Human brain transcriptomics: towards understanding multiple system atrophy James Dominic Mills Athesissubmittedinfulfilmentoftherequirementsforthedegreeof Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia October 28, 2015 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Mills First name: James Other name/s: Dominic Abbreviation for degree as given In the University calendar: BIOC8608 School: The School of Biotechnology and Biomolecular Sciences Faculty: Science Title: Human brain transcriptomlcs: towards understanding multiple system atrophy Abstract 350 words maximum: (PLEASE TYPE) The human brain is a remarkably complex organ. It is a heterogeneous collection of billions of neurons and glial cells that are interconnected to form a finely tuned network capable of higher cognition. It is thought that the transcriptome may hold the key to understanding the complexity of the human brain. Next-generation sequencing allows the brain's transcriptome to be probed at an unmatched resolution. This has uncovered a myriad of RNA elements. including a number of long intervening non-coding RNAs (lincRNAs) that appear to be vital to the development and fun ction of the brain. The complexity of the human brain also makes it prone to a variety of different neurodegenerative diseases. One such disorder is multiple system atrophy (MSA). MSA is a sporadic, rapidly progressing neurodegenerative disease. Currently no treatment exists and very little is known about the molecular basis of MSA. In an attempt to understand the complexity of the human brain, transcriptome profiling of grey matter (GM) and white matter (WM) from lhe prefrontal cortex was performed. This revealed high expression of numerous lincRNAs, including the oligodendrocyte maturation-associated lincRNA (OLMALINC) in WM. To further establish the role of OLMALINC In the human brain the transcript was knocked down in neurons and oligodendrocytes. This revealed that OLMALINC plays a role in oligodendrocyte maturation. Next transcriptome profiling of MSA brain tissue was carried out. Genes that were differentially expressed between the healthy and MSA brain included. a1-hemoglobin (HBA 1), a2-hemoglobin (HBA2) and 13-hemoglobin (HBB). This suggests that perturbation of iron metabolism may be involved in MSA pathology. A number of differentially expressed lincRNAs between MSA GM and MSA WM were also Identified. One of these lincRNAs. linc00320 was further investigated to establish if it played role in MSA pathology. Together. using next-generation sequencing and bioinformatic tools this thesis provides a comprehensive transcriptional analysis of the human brain, followed by the most detailed transcriptome analysis of MSA. The transcriptome wide profiling analyses are also coupled with comprehensive analyses of OLMALINC and linc00320, detailing their importance In human brain physiology. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known. subject to the provisions of the Copyright Act 1968. I retain all property rights. such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. 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FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS Copyright Statem ent 'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in t he University libraries in all forms of media, now or here after known, subject to t he provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University :Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtnined permission to use copyright material; where perm ission has not been granted I have npplied/ will apply for n partial restriction of the digital copy of my t hesis or dissertation.' Signed Date Authent icity Statem ent 'I certify that the Library deposit digital copy is a direct equivalent of the final offi cially approved version of my thesis. No emendation of content has occurred and if t here are any minor variations in formatting, they arc the result of the conversion to digital format.' Signed Date Originality Statement 'I hereby declare that t his submission is my own work and to the best of my knowl­ edge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSvV or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that t he intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expres­ sion is acknowledged.· Signed Date Contents Acknowledgements IV Abstract V List of figures VIII Abbreviations IX 1 Introduction 1 1.1 The prefrontal cortex of the human brain . 1 1.2 Thetranscriptomeandorganismcomplexity . 3 1.3 Non-codingRNAs............................. 4 1.4 Conservation and tissue-specificity of long non-coding RNAs . 8 1.5 RNA-sequencing ............................. 10 1.6 Multiple system atrophy . 13 1.7 Multiple system atrophy and transcriptomics . 16 1.8 Aimsofthisstudy ............................ 17 1.9 Thesis synopsis . 18 I 1.10 Review article: Strand-specific RNA-Seq provides greater resolution of transcriptome profiling . 19 2 Transcriptome profiling of the healthy human brain using RNA- Seq 30 2.1 Primary research article: Unique transcriptome patterns of the white and grey matter corroborate structural and functional heterogeneity in the human frontal lobe . 31 3 The role of long intervening non-coding RNAs in healthy brain development and function 51 3.1 Primary research article: High expression of long intervening non- coding RNA OLMALINC in the human cortical white matter is as- sociated with regulation of oligodendrocyte maturation . 52 4 Transcriptome profiling of multiple system atrophy brain tissue using RNA-Seq 66 4.1 Primary research article: Transcriptome analysis of grey and white matter cortical tissue in multiple system atrophy . 67 5 Investigation of a long intervening non-coding RNA that may be involved in the pathology of multiple system atrophy 85 5.1 Primary research article: Long intervening non-coding RNA 00320 is human brain-specific and highly expressed in the cortical white matter . 86 II 6 Conclusions and future directions 101 6.1 Summary . 101 6.2 The use of post-mortem brain tissue in transcriptome profiling . 104 6.3 Selection of the superior frontal gyrus . 107 6.4 The complexity of the transcriptome of the human brain . 108 6.5 Brain lincRNAs show low levels of sequence and expression conservation . 110 6.6 Insights into the pathology of multiple system atrophy . 111 6.7 Futuredirections ............................. 112 References 116 Appendices 133 A Publications with non-first authorship related to this thesis 133 A.1 The role of transcriptional control in multiple system atrophy . 134 A.2 Pathway analysis of the human brain transcriptome in disease . 142 A.3 Conservation and tissue-specific transcription patterns of long non- codingRNAs ............................... 152 III Acknowledgements First and foremost I o↵er my sincerest gratitude to my supervisor Dr. Michael Janitz. His enthusiasm, commitment and support made the completion of this thesis possible. I could not have asked for a better mentor throughout my PhD. You have had a profound influence on my career path. Next, I would like to thank all of my co-authors who contributed to the papers presented in this thesis including; Prof. Glenda Halliday, Dr. Paul Waters, Dr. Scott Kim, Dr. Yoshihiro Kawahara, Prof. Eleonora Aronica, Bei Jun Chen, Tomas Kavanagh, Jieqiong Chen and Avanita Prabowo. To all of my friends and family throughout Australia and the world, thanks for your support. Last but in no way the least, I would like to thank the donors who made these studies possible. I implore everyone who reads this to donate their body to science. IV Abstract The human brain is a remarkably complex organ. It is a heterogeneous collection of billions of neurons and glial cells that are interconnected to form a finely tuned network capable of higher cognition. It is thought that the transcriptome may hold the key to understanding the complexity seen in the human brain. Next-generation sequencing allows the brain’s transcriptome to be probed at an unmatched resolu- tion. This has uncovered a myriad of RNA elements, including RNA that does not code for protein, known as non-coding RNA (ncRNA).