This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Article: New Research | Disorders of the Nervous System Brain region-specific gene signatures revealed by distinct astrocyte subpopulations unveil links to glioma and neurodegenerative diseases Raquel Cuevas Diaz Duran1,2,3, Chih-Yen Wang4, Hui Zheng5,6,7, Benjamin Deneen8,9,10,11 and Jia Qian Wu1,2 1The Vivian L. Smith Department of Neurosurgery, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA 2Center for Stem Cell and Regenerative Medicine, UT Brown Foundation Institute of Molecular Medicine, Houston, TX 77030, USA 3Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Ave. Morones Prieto 3000, Monterrey, N.L., 64710, Mexico 4Department of Life Sciences, National Cheng Kung University, Tainan City, 70101, Taiwan 5Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA 6Medical Scientist Training Program, Baylor College of Medicine, Houston, TX 77030, USA 7Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 8Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA 9Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA 10Neurological Research Institute at Texas’ Children’s Hospital, Baylor College of Medicine, Houston, TX 77030, USA 11Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA https://doi.org/10.1523/ENEURO.0288-18.2019 Received: 19 July 2018 Revised: 16 January 2019 Accepted: 12 February 2019 Published: 7 March 2019 J.Q.W. and B.D. designed research; J.Q.W., R.C.-D.D., C.-Y.W., and B.D. wrote the paper; R.C.-D.D. and C.- Y.W. performed research; R.C.-D.D. analyzed data; H.Z. and B.D. contributed unpublished reagents/analytic tools. Funding: http://doi.org/10.13039/100000002HHS | National Institutes of Health (NIH) R01 NS088353 1RF1AG054111 R01NS071153 Funding: The Staman Ogilvie Fund-Memorial Hermann Foundation Funding: Mission Connect Funding: http://doi.org/10.13039/100000890National Multiple Sclerosis Society (National MS Society) RG-1501-02756 Authors declare no competing financial interests. Correspondence should be addressed to Benjamin Deneen, [email protected] or Jia Qian Wu, [email protected] Cite as: eNeuro 2019; 10.1523/ENEURO.0288-18.2019 Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 Cuevas- et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. 1 Brain region-specific gene signatures revealed by distinct astrocyte 2 subpopulations unveil links to glioma and neurodegenerative diseases 3 4 Abbreviated Title: Astrocyte signatures linked to glioma and diseases 5 6 Raquel Cuevas-Diaz Duran1-3, Chih-Yen Wang4, Hui Zheng5-7, Benjamin Deneen8-11*, and 7 Jia Qian Wu1,2* 8 9 1 The Vivian L. Smith Department of Neurosurgery, McGovern Medical School, The 10 University of Texas Health Science Center at Houston, Houston, TX 77030, USA 11 2 Center for Stem Cell and Regenerative Medicine, UT Brown Foundation Institute of 12 Molecular Medicine, Houston, TX 77030, USA 13 3 Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Ave. Morones 14 Prieto 3000, Monterrey, N.L., 64710, Mexico 15 4 Department of Life Sciences, National Cheng Kung University, Tainan City, 70101, 16 Taiwan 17 5 Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA 18 6 Medical Scientist Training Program, Baylor College of Medicine, Houston, TX 77030, 19 USA 20 7 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 21 77030, USA 22 8 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, 23 USA 24 9 Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA 25 10 Neurological Research Institute at Texas’ Children’s Hospital, Baylor College of 26 Medicine, Houston, TX 77030, USA 27 11 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, 28 USA 29 30 Author contributions 31 JQW and BD designed research. RCDD and CYW performed research. RCDD analyzed 32 data. HZ and BD contributed reagents/materials. RCDD, CYW, BD, and JQW participated 33 in writing the manuscript. All authors have read and approved the final manuscript. 1 34 35 Corresponding authors 36 * To whom correspondence should be addressed. Tel: +1 (713) 500-3421; Fax: +1 (713) 37 500-2424; Email: [email protected] 38 39 Number of 40 Figures: 6 41 Tables: 4 42 Multimedia: 0 43 Extended Data Figures: 11 44 Extended Data Tables: 5 45 46 Number of words in 47 Abstract: 206 48 Significance Statement: 64 49 Introduction: 748 50 Discussion: 2067 51 52 Acknowledgements 53 The authors would like to thank Shun-Fen Tzeng for supporting Chih-Yen and 54 Ms. Mary Ann Cushman for editing the manuscript. 55 56 Conflicts of Interest 57 Authors declare no competing financial interests. 58 59 Funding sources 60 JQW and RCDD were supported by grants from the National Institutes of Health R01 61 NS088353; The Staman Ogilvie Fund-Memorial Hermann Foundation; Mission Connect, a 62 program of the TIRR Foundation. BD was supported by grants from the National Institutes 63 of Health 1RF1AG054111 and R01NS071153; National MS Society RG-1501-02756. 64 2 65 Abstract 66 Currently, there are no effective treatments for glioma or for neurodegenerative diseases 67 due, in part, to our limited understanding of the pathophysiology and cellular heterogeneity 68 of these diseases. Mounting evidence suggests that astrocytes play an active role in the 69 pathogenesis of these diseases by contributing to a diverse range of pathophysiological 70 states. In a previous study, five molecularly distinct astrocyte subpopulations from three 71 different brain regions were identified. To further delineate the underlying diversity of 72 these populations, we obtained mouse brain region-specific gene signatures for both 73 protein-coding and long non-coding RNA (lncRNA) and found that these astrocyte 74 subpopulations are endowed with unique molecular signatures across diverse brain regions. 75 Additional gene set and single-sample enrichment analyses revealed that gene signatures of 76 different subpopulations are differentially correlated with glioma tumors that harbor distinct 77 genomic alterations. To the best of our knowledge, this is the first study that links 78 transcriptional profiles of astrocyte subpopulations with glioma genomic mutations. 79 Furthermore, our results demonstrated that subpopulations of astrocytes in select brain 80 regions are associated with specific neurodegenerative diseases. Overall, the present study 81 provides a new perspective for understanding the pathophysiology of glioma and 82 neurodegenerative diseases and highlights the potential contributions of diverse astrocyte 83 populations to normal, malignant, and degenerative brain functions. 84 85 Significance Statement 86 Mounting evidence suggests that astrocytes play an active role in disease 87 pathogenesis by contributing to a diverse range of pathophysiological states. The present 88 study identifies new layers of astrocyte diversity and new correlations between distinct 3 89 astrocyte subpopulations and disease states. Understanding the heterogeneity of astrocytes 90 in different physiological conditions and diseases will provide new avenues for improved 91 diagnostics and therapeutics targeting specific astrocyte subpopulations. 92 93 Introduction 94 Astrocytes make up about 40% of all cells in the human brain (Herculano-Houzel, 2014; 95 Zhang et al., 2016) and play essential roles in brain function by controlling extracellular 96 neurotransmitter and potassium ion levels, regulating the blood-brain-barrier, and 97 promoting synapse formation and function (Barres, 2008). Previously, astrocytes were 98 thought to be a homogeneous population of cells that tile the CNS and support neuronal 99 survival. However, recent genome-wide gene expression studies have shown that astrocytes 100 are highly heterogeneous (Cahoy et al., 2008; Doyle et al., 2008; Yeh et al., 2009). A recent 101 study demonstrated astrocyte heterogeneity in the brain by comparing gene expression 102 profiles of FACS-sorted Aldh1l1-GFP+ subpopulations and identified five astrocyte 103 subpopulations (A-E) across three brain regions (olfactory bulb, cortex, and brain stem). 104 Moreover, these astrocyte subpopulations exhibited functional differences related to 105 proliferation, migration, and synapse formation. (Lin et al., 2017). 106 Functional heterogeneity of astrocytes has been described under both normal 107 physiological conditions and diverse disease states (Denis-Donini et al., 1984; Hill et al., 108 1996; Grass et al., 2004; White et al., 2010; Morel et al., 2017). During disease progression, 109 astrocyte populations respond to pathological conditions through a transformation called 110 reactive astrogliosis. Morphological and gene expression differences have been