Genetic Regulation of Neuronal Progranulin Reveals a Critical Role for the Autophagy-Lysosome Pathway
Total Page：16
File Type：pdf, Size：1020Kb
This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease Genetic Regulation of Neuronal Progranulin Reveals a Critical Role for the Autophagy-Lysosome Pathway Lisa P. Elia1,2, Amanda R. Mason3, Amela Alijagic2 and Steven Finkbeiner1,2,4 1Center for Systems and Therapeutics and Taube/Koret Center for Neurodegenerative Disease Research, San Francisco, CA 2The J. David Gladstone Institutes, San Francisco, CA 3Keck School of Medicine of USC, Los Angeles, CA 4Departments of Neurology and Physiology, UCSF, San Francisco, CA https://doi.org/10.1523/JNEUROSCI.3498-17.2019 Received: 23 March 2018 Revised: 16 January 2019 Accepted: 16 January 2019 Published: 29 January 2019 Author contributions: L.P.E. designed research; L.P.E. and A.A. performed research; L.P.E. analyzed data; L.P.E. wrote the first draft of the paper; L.P.E., A.R.M., and S.F. edited the paper; L.P.E. wrote the paper. Conflict of Interest: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Robert V. Farese (Harvard Medical School), Andrew D. Nguyen (Harvard Medical School), and Ping Zhou (Gladstone Institute of Cardiovascular Disease) for kindly providing the GRN+/- and GRNR493X/+ mice, and the progranulin rabbit polyclonal antibody. Psammaplysene A (PSA) was a gift from R.G. Kalb (Mojsilovic- Petrovic et al., 2009). We thank Laura Mitic (UCSF), and members of the Finkbeiner lab for discussion and comments on these experiments. We thank Megan Laurance (UCSF) for assistance with Ingenuity Pathway Analysis. We also thank Hui Wang for animal husbandry, Gary Howard for editorial assistance, and Kelley Nelson for administrative assistance. Study funding: 1R21NS093236-01A1, 1P01AG054407, 1RF1AG058476, the Consortium for Frontotemporal Dementia Research, the Taube/Koret Center for Neurodegenerative Disease Research, and the J. David Gladstone Institutes. The Gladstone Institutes received support from a National Center for Research Resources Grant RR18928. Corresponding authors: Lisa P. Elia and Steven Finkbeiner, Gladstone Institutes, 1650 Owens St., San Francisco, California 94158, Telephone: (415) 734-2508; FAX: (415) 355-0824; E-mail: [email protected], [email protected] Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.3498-17.2019 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 the authors 1 Genetic Regulation of Neuronal Progranulin Reveals a Critical Role for the 2 Autophagy-Lysosome Pathway 3 Abbreviated title: Genetic modulators of neuronal progranulin levels 4 5 Author names and affiliations: 6 Lisa P. Elia*1,2, Amanda R. Mason3, Amela Alijagic 2, Steven Finkbeiner*1,2,4 7 8 9 1 Center for Systems and Therapeutics and Taube/Koret Center for Neurodegenerative 10 Disease Research, San Francisco, CA 11 2 The J. David Gladstone Institutes, San Francisco, CA 12 3 Keck School of Medicine of USC, Los Angeles, CA 13 4 Departments of Neurology and Physiology, UCSF, San Francisco, CA 14 15 *Corresponding authors: 16 Lisa P. Elia and Steven Finkbeiner, Gladstone Institutes, 1650 Owens St., San 17 Francisco, California 94158, Telephone: (415) 734-2508; FAX: (415) 355-0824; E-mail: 18 [email protected], [email protected] 19 Number of pages: 46 20 Number of figures: primary figures (8), extended data figures (3), tables (2) 21 Number of words: Abstract (207), Significance Statement (120), Introduction (941), 22 Discussion (836) 23 1 24 Conflict of interest: The authors declare that they have no conflicts of interest with the 25 contents of this article. The content is solely the responsibility of the authors and does 26 not necessarily represent the official views of the National Institutes of Health. 27 28 Acknowledgments: We thank Robert V. Farese (Harvard Medical School), Andrew D. 29 Nguyen (Harvard Medical School), and Ping Zhou (Gladstone Institute of 30 Cardiovascular Disease) for kindly providing the GRN+/- and GRNR493X/+ mice, and the 31 progranulin rabbit polyclonal antibody. Psammaplysene A (PSA) was a gift from R.G. 32 Kalb (Mojsilovic-Petrovic et al., 2009). We thank Laura Mitic (UCSF), and members of 33 the Finkbeiner lab for discussion and comments on these experiments. We thank 34 Megan Laurance (UCSF) for assistance with Ingenuity Pathway Analysis. We also 35 thank Hui Wang for animal husbandry, Gary Howard for editorial assistance, and Kelley 36 Nelson for administrative assistance. Study funding: 1R21NS093236-01A1, 37 1P01AG054407, 1RF1AG058476, the Consortium for Frontotemporal Dementia 38 Research, the Taube/Koret Center for Neurodegenerative Disease Research, and the J. 39 David Gladstone Institutes. The Gladstone Institutes received support from a National 40 Center for Research Resources Grant RR18928. 41 42 43 44 45 46 2 47 Abstract 48 Deficient progranulin levels cause dose-dependent neurological syndromes: 49 haploinsufficiency leads to frontotemporal lobar degeneration (FTLD), and nullizygosity 50 produces adult-onset neuronal ceroid lipofuscinosis. Mechanisms controlling 51 progranulin levels are largely unknown. To better understand progranulin regulation, we 52 performed a genome-wide RNAi screen using an ELISA-based platform to discover 53 genes that regulate progranulin levels in neurons. We identified 830 genes that raise or 54 lower progranulin levels by at least 1.5-fold in Neuro2a cells. When inhibited by siRNA 55 or some by submicromolar concentrations of small-molecule inhibitors, 33 genes of the 56 druggable genome increased progranulin levels in mouse primary cortical neurons; 57 several of these also raised progranulin levels in FTLD-model mouse neurons. “Hit” 58 genes regulated progranulin by transcriptional or post-transcriptional mechanisms. 59 Pathway analysis revealed enrichment of hit genes from the autophagy-lysosome 60 pathway (ALP), suggesting a key role for this pathway in regulating progranulin levels. 61 Progranulin itself regulates lysosome function. We found progranulin-deficiency in 62 neurons increased autophagy and caused abnormally enlarged lysosomes, and 63 boosting progranulin levels restored autophagy and lysosome size to control levels. Our 64 data link the ALP to neuronal progranulin: progranulin levels are regulated by 65 autophagy, and in turn, progranulin regulates the ALP. Restoring progranulin levels by 66 targeting genetic modifiers reversed FTLD functional deficits, opening up potential 67 opportunities for future therapeutics development. 68 69 3 70 Significance statement 71 Progranulin regulates neuron and immune functions, and is implicated in aging. Loss of 72 one functional allele causes haploinsufficiency and leads to frontotemporal lobar 73 degeneration (FTLD), the second leading cause of dementia. Progranulin gene 74 polymorphisms are linked to Alzheimer’s disease (AD) and complete loss of function 75 causes neuronal ceroid lipofuscinosis. Despite the critical role of progranulin levels in 76 neurodegenerative disease risk, almost nothing is known about their regulation. We 77 performed an unbiased screen and identified specific pathways controlling progranulin 78 levels in neurons. Modulation of these pathways restored levels in progranulin-deficient 79 neurons and reversed FTLD phenotypes. We provide a new comprehensive 80 understanding of the genetic regulation of progranulin levels, and identify potential 81 targets to treat FTLD and other neurodegenerative diseases, including AD. 82 83 Introduction 84 Progranulin is a widely expressed secreted protein encoded by the GRN gene. In the 85 brain, progranulin is expressed primarily by neurons and microglia, involved in neuronal 86 survival, neurite outgrowth, and synaptogenesis, and a key regulator of 87 neuroinflammation (Ahmed et al., 2007; Chitramuthu et al., 2010; Cenik et al., 2012; 88 Gass et al., 2012; Petkau et al., 2012; Petoukhov et al., 2013; Petkau and Leavitt, 89 2014). Over 60 mutations leading to haploinsufficiency of GRN have been identified and 90 result in decreased progranulin levels. People with GRN haploinsufficiency develop the 91 fatal neurodegenerative disease frontotemporal lobar degeneration (FTLD), the second 92 most common cause of early onset dementia after Alzheimer’s disease (AD) (Rabinovici 4 93 and Miller, 2010; Rademakers et al., 2012; Riedl et al., 2014). Rare individuals 94 nullizygous for GRN develop neuronal ceroid lipofuscinosis (NCL), with onset much 95 earlier than FTLD (Smith et al., 2012; Gotzl et al., 2014). Recently, polymorphisms that 96 decrease GRN expression were linked to increased risk for AD, and boosting levels of 97 progranulin in mouse models of AD suppresses neuroinflammatory-, pathological-, and 98 cognitive-based disease phenotypes (Pereson et al., 2009; Hsiung et al., 2011; 99 Kamalainen et al., 2013; Perry et al., 2013; Minami et al., 2014; Chen et al., 2015; 100 Hosokawa et al., 2015; Xu et al., 2016). Thus, differing levels of progranulin deficiency 101 in the brain lead to different neurodegenerative syndromes, showing that loss of 102 progranulin function contributes to disease development. 103 104 Although progranulin