving on my thesis committee. I would also like to thank all the past and present members of the Lobel laboratory for being like a family to me. They help to provide a perfect environment for the great science they strive to produce. I am proud to be part of these exemplary people. Finally, I would like to thank my husband Ahmed Mohamed for his support, love and care. You are the reason I did this. I would also like to thank my wonderful parents Mohamed El-Banna and Hanan EL-Banna, for being so understanding and helpful with the two little ones. Thanks to my two little ones, Yasin and Ameen, who serve as my inspiration, and to my wonderful sisters for their great help. A special thanks to Patricia Soudani for all her love and support and help with everything. v Table of Contents Title page Abstract ii Dedication iv Acknowledgements v Table of Contents vi List of Tables & Figures vii Abbreviations ix Introduction 1 1. Neuronal Ceroid-Lipofuscinosis (NCLs) 3 2. Classical Late-Infantile Neuronal Ceroid Lipofuscinosis (cLINCL) 5 Biochemical Features and Neuropathology 3. Molecular Genetics of CLN2 7 4. Genetically Engineered Mouse Models of cLINCL; 8 the Cln2-targeted mouse and the Cln2 hypomorphs 4a. Cln2-targeted mouse model 8 4b. Cln2-hypomorph mouse model 11 5. Potential avenues for therapeutic intervention in cLINCL disease 13 using our mouse models 6. Niemann-Pick C disease 15 7. NPC Genes 17 vi 7a. NPC1 17 7b. NPC2 18 8. NPC proteins are required for the egress of cholesterol 19 from late endosomes/lysosomes 9. NPC disease primarily affects the nervous system 23 10. Potential avenues for therapeutic intervention in NPC disease 25 11. The NPC mouse models 27 11a. Npc1 Mouse 27 11b. Npc2 mouse 28 12. Rationale 34 12a. Overview of Immunohistochemistry 37 12b. Intracardiac Perfusion 38 12c. Fixative/post-fixative 40 - CLN2 41 - NPC 42 12d. Preparation of tissues for cryo-sectioning 43 12e. Cryo-stat sectioning/slide preparation 44 12f. Tissue antibody staining procedures 46 12g. Methods for Improving Signal: Noise Ratio 48 12h. Detection/Secondary Antibody 51 13. Analysis/Conclusions 53 Appendix 60 Bibliography 63 vii List of Tables Table 1. Genetic Classification: Human Neuronal Ceroid Lipofuscinoses 4 Table 2. Perfusion Fixation & Corresponding Post-Fixation 43 Table 3. Candidate Antibodies Tested 47 Table 4: Blocking Buffers Used for Optimization 49 Table 5. The Use of GSH in Signal: Noise Optimization 50 Figures Fig 1. The Lysosome 1 Fig 2. CLN2 hypomorph construct 10 Fig. 3. Strain dependence of survival in CLN2 mice 11 Fig 4. Survival analysis of CLN2 mutants 12 Fig. 5. The NPC1 protein 17 Fig. 6. Intracellular LDL derived cholesterol transport 19 Fig. 7. LDL receptor in mammalian cells/The role of NPC2 22 Fig. 8. Targeted disruption of the murine NPC2 gene 29 Fig. 9. Growth and survival of NPC2 hypo in the presence or absence of NPC1 31 Fig. 10. Summary of immunohistochemical process 38 Fig. 11. Optimized detection of TPP1 in mouse cerebellum and hippocampus 57 Fig. 12 NPC1 (Novus Biologicals) Antibody optimization in mouse cerebellum 58 Fig 13. NPC2 (HL5873) Antibody optimization in mouse cerebellum 59 viii Abbreviations AAV Adeno-Associated Virus ANCL Adult Neuronal Ceroid Lipofuscinosis CE Cholesterol Ester cLINCL Classical Late Infantile Neuronal Ceroid Lipofuscinosis CLN2 Ceroid Lipofuscinosis, Neuronal 2 CNS Central Nervous System CT Computed Tomography DNA Deoxyribonucleic acid EEG Electroencephalogram ERG Electroretinogram ES Embryonic Stem Cells FA Unesterified Fatty Acid Glut Gluteraldehyde GM2 Ganglioside 2 GM3 Ganglioside 3 GSH Glutathione HIER Heat Induced Epitope Retrieval HMG-CoA 3- hydroxy-3-methylglutaryl CoA reductase IgG Immunoglobulin G INCL Infantile Neuronal Ceroid Lipofuscinosis ix JNCL Juvenile Neuronal Ceroid Lipofuscinosis KCl Potassium Chloride LDL Low Density Lipoprotein LINCL Late Infantile Neuronal Ceroid Lipofuscinosis MAW Methanol/Acetone/Water NaCl Sodium Chloride NCL Neuronal Ceroid Lipofuscinosis NPA Niemann-Pick type A NPB Niemann-Pick type B NPC Niemann-Pick type C NPC1 Niemann-Pick type C-1 NPC2 Niemann-Pick type C-2 OCT Optimal Cutting Temperature PBS Phosphate Buffered Saline PFA Paraformaldehyde SAP Saposins SCAP Sterol regulatory element- binding protein cleavage activating protein SCMAS Subunit c of the mitochondrial ATP synthase SDS Sodium Dodecyl Sulfate TPP1 Tripeptidyl-peptidase 1 VER Visual Evoked Response VLINCL Variant Late Infantile Neuronal Ceroid Lipofuscinosis x 1 Introduction Lysosomal storage disorders are comprised of a group of over forty genetic disorders in humans that result from defects in the function of the lysosome (Winchester et al., 2000). Lysosomes are organelles within the cytoplasm of the cell that contain enzymes, the majority of which are acid hydrolases, that break down substrates, such as lipids and proteins, into peptides, monosaccharides, amino acids, fatty acids, and nucleic acids (Figure 1). Diseases are due to the lack of one of these important enzymes thereby resulting in an accumulation of material within lysosomes. Figure 1: From http://micro.magnet.fsu.edu/cells/lysosomes/lysosomes.html Lysosomal storage diseases are classified by type of primary stored material involved. Diseases include lipid storage disorders or lipidoses, such as Gaucher’s and Niemann- Pick diseases, gangliosidosis (Tay-Sachs disease), glycoprotein storage disorders, and mucolipidoses. There currently are no cures for these diseases; however there have been 2 studies involving bone marrow transplantation and enzyme replacement therapies that show some progress in treatment options (Bruni et al., 2007; Clarke and Iwanochko, 2005). Gene therapy may also be an option in the near future (Passini et al., 2006; Ponder and Haskins, 2007). In our laboratory we currently have mouse models for classical late infantile neuronal ceroid lipofuscinosis (cLINCL) and Niemann-Pick C (NPC) types 1 and 2. Investigation of lysosomal storage disorders such as cLINCL and the NPC types 1 & 2 require the utilization of mouse models in order to aid in elucidating the specific localization of proteins associated with these diseases. Key to understanding the NPC and TPP1 proteins is to have definitive