This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular Proteolytic Processing of Neurexins by Presenilins Sustains Synaptic Vesicle Release Emilia Servián-Morilla1,2, Estefanía Robles-Lanuza1,2, Ana C. Sánchez-Hidalgo1,2, Rafael J. Camacho- Garcia1,2, Juan A. Paez-Gomez1,2, Fabiola Mavillard1,2,3, Carlos A. Saura3,4, Amalia Martinez-Mir1 and Francisco G. Scholl1,2 1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Sevilla, Spain 2Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, 41009 Sevilla, Spain 3Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 4Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain DOI: 10.1523/JNEUROSCI.1357-17.2017 Received: 18 May 2017 Revised: 3 November 2017 Accepted: 26 November 2017 Published: 11 December 2017 Author contributions: E.S.-M., E.R.-L., and F.G.S. designed research; E.S.-M., E.R.-L., A.C.S.-H., and R.J.C.- G. performed research; E.S.-M., E.R.-L., A.C.S.-H., J.A.P.-G., A.M.-M., and F.G.S. analyzed data; F.M. and C.A.S. contributed unpublished reagents/analytic tools; A.M.-M. and F.G.S. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was funded by grants from Junta de Andalucía (P11-CVI-7599) and MINECO (BFU2015-71464-R), co-funded by FEDER funds. E. S-M was a recipient of a fellowship from Junta de Andalucía. E. R-L is a fellow from V Plan Propio de Investigación (Universidad de Sevilla) and A. C. S-H received a fellowship from Junta de Andalucía. Part of the study was performed at CITIUS (Universidad de Sevilla). We thank J. Shen for providing fPS1/fPS1 and PS2 -/- mice and Cre constructs, D. Selkoe for PS1 vectors, L. Lagnado for the gift of the SypHy construct, and F.J. Morón from the IBiS Genomics Service for technical support. The authors wish to thank Dr. Rafael Fernández-Chacón for discussion and critical reading of the manuscript. Correspondence should be addressed to Francisco G. Scholl, Instituto de Biomedicina de Sevilla (IBiS), Avda Manuel Siurot s/n, 41013 Sevilla, Spain. E-mail address: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1357-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/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 © 2017 the authors 1 Proteolytic Processing of Neurexins by Presenilins Sustains Synaptic 2 Vesicle Release 3 Abbreviated title: Processing of Neurexins and Presynaptic Release 4 Emilia Servián-Morilla1,2, Estefanía Robles-Lanuza1,2*, Ana C. Sánchez- 5 Hidalgo1,2*, Rafael J. Camacho-Garcia1,2, Juan A. Paez-Gomez1,2, Fabiola 6 Mavillard1,2,3, Carlos A. Saura3,4, Amalia Martinez-Mir1, Francisco G. Scholl1,2, 7 8 1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del 9 Rocío/CSIC/Universidad de Sevilla, 41013 Sevilla, Spain 10 2Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, 11 Universidad de Sevilla, 41009 Sevilla, Spain 12 3Centro de Investigación Biomédica en Red sobre Enfermedades 13 Neurodegenerativas (CIBERNED), Madrid, Spain 14 4Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, 15 Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 16 *E.R.-L., A.C.S.-H. contributed equally to this work 17 Correspondence should be addressed to Francisco G. Scholl, Instituto de 18 Biomedicina de Sevilla (IBiS), Avda Manuel Siurot s/n, 41013 Sevilla, Spain. E- 19 mail address: [email protected] 20 21 Number of pages: 44 22 Number of figures: 9 23 Number of words for Abstract: 194 24 Number of words for Introduction: 650 25 Number of words for Discussion: 1417 1 26 The authors declare no competing financial interests 27 28 Acknowledgements 29 This work was funded by grants from Junta de Andalucía (P11-CVI-7599) and 30 MINECO (BFU2015-71464-R), co-funded by FEDER funds. E. S-M was a 31 recipient of a fellowship from Junta de Andalucía. E. R-L is a fellow from V Plan 32 Propio de Investigación (Universidad de Sevilla) and A. C. S-H received a 33 fellowship from Junta de Andalucía. Part of the study was performed at CITIUS 34 (Universidad de Sevilla). We thank J. Shen for providing fPS1/fPS1 and PS2 -/- 35 mice and Cre constructs, D. Selkoe for PS1 vectors, L. Lagnado for the gift of 36 the SypHy construct, and F.J. Morón from the IBiS Genomics Service for 37 technical support. The authors wish to thank Dr. Rafael Fernández-Chacón for 38 discussion and critical reading of the manuscript. 2 39 Abstract 40 Proteolytic processing of synaptic adhesion components can accommodate the 41 function of synapses to activity-dependent changes. The adhesion system 42 formed by Neurexins (Nrxns) and Neuroligins (Nlgns) bi-directionally 43 orchestrate the function of pre- and postsynaptic terminals. Previous studies 44 have shown that Presenilins (PS), components of the gamma-secretase 45 complex frequently mutated in familial Alzheimer’s disease, clear from 46 glutamatergic terminals the accumulation of Neurexin C-terminal fragments 47 (Nrxn-CTF) generated by ectodomain shedding. Here, we characterized the 48 synaptic consequences of the proteolytic processing of Nrxns in cultured 49 hippocampal neurons from mice and rats of both sexes. We show that 50 activation of presynaptic Nrxns with postsynaptic Neuroligin-1 (Nlgn1) or 51 inhibition of ectodomain shedding in axonal Nrxn1-beta increases presynaptic 52 release at individual terminals, likely reflecting an increase in the number of 53 functional release sites. Importantly, inactivation of PS inhibits presynaptic 54 release downstream of Nrxn activation, leaving synaptic vesicle recruitment 55 unaltered. Glutamate-receptor signaling initiates the activity-dependent 56 generation of Nrxn-CTF, which accumulates at presynaptic terminals lacking PS 57 function. Notably, the sole expression of Nrxn-CTF decreases presynaptic 58 release and calcium flux, recapitulating the deficits due to loss of PS function. 59 Our data indicate that inhibition of Nrxn processing by PS is deleterious to 60 glutamatergic function. 61 1 62 Significance statement 63 To gain insight into the role of Presenilins in excitatory synaptic function, we 64 address the relevance of the proteolytic processing of presynaptic Neurexins in 65 glutamatergic differentiation. Using synaptic fluorescence probes in cultured 66 hippocampal neurons we report that trans-synaptic activation of Neurexins 67 produces a robust increase in presynaptic calcium levels and neurotransmitter 68 release at individual glutamatergic terminals by a mechanism that depends on 69 normal PS activity. Abnormal accumulation of Neurexin C-terminal fragment 70 resulting from impaired Presenilin activity inhibits presynaptic calcium signal 71 and neurotransmitter release, assigning synaptic defects to Neurexins as a 72 specific Presenilin substrate. These data may provide links into how loss of 73 Presenilin activity inhibits glutamatergic synaptic function in Alzheimer’s disease 74 patients. 75 2 76 Introduction 77 Interaction between synaptic adhesion proteins mediates the formation and 78 function of synaptic contacts, as they organize supramolecular complexes at 79 both sides of the synapse. Proteolytic processing of synaptic proteins is a post- 80 translational mechanism that regulates the function of synapses upon activity- 81 dependent changes, whereas impairment of normal proteolytic processing often 82 results in synaptic dysfunction (Saura et al., 2004; Nagy et al., 2006; Huntley, 83 2012; Prox et al., 2013). Neurexins (Nrxn1, 2, 3) are presynaptic membrane 84 proteins that link neurotransmitter release with adhesion to postsynaptic 85 partners. The use of different promoters and alternative splicing at the 86 extracellular domain originate over one thousand Nrxn isoforms (Tabuchi and 87 Sudhof, 2002; Schreiner et al., 2014; Fuccillo et al., 2015; Schreiner et al., 88 2015). The highly polymorphic extracellular domain of Nrxns engages in trans- 89 synaptic interactions with postsynaptic proteins, whereas the conserved 90 cytoplasmic domain recruits synaptic ligands and regulates neurotransmitter 91 release (Boucard et al., 2005; Chih et al., 2006; Ko et al., 2009; Rabaneda et 92 al., 2014; Aoto et al., 2015). Neuroligins (Nlgn1, 2, 3, 4X, 4Y) are postsynaptic 93 partners of Nrxns that regulate several aspects of glutamatergic and GABAergic 94 differentiation (Ichtchenko et al., 1995; Baudouin and Scheiffele, 2010). At 95 glutamatergic synapses, Nlgn1 increases synapse density and stimulates 96 neurotransmitter release probability by a mechanism that depends on trans- 97 synaptic interaction with Nrxns (Dean et al., 2003; Chih et al., 2006; Futai et al., 98 2007). Mutations in NLGN3, NLGN4X and NRXN1 genes have been identified 99 in neurodevelopmental disorders, including autism and schizophrenia (Jamain 100 et al., 2003; Laumonnier et al., 2004; Kim et al., 2008; Rujescu et al., 2009; 3 101 Camacho-Garcia et al., 2012; Camacho-Garcia et al., 2013; Lowther et al., 102 2017), suggesting a causative role for synaptic dysfunction of Nrxns and Nlgns 103 in brain diseases. 104 Apart from their role in neurodevelopmental disorders, recent findings 105 suggest a malfunctioning of the Nrxn-Nlgn1 glutamatergic pathway in age- 106 associated diseases such as Alzheimer’s disease (AD) (Martinez-Mir et al., 107 2013; Bie et al., 2014; Sindi et al., 2014; Tristan-Clavijo et al., 2015). We and 108 others have shown that Presenilins (PS1, 2), the catalytic subunit of the 109 gamma-secretase complex mutated in familial AD (FAD), cleave the C-terminal 110 fragment (CTF) of Nlgn1 and Nrxn isoforms (Bot et al., 2011; Saura et al., 2011; 111 Peixoto et al., 2012; Suzuki et al., 2012).