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Regulatory Elements Inserted Into Aavs Confer Preferential Activity in Cortical Interneurons

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Regulatory Elements Inserted Into Aavs Confer Preferential Activity in Cortical Interneurons Research Article: Methods/New Tools | Novel Tools and Methods Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons https://doi.org/10.1523/ENEURO.0211-20.2020 Cite as: eNeuro 2020; 10.1523/ENEURO.0211-20.2020 Received: 21 May 2020 Revised: 13 October 2020 Accepted: 15 October 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Rubin 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. 1 Regulatory Elements Inserted into AAVs Confer Preferential Activity in 2 Cortical Interneurons 3 4 Abbreviated title: Cortical Interneuron-Specific Enhancer AAVs 5 Anna N Rubin1*, Ruchi Malik2*, Kathleen KA Cho2, Kenneth J Lim1, Susan Lindtner1, Sarah E 6 Robinson Schwartz2, Daniel Vogt1, Vikaas S Sohal2†, John LR Rubenstein1† 7 1 Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, 8 UCSF Weill Institute for Neurosciences, University of California San Francisco, San Francisco, 9 CA 94158, USA 10 11 2 Center for Integrative Neuroscience, Department of Psychiatry, UCSF Weill Institute for 12 Neurosciences, Kavli Institute for Fundamental Neuroscience, University of California San 13 Francisco, San Francisco, CA 94158, USA 14 15 * These authors contributed equally to this work 16 † Co-corresponding authors 17 Author contributions 18 A.N.R., R.M., D.V., V.S.S. and J.L.R.R. Designed research; A.N.R., R.M., K.K.A.C, S.L. and 19 S.E.R. Performed research; A.N.R., R.M., K.K.A.C and K.J.L. Analyzed data; A.N.R., R.M., 20 V.S.S. and J.L.R.R. Wrote the paper. 21 Correspondence should be addressed to: 22 Vikaas S Sohal 23 UCSF Department of Psychiatry, Box 0444 24 675 Nelson Rising Lane, 415C 25 San Francisco, CA 94143 26 Email: [email protected] 27 28 John LR Rubenstein 29 UCSF Department of Psychiatry, Box 2611 30 1550 Fourth St., Floor 2 31 San Francisco, CA 94143 32 Email: [email protected] 33 34 Number of Figures: 6 main, 6 extended data 35 Number of Tables: 2 (in methods section) 36 Number of Multimedia Files: 1 (extended data 2-2 excel file) 37 Number of words for Abstract: 196 38 Number of words for Significance Statement: 120 39 Number of words for Introduction: 750 40 Number of words for Discussion: 1640 41 Acknowledgements 42 Confocal microscopy was performed at the UCSF Nikon Imaging Center, supported by NIH S10 43 Shared Instrumentation grant 1S10OD017993-01A1. Cell sorting was performed at the UCSF 1 44 HDFCC Laboratory for Cell Analysis, using instrumentation supported by NIH grant 45 P30CA082103. Next-generation sequencing was carried out by the Center for Advanced 46 Technology at UCSF. 47 Current affiliations 48 D.V.: Department of Pediatrics and Human Development, 400 Monroe Ave. NW, Grand Rapids, 49 MI 49503 USA; Neuroscience Program, Michigan State University, East Lansing, MI USA. 50 Conflict of Interest 51 J.L.R.R. is a co-founder, stockholder, and currently on the scientific board of Neurona, a 52 company studying the potential therapeutic use of interneuron transplantation. The remaining 53 authors declare no conflict of interest. 54 Funding sources 55 J.L.R.R. and V.S.: U01MH105948 from NIMH; J.L.R.R.: R01 Grant #MH081880 from NIMH. 56 R.M. from NARSAD Young Investigator Grant (Leichtung family Investigator, Brain and 57 Behavior Research Foundation). 58 59 2 60 Abstract 61 Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric 62 conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how 63 the development, physiology and functions of CINs influence cortical circuit activity and behavior 64 in model organisms such as mice and primates. While transgenic driver lines are powerful tools 65 for studying CINs in mice, this technology is limited in other species. An alternative approach is 66 to use viral vectors such as AAV, which can be used in multiple species including primates and 67 also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory 68 enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell 69 types. The present study describes the systematic genome-wide identification of putative REs 70 (pREs) that are preferentially active in immature CINs by histone modification ChIP-seq. We 71 evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, 72 and found that they drove CIN-specific reporter expression in adult mice. We also showed that 73 the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic 74 rescue of behavioral deficits in the Dlx5/6+/- mouse model of fast-spiking CIN dysfunction. 75 Significance Statement 76 Uncovering the contribution of cortical interneuron (CIN) dysfunction to neuropsychiatric 77 conditions is pivotal to generating therapies for these debilitating disorders. To this end, it is 78 important to study the development, physiology and function of CINs in model organisms. While 79 transgenic driver lines are powerful tools to study CINs in mice, this technology is limited in 80 other species. As an alternative, gene regulatory enhancer elements (REs) can be used in viral 81 vectors to drive expression in multiple species including primates. The present study describes 82 the genome-wide identification of REs preferentially active in CINs and the use of these RE 3 83 AAVs for CIN-specific targeting in adult mice. The methodology and novel REs described here 84 provide new tools for studying and targeting CINs. 85 Introduction 86 GABAergic cortical interneurons (CINs) constitute a diverse population of biochemically, 87 physiologically and morphologically distinct neuronal subtypes that have specialized functions 88 within cortical circuitry (Fishell and Kepecs, 2019; Tremblay et al., 2016). Based on their 89 neurochemical and cellular properties, CINs can be broadly divided into three major subtypes: 90 1) parvalbumin (PV) expressing CINs, which have fast spiking physiological properties and 91 innervate the perisomatic or axonal regions of excitatory pyramidal neurons (PNs); 2) 92 somatostatin (SST) expressing CINs, which typically have regular spiking properties and target 93 distal dendritic regions of PNs; and 3) vasoactive peptide (VIP) expressing CINs, which have 94 irregular spiking properties and cause disinhibition of PNs by preferentially targeting other CINs. 95 Multiple lines of evidence highlight that these CIN subtypes play dissociable roles in cortical 96 circuits. In particular, fast spiking PV+ CINs promote cortical gamma oscillations, which are 97 necessary for cognitive functions such as attention, working memory, and cognitive flexibility 98 (Cardin et al., 2009; Cho et al., 2015; Sohal et al., 2009), and SST+ CINs play important roles in 99 cortical beta oscillations, input integration and sensory processing (Chen et al., 2017; Yavorska 100 and Wehr, 2016). 101 102 Our understanding of the development and functional heterogeneity of CINs owes heavily to the 103 use of transgenic mouse lines expressing Cre recombinase in molecularly specified CIN 104 subtypes (Taniguchi et al., 2011). Although these Cre-driver lines have been extremely valuable 105 to the field, the current reliance on them has some limitations. First, while Cre-driver lines can 106 be used in combination with other recombinases (such as Flp) to label different subtypes of 107 neurons, the ability to manipulate and record from multiple classes of CINs simultaneously or 4 108 study interactions of CINs with specific types of PNs has been lacking. Second, the Cre-driver 109 lines are not able to distinguish between additional subclasses within the PV, SST and VIP CIN 110 subtypes, which have distinct physiological properties and molecular markers (Nigro et al., 111 2018). Third, while Cre-driver lines are widely available for mice, this technology is currently 112 unsuitable for research in other species such as non-human primates. 113 114 A large number of studies implicate loss or alteration of the activity of CIN subtypes, particularly 115 PV+ fast spiking CINs, in neurological diseases such as schizophrenia, epilepsy and autism 116 (Cho and Sohal, 2014; Hashemi et al., 2017; Jiang et al., 2016; Lewis et al., 2012; Malik et al., 117 2019; Vogt et al., 2015; 2017). To develop targeted treatments for these disorders, it is 118 imperative to develop technologies that will allow manipulation of the activity of CIN subtypes in 119 humans. 120 121 The use of recombinant AAV vectors has become a widespread technique for labeling neurons 122 as well as monitoring and modifying their electrophysiological activity (Betley and Sternson, 123 2011). For instance, AAV1, AAV5 and AAV9 disperse throughout the brain readily and can 124 infect most neurons (Burger et al., 2004; Nathanson et al., 2009b; Taymans et al., 2007; 125 Watakabe et al., 2015). AAVs have the added benefit of being translatable across species and 126 are becoming a promising avenue for human therapeutic intervention (Haggerty et al., 2020). 127 128 In spite of these advantages, limited viral genome capacity has hampered the generation of 129 AAVs to target specific CIN subclasses. Due to their small size (often <1kb) and specific 130 patterns of activity, gene regulatory elements (REs) such as enhancers have proven to be a 131 good tool for driving CIN-specific expression by viral methods.
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