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Understanding the Molecular Pathobiology of Acid Ceramidase Deficiency

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Understanding the Molecular Pathobiology of Acid Ceramidase Deficiency By Fabian Yu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Fabian PS Yu 2018 Understanding the Molecular Pathobiology of Acid Ceramidase Deficiency Fabian Yu Doctor of Philosophy Institute of Medical Science University of Toronto 2018 Abstract Farber disease (FD) is a devastating Lysosomal Storage Disorder (LSD) caused by mutations in ASAH1, resulting in acid ceramidase (ACDase) deficiency. ACDase deficiency manifests along a broad spectrum but in its classical form patients die during early childhood. Due to the scarcity of cases FD has largely been understudied. To circumvent this, our lab previously generated a mouse model that recapitulates FD. In some case reports, patients have shown signs of visceral involvement, retinopathy and respiratory distress that may lead to death. Beyond superficial descriptions in case reports, there have been no in-depth studies performed to address these conditions. To improve the understanding of FD and gain insights for evaluating future therapies, we performed comprehensive studies on the ACDase deficient mouse. In the visual system, we reported presence of progressive uveitis. Further tests revealed cellular infiltration, lipid buildup and extensive retinal pathology. Mice developed retinal dysplasia, impaired retinal response and decreased visual acuity. Within the pulmonary system, lung function tests revealed a decrease in lung compliance. Mice developed chronic lung injury that was contributed by cellular recruitment, and vascular leakage. Additionally, we report impairment to lipid homeostasis in the lungs. ii To understand the liver involvement in FD, we characterized the pathology and performed transcriptome analysis to identify gene and pathway changes. We revealed progressive liver injury, inflammation and fibrosis. RNAseq analyses on hepatocytes revealed activation of pathways in inflammation response and cellular recruitment and deactivation of pathways related to lipid metabolism. MCP-1 is an inflammatory chemokine that is dramatically elevated in ACDase deficient mice and humans. To understand the role of MCP-1 in FD, we created and characterized the Asah1P361R/P361R;MCP-1-/- double mutant mice. Ablation of MCP-1 attenuated disease and provided a modest extension of life from ~9 weeks to ~14 weeks of age. The greatest reduction in inflammation was detected in the lung. Decreased inflammation in the Asah1P361R/P361R;MCP- 1-/- mice also resulted in less ceramide accumulation in the lung and liver. Taken together, targeting MCP-1 though not effective in all organs may provide a benefit in combination with other therapies until a cure is available for this debilitating disorder. iii Acknowledgments I would like to first and foremost thank my supervisor Dr. Jeffrey Medin, for being encouraging and giving me intellectual freedom to pursue my projects. Thanks to my committee members Dr. Razqallah Hakem and Dr. Mingyao Liu for their guidance and intellectual input. I would also like to acknowledge all my colleagues and collaborators for their scientific and intellectual contributions. I am thankful for the generous funding to study and travel to conferences provided during my PhD from the Institute of Medical Science, the University of Toronto School of Graduate Studies, the University Health Network Office of Research Trainees, the WORLD Symposium, and the Midwest Athletes Against Childhood Cancer Fund. For my supportive Parents. Facta, non verba iv Contributions Chapter 3 Dr. Iris Kassem performed slit-lamp analyses (Figure 9, Figure 15). Ben Sajdak and Alexander Salmon performed fundus photography, OCT, cSLO and analyzed OCT images for retinal thickness (Figure 9, Figure 10, Figure 11 and Figure 17). Dr. Daniel Lipinski and I performed ERG analyses (Figure 12 and Figure 13). Dr. Jakub Sikora and Jirí Gurka prepared tissue for electron microscopy and performed ultrastructure analyses (Figure 20, Figure 21 and Figure 22). Children’s Hospital of Wisconsin (CHW) Children’s Research Institute (CRI) Histology Core performed immunohistochemistry staining (Figure 18, Figure 19, Figure 21 and Figure 23). Medical University of South Carolina (MUSC) Lipidomic Core identified sphingolipids in the retina (Figure 25). Chapter 4 Figures and text adapted from Yu et al., 2017 with permission from American Journal of Physiology- Lung Cellular and Molecular Physiology. Diana Islam performed the lung mechanics tests and blood oxygenation analyses (Figure 26 and Figure 27). Dr. Wolfgang Kuebler assisted with pulse oximeter measurements (Figure 27). The Centre for Modeling Human Disease (CMHD) performed the histological and immunohistochemistry staining (Figure 28, Figure 31 and Figure 36). Dr. Jakub Sikora and Jirí Gurka prepared lungs for electron microscopy and performed ultrastructure analyses (Figure 30 and Figure 31). Lucía López- Vásquez and I performed the Evans Blue dye experiment (Figure 33). Dr. Shaalee Dworski and I performed the cytokine analyses on BALF samples (Figure 34). Dr. Thierry Levade and I identified the phospholipids and sphingolipids in BALF and lung tissue (Figure 37 and Figure 38). v Chapter 5 The Centre for Modeling Human Disease (CMHD) performed the histological and immunohistochemistry staining (Figure 40, Figure 44 and Figure 45). Dr. Jakub Sikora performed TEM analyses (Figure 41, Figure 42 and Figure 43). Dr. Patricia Turner performed liver injury assessment (Table 10). Medical University of South Carolina (MUSC) Lipidomics Core identified sphingolipids liver and hepatocytes (Figure 46, Figure 47 and Figure 49). Dr. Shauna Rasmussen, Dr. Salvatore Molino and I, performed the liver perfusion and hepatocyte isolation. Everett Tate performed FACS analyses (Figure 48, Figure 49, Figure 50, Figure 51 and Figure 52). Dr. Salvatore Molino performed RNA extraction, and qPCR. Transcriptome analyses was performed by the MCW Sequencing library. Dr. Salvatore Molino performed bioinformatic and pathway analyses (Figure 50, Figure 51 and Figure 52). Chapter 6 Figures and text adapted from Yu et al., 2018 with permission from Scientific Reports. Dr. Shaalee Dworski and I performed cytokine analyses on serum samples (Figure 66 and Figure 67), and flow cytometry analyses (Figure 57, Figure 58 and Figure 59). CMHD and CHW- CRI performed the histological and immunohistochemistry staining (Figure 57, Figure 58, Figure 59, Figure 60 and Figure 62). Chapter 7 The List of ASAH1 mutations (Table 12) were curated by Dr. Samuel Amintas, Dr. Thierry Levade and myself. The predicted 3D structure of the mouse and human ACDase (Figure 68 was developed by Zi Jian Xiong). I annotated it based on color-coded mutations on PyMOL. vi Table of Contents Acknowledgments ...................................................................................................................... iv Contributions ............................................................................................................................... v Table of Contents ...................................................................................................................... vii List of Abbreviations ................................................................................................................. xvi List of Tables .......................................................................................................................... xxiii List of Figures ......................................................................................................................... xxiv Chapter 1 Literature Review ....................................................................................................... 1 1.1 Lysosome and Lysosomal Storage Disorders ................................................................. 1 1.1.1 Discovery of the lysosome .................................................................................. 1 1.1.2 Function of the lysosome .................................................................................... 2 1.1.3 Overview of Lysosomal Storage Disorders .......................................................... 5 1.1.4 Prevalence and affected populations ................................................................... 8 1.1.5 Disease screening ............................................................................................... 9 1.1.6 Diagnosis and screening ................................................................................... 10 1.1.7 Overview of treatment and therapy .................................................................... 11 1.1.8 Hematopoietic stem cell transplantation ............................................................ 12 1.1.9 Enzyme replacement therapy ............................................................................ 13 1.1.10 Substrate reduction therapy .............................................................................. 15 1.1.11 Gene therapy .................................................................................................... 16 1.2 Sphingolipids ................................................................................................................ 19 1.2.1 Overview of sphingolipids .................................................................................. 19 1.2.2 Structure ..........................................................................................................
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  • Generation of Sphingosine-1-Phosphate Is Enhanced in Biliary Tract Cancer Patients and Is Associated with Lymphatic Metastasis

    Generation of Sphingosine-1-Phosphate Is Enhanced in Biliary Tract Cancer Patients and Is Associated with Lymphatic Metastasis

    www.nature.com/scientificreports OPEN Generation of sphingosine- 1-phosphate is enhanced in biliary tract cancer patients and Received: 5 April 2018 Accepted: 4 July 2018 is associated with lymphatic Published: xx xx xxxx metastasis Yuki Hirose1, Masayuki Nagahashi1, Eriko Katsuta2, Kizuki Yuza1, Kohei Miura1, Jun Sakata1, Takashi Kobayashi1, Hiroshi Ichikawa1, Yoshifumi Shimada1, Hitoshi Kameyama1, Kerry-Ann McDonald2, Kazuaki Takabe 1,2,3,4,5 & Toshifumi Wakai1 Lymphatic metastasis is known to contribute to worse prognosis of biliary tract cancer (BTC). Recently, sphingosine-1-phosphate (S1P), a bioactive lipid mediator generated by sphingosine kinase 1 (SPHK1), has been shown to play an important role in lymphangiogenesis and lymph node metastasis in several types of cancer. However, the role of the lipid mediator in BTC has never been examined. Here we found that S1P is elevated in BTC with the activation of ceramide-synthetic pathways, suggesting that BTC utilizes SPHK1 to promote lymphatic metastasis. We found that S1P, sphingosine and ceramide precursors such as monohexosyl-ceramide and sphingomyelin, but not ceramide, were signifcantly increased in BTC compared to normal biliary tract tissue using LC-ESI-MS/MS. Utilizing The Cancer Genome Atlas cohort, we demonstrated that S1P in BTC is generated via de novo pathway and exported via ABCC1. Further, we found that SPHK1 expression positively correlated with factors related to lymphatic metastasis in BTC. Finally, immunohistochemical examination revealed that gallbladder cancer with lymph node metastasis had signifcantly higher expression of phospho-SPHK1 than that without. Taken together, our data suggest that S1P generated in BTC contributes to lymphatic metastasis. Biliary tract cancer (BTC), the malignancy of the bile ducts and gallbladder, is a highly lethal disease in which a strong prognostic predictor is lymph node metastasis1–5.