Research Article: New Research | Development Erythropoietin Stimulates GABAergic Maturation in the Mouse Hippocampus https://doi.org/10.1523/ENEURO.0006-21.2021 Cite as: eNeuro 2021; 10.1523/ENEURO.0006-21.2021 Received: 7 January 2021 Accepted: 9 January 2021 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 © 2021 Khalid 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 Erythropoietin Stimulates GABAergic Maturation in the Mouse Hippocampus 2 Abbreviated Title: EPO and hippocampal GABAergic maturation 1,3,+ 1,+ 2 1,2 3 Kasifa Khalid , Julia Frei , Mostafa A. Aboouf , Christina Koester-Hegmann , Max 4 Gassmann2, Jean-Marc Fritschy1,3 and Edith M. Schneider Gasser1,2,3 5 1Institute of Pharmacology and Toxicology, Neuroprotection Group, University of Zurich, 6 Winterthurerstrasse 190, 8057 Zurich, Switzerland 7 2Institute of Veterinary Physiology, Vetsuisse Faculty, and Zurich Center for Integrative 8 Human Physiology, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland 9 3Neuroscience Centre Zurich, University of Zurich and ETH Zurich, 8057 Zurich, Switzerland 10 +Equal contribution 11 Author contributions: EMSG designed research; KK, JF, MAA, CK-H, and EMSG 12 performed research and analyzed data; MG provided transgenic Tg21 mice and expertise 13 with those; EMSG and J-MF wrote the manuscript. 14 Correspondence should be addressed to: [email protected] 15 Number of figures: 8 Number of words for abstract: 203 16 Tables: 1 Number of words significant statement: 98 17 Number of words introduction: 830 18 Number of Words discussion: 1621 19 Acknowledgements: We thank Prof. Christian Grimm for scientific feedback; Dr. Lena Rubi 20 and Mr. Mohammad Hleihil for their support with electrophysiology; Dr. Tatjana Haenggi for 21 genotyping the animals; Fabio Valeri for his support with statistics; and Barbara Ellis, Dr. 22 Christina Boyle and Dr. Robert Ganley for proofreading this manuscript. 23 Conflict of interests: The authors declare no conflict of interests. 24 Funding sources: This work was supported by University of Zürich and Swiss National 25 Science Foundation MHV Grant PMPDP3_145480 to E.M.S.G. and the Baugenossenschaft 26 Zurlinden. 1 27 Abstract 28 Several neurodevelopmental disabilities are strongly associated with alterations in 29 GABAergic transmission, and therapies to stimulate its normal development are lacking. 30 Erythropoietin (EPO) is clinically used in neonatology to mitigate acute brain injury, and to 31 stimulate neuronal maturation. Yet it remains unclear whether EPO can stimulate maturation 32 of the GABAergic system. Here, with the use of a transgenic mouse line that constitutively 33 overexpresses neuronal EPO (Tg21), we show that EPO stimulates postnatal GABAergic 34 maturation in the hippocampus. We show an increase in hippocampal GABA- 35 immunoreactive neurons, and postnatal elevation of interneurons expressing parvalbumin 36 (PV), somatostatin (SST) and neuropeptide Y (NPY). Analysis of perineuronal net formation 37 and innervation of glutamatergic terminals onto PV+ cells, shows to be enhanced early in 38 postnatal development. Additionally, an increase in GABAAergic synapse density and 39 inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells from Tg21 mice is observed. 40 Detection of erythropoietin receptor (EPOR) mRNA was observed to be restricted to 41 glutamatergic pyramidal cells and increased in Tg21 mice at postnatal day 7, along with 42 reduced apoptosis. Our findings show that EPO can stimulate postnatal GABAergic 43 maturation in the hippocampus, by increasing neuronal survival, modulating critical plasticity 44 periods, and increasing synaptic transmission. Our data supports EPO’s clinical use to 45 balance GABAergic dysfunction. 46 Significance Statement 47 Using a mouse model that overexpresses recombinant human EPO in the CNS, we 48 observed stimulation of the postnatal maturation of GABAergic transmission in the 49 hippocampus, notably accelerated maturation of PV+ interneurons, enhanced glutamatergic 50 inputs onto these interneurons, and enhanced inhibitory postsynaptic currents (IPSCs) onto 51 pyramidal cells. We show that EPORs are expressed on pyramidal cells, therefore the impact 52 of EPO on GABAergic maturation is likely to be indirect. Our data show that EPO can 2 53 modulate hippocampal network maturation and support ongoing trials of the use of EPO in 54 clinical neonatology to stimulate neuronal maturation after perinatal brain injury. 55 Introduction 56 Perinatal brain injury (PBI) might lead to psychiatric disorders associated with alterations in 57 GABAergic transmission (Marin 2012, Cunha-Rodrigues, Balduci et al. 2018, Lacaille, 58 Vacher et al. 2019). A significant reduction of several markers of GABAergic transmission, 59 including glutamic acid decarboxylase (GAD), GABA, GABAA receptors (GABAARs), and 60 perturbed parvalbumin (PV) and somatostatin (SST) expression in cortical interneurons, 61 have been reported in the neonatal brain after injury (Robinson, Li et al. 2006, Komitova, 62 Xenos et al. 2013). In addition, postmortem samples from human preterm infants with brain 63 injury, as well as the hippocampus of rat models of prematurity, showed reduced potassium- 64 chloride co-transporter 2 (KCC2) expression (Jantzie, Getsy et al. 2014). Perturbations of the 65 GABAergic system in PBI might disrupt the excitatory/inhibitory balance and lead to long- 66 lasting deficits in brain function (Komitova, Xenos et al. 2013). Therefore, there is an urgent 67 need for new therapeutic strategies protecting the GABAergic system in clinical neonatology. 68 Erythropoietin (EPO), the erythropoietic hormone (Farrell and Lee 2004). is a leading therapy 69 in neonatology as a neuroprotective agent (Juul, Mayock et al. 2015, Juul and Pet 2015, 70 Natalucci, Latal et al. 2016). EPO signaling, leads to activation of several downstream 71 pathways including the STAT5, ERK1/2 and PI3K/Akt pathways (Lombardero, Kovacs et al. 72 2011). EPO’s immediate neuroprotective effects are anti-apoptotic, anti-inflammatory, and 73 anti-oxidative (Noguchi, Asavaritikrai et al. 2007, Rangarajan and Juul 2014). In the long- 74 term, EPO stimulates angiogenesis (Zhu, Bai et al. 2014), neurogenesis (Castaneda- 75 Arellano, Beas-Zarate et al. 2014) and oligodendrogenesis (Jantzie, Miller et al. 2013, Juul, 76 Mayock et al. 2015). EPO has also been shown to restore deficits in potasium-chloride 77 cotransporter 2 (KCC2) expression (Jantzie, Getsy et al. 2014), and enhance synaptic 78 plasticity and cognition (Adamcio, Sargin et al. 2008, Kamal, Al Shaibani et al. 2011, Sargin, 79 El-Kordi et al. 2011, Almaguer-Melian, Merceron-Martinez et al. 2015), while facilitating 3 80 inhibitory synaptic transmission (Wojtowicz and Mozrzymas 2008, Roseti, Cifelli et al. 2020). 81 Nevertheless, it is not yet established whether EPO promotes the development of GABAergic 82 neurons and GABAergic neurotransmission. 83 EPO and its receptor (EPOR) are expressed in human and mouse brain (Digicaylioglu, 84 Bichet et al. 1995, Marti, Wenger et al. 1996). Specifically, they are reported to be expressed 85 in the embryonic neocortex in areas close to ventricles and deeper layers, regulating radial 86 migration and laminar positioning of granular neurons (Constanthin, Contestabile et al. 87 2020). Here we report that EPORs are highly expressed postnatally in the cornu ammonis 88 (CA)1 hippocampus from mice, increasing their expression to reach a zenith towards 89 adulthood. Additionally, we showed in a transgenic mouse line constitutively overexpressing 90 human EPO in the CNS, without hematopoietic changes (Tg21) (Wiessner, Allegrini et al. 91 2001), a strong activation of the AKT pathways in the postnatal CA1 hippocampus (Jacobs 92 R. A. et al, Comm biology, in revision). AKT phosphorylation has a strong antiapoptotic 93 action and can increase the number of GABAARs on the plasma membrane increasing 94 synaptic transmission in neurons (Wang, Liu et al. 2003). Therefore, we hypothesize an 95 important role for EPO signaling in postnatal hippocampal GABAergic maturation. 96 During the second postnatal week, GABAergic transmission in the cornu ammonis (CA) 97 hippocampus switches from excitatory, due to elevated intracellular chloride concentration, to 98 inhibitory at around postnatal days (P)13-15 (Tyzio, Holmes et al. 2007), an age that 99 coincides with a peak in synaptogenesis, and the formation of adult neuronal networks (Ben- 100 Ari, Gaiarsa et al. 2007). GABAergic transmission is essential for establishing critical periods 101 of enhanced synaptic plasticity during development (Hensch and Bilimoria 2012). 102 Perineuronal nets (PNNs) are specialized extracellular matrix (ECM) structures composed of 103 chondroitin sulfate proteoglycans that are responsible for synaptic stabilization, a process 104 that influences the closing of critical periods of plasticity. In the hippocampus PNNs are found 105 around the somata and proximal dendrites