Proteolytic Processing of Neurexins by Presenilins Sustains Synaptic Vesicle Release

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.

Research Articles: Cellular/Molecular

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research; E.S.-M., E.R.-L., A.C.S.-H., J.A.P.-G., A.M.-M., and F.G.S. analyzed data; F.M. and C.A.S. contributed unpublished reagents/analytic tools; A.M.-M. and F.G.S. wrote the paper.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

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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,

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]

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

26 The authors declare no competing financial interests

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.

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.

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.

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;

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). Nrxn-CTF accumulates at

112 glutamatergic terminals of cultured hippocampal neurons defective in

113 PS/gamma-secretase activity and at presynaptic fractions of PS1 and PS2

114 conditional double knock-out (PS cDKO) mice that lack PS expression in

115 forebrain glutamatergic neurons (Saura et al., 2004; Saura et al., 2011). FAD-

116 mutations in PS1 are thought to act by a partial loss of function mechanism

117 (Shen and Kelleher, 2007; Xia et al., 2015; Sun et al., 2017). Interestingly,

118 several FAD-linked PS1 mutants fail to clear Nrxn-CTF (Bot et al., 2011; Saura

119 et al., 2011). Thus, based on the accumulation of Nrxn-CTF by loss of PS

120 function, an unexplored hypothesis is that failure of the proteolytic processing of

121 Nrxns mediates synaptic defects at glutamatergic terminals lacking PS activity.

122 In this study, we show that proteolytic processing of Nrxns by PS is

123 required to maintain synaptic vesicle release at glutamatergic terminals. Using

124 fluorescent synaptic probes, we found that Nlgn1 increases presynaptic release

125 and evoked-calcium levels at contacting terminals. Genetic inactivation of PS or

126 inhibition of PS/gamma-secretase activity decreases synaptic vesicle release

127 and presynaptic calcium levels at glutamatergic terminals induced by the Nrxn-

128 Nlgn1 complex. However, synapse formation and recruitment of synaptic

129 vesicles induced by Nlgn1 were not affected by inhibition of PS function. We

130 found that the production of Nrxn-CTF is initiated by an activity-dependent

131 mechanism that requires NMDA/AMPA receptor function. By mutational

132 screening, we identified a conserved region in the juxtamembrane domain of

133 Nrxn1 responsible for the generation of Nrxn-CTF. Importantly, the sole

134 expression of exogenous Nrxn-CTF at presynaptic terminals decreased evoked

135 presynaptic release and activity-dependent calcium levels in neurons with

136 unaltered PS function, thereby mimicking the effect of the loss of PS function at

137 glutamatergic synapses.

138

139 Materials and Methods

140 DNA AND LENTIVIRAL VECTORS

141 For expression experiments, pCAG-GS and lentiviral vectors encoding DsRed,

142 HA-Nrxn1-beta, HA-Neuroligin1(AB), SypHy, hPS1 and PS1 D385A were

143 previously described (Taniguchi et al., 2007; Saura et al., 2011; Rabaneda et

144 al., 2014). Mutants harboring deletions in the juxtamembrane domain of human

145 Nrxn1 were generated by PCR with specific forward primers containing a SacI

146 site and a common reverse primer containing a BglII site at 5’ ends. PCR

147 products were digested with SacI-BglII and cloned into human HA-Nrxn1-beta

148 (Camacho-Garcia et al., 2012). For the generation of HA-Nrxn-CTF, a PCR-

149 fragment coding for the 85 C-terminal residues of human Nrnx-1 was inserted in

150 frame downstream of a Nlgn1 signal peptide followed by an HA-tag and cloned

151 into pCAG-GS. An HA-Nrxn-CTF'PDZ construct lacking the three C-terminal

152 residues of human Nrxn1 was generated by PCR with specific primers. The

153 lentiviral vectors driving Cre and 'Cre expression under the ubiquitin C

154 promoter were previously described to efficiently inactivate PS1 in fPS1/fPS1;

155 PS2 -/- primary neurons (Ho et al., 2006; Watanabe et al., 2009). For the

156 generation of the SyGCaMP3 construct, the DNA fragment coding for GCaMP2

157 in SyGCaMP2 vector (Addgene, reference #26124, Cambridge, Massachusetts,

158 USA) (Dreosti et al., 2009) was exchanged by a fragment coding for GCaMP3

159 (Addgene, reference #22692) (Tian et al., 2009). For lentiviral expression, HA-

160 Nrxn-CTF, HA-Nrxn-CTF'PDZ and SyGCaMP3 constructs were inserted in a

161 lentiviral vector containing the human synapsin promoter (Gascon et al., 2008).

162 Generation of recombinant lentiviral particles and infection of hippocampal

163 cultures with concentrated lentivirus were performed as previously described

164 (Gascon et al., 2008).

165

166 CELL CULTURE, TRANSFECTION AND IMMUNOFLUORESCENCE

167 Hippocampal cultures were obtained from E18-E19 rat brains or from E18-E19

168 fPS1/fPS1; PS2 -/- mouse embryos of either sex (RRID:MGI:3777984,

169 RRID:MGI:2664243). Dissociated cells were plated at 35.000 cells/cm2 on poly-

170 D-lysine (Sigma-Aldrich, St. Louis, MO, USA) coated glass coverslips and

171 maintained in neurobasal medium (Invitrogen, Carlsbad, CA, USA)

172 supplemented with B27 (Thermo Fisher Scientific, Boston, MA, USA), glutamax

173 and penicillin/streptomycin (Invitrogen). HEK293T cultures (RRID:CVCL_0063)

174 were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal

175 bovine serum (HyClone, Logan, UT, USA), glutamax and

176 penicillin/streptomycin. Cultured hippocampal neurons and HEK293T cells were

177 transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer’s

178 recommendations. For immunofluorescence experiments, cultures were fixed

179 with 4% paraformaldehyde (PFA) and permeabilized with 0.05% Triton X-100

180 (Sigma-Aldrich). The following primary antibodies were used: rat anti-HA

181 (1:1,000; clone 3F10; Roche, Basel, Switzerland; RRID:AB_390918), mouse

182 anti-CASK (1:200; BD Transduction Lab, Franklin Lakes, NJ, USA;

183 RRID:AB_398103) and rabbit anti-VGluT1 (1:1,500; Synaptic Systems,

184 Göttingen, Germany; RRID:AB_887877). A chicken anti cyto-neurexin antibody

185 has been previously described (Dean et al., 2003). As secondary antibodies,

186 preabsorbed donkey antibodies conjugated with Cy2, Cy3 and Cy5

187 fluorophores were used (1:800; Jackson ImmunoResearch, West Grove, PA,

188 USA; RRID:AB_2340674 rat cy2; RRID:AB_2340667 rat cy3;

189 RRID:AB_2340672 rat cy5; RRID:AB_2315777 mouse cy3; RRID:AB_2340607

190 rabbit cy5; RRID:AB_2340365 chicken cy5). Images were acquired on a Leica

191 TCS SP2 laser scanning spectral confocal microscope. Maximal projections of

192 Z-stacked images were analyzed with ImageJ software (RRID:SCR_003070).

193 Synaptic markers were quantified as the average intensity and the relative

194 number of pixels per cell area above a threshold value. Thresholds were set

195 such that most of the specific signal was selected. Masks covering the area of

196 interest were created by drawing a line around the transfected cells. The

197 intensity and number of positive pixels normalized to the cell area were

198 averaged in each experimental condition.

199

200 BIOCHEMICAL ANALYSIS

201 Cell cultures were homogenized in lysis buffer (50 mM Tris-HCl pH 7.4; 100 mM

202 NaCl; 5 mM MgCl2; 1% Triton X-100 and 0.1% SDS) containing a protease

203 inhibitor cocktail (Roche). When indicated, cells were treated with GM6001

204 (Calbiochem, San Diego, CA, USA) and N-[N-(3,5-difluorophenacetyl-L-alanyl]-

205 S-phenylglycine t-butyl ester (DAPT; Sigma-Aldrich). For immunoprecipitation

206 experiments, cell lysates or conditioned media were incubated with the HA

207 antibody. The immune complexes were isolated with Protein-G Sepharose and

208 resolved in SDS-PAGE gels. Western-blot experiments of immunoprecipitates

209 or lysates containing equal-protein loading were performed using the following

210 primary antibodies: rat anti-HA (1:500); mouse anti-CASK (1:200); chicken anti

211 cyto-neurexin (1:200); rabbit anti-neurexin 1/2/3 against the common

212 cytoplasmic tail (1:500; Synaptic Systems; RRID:AB_10697815); rabbit anti-

213 presenilin 1 N-terminal (1-65) (1:5,000; Calbiochem, Merck Millipore, Burlington,

214 MA, USA; RRID:AB_10682152) and rabbit anti-α actin (1:1,000; Sigma-Aldrich;

215 RRID:AB_476693). Immunoreactivity was detected with secondary antibodies

216 conjugated with horseradish peroxidase (1:5,000; Jackson ImmunoResearch;

217 RRID:AB_2313567 rabbit; RRID:AB_2339290 chicken; RRID:AB_2338128 rat;

218 RRID:AB_10015289 mouse). Chemiluminiscence was developed using Immun-

219 Star WesternC Kit (Bio-Rad, Hercules, CA, USA) or SuperSignal West Femto

220 Maximum Sensitivity Substrate (Thermo Fisher Scientific) on an ImageQuant

221 LAS4000 Mini (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

222

223 CELL IMAGING

224 Imaging experiments in hippocampal neurons expressing synaptic probes were

225 performed as previously described (Rabaneda et al., 2014). Briefly, cultures

226 were perfused with extracellular solution (2.5 mM KCl, 119 mM NaCl, 2 mM

227 CaCl2, 2 mM MgCl2, 25 mM HEPES, 30 mM glucose, pH 7.4) for SypHy or (2.5

228 mM KCl, 136 mM NaCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM HEPES, 10 mM

229 glucose, pH 7.4) for SyGCamP3, containing 0.05 mM CNQX and 0.01 mM APV

230 (Ascent Scientific, Cambridge, UK). Electrical field stimulation was delivered

231 with a 2100 isolated pulse stimulator (AM systems, Sequim, WA, USA).

232 Fluorescence images were acquired on an Axio Observer.A1 inverted

233 microscopy (Zeiss, Oberkochen, Germany) with a 63x Zeiss Plan-Apochromat

234 oil immersion objective. Images were captured with a Hamamatsu Orca R2

235 camera controlled by Micro-Manager software (Edelstein et al., 2010,

236 RRID:SCR_000415). Neurons were stimulated with 5 trains of 1, 3 and 5 AP for

237 SyGCaMP3 experiments or with 40 and 300 AP for SypHy experiments, both

238 delivered at 20 Hz using a biphasic pulse of 10 mA and 5 ms of duration. Time-

239 lapse images were acquired at 4 fps (SyGCaMP3) or 1 fps (SypHy), 672 x 512

240 pixels (binned 2 x 2), with an exposure time of 100 ms. Routinely, 3-5 fields of

241 data sets were recorded from each coverslip. Responding synapses were

242 selected manually as Region of Interest (ROI) with Time Series Analyzer plugin

243 of ImageJ software (RRID:SCR_014269). The standard size of the selected

244 ROIs was 1 μm2. Background noise within each experiment was quantified as

245 the average intensity of 5 non-responding regions and subtracted from each

246 responding ROI. The increase in fluorescence (ΔF) was calculated in the

247 selected ROIs.

248

249 EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS

250 Experimental design for imaging approaches using fluorescent synaptic probes

251 is summarized in Figures 1, 2, 7 and 8. In Figure 1E-G (12 experiments

252 obtained from 2 independent cultures), n = 355 for internal control synapses, n

253 = 407 for Nlgn1 synapses, n = 200 for internal 'Cre synapses, n = 322 for

254 'Cre synapses, n = 271 for internal PS cDKO synapses, n = 443 for

255 Nlgn1 PS cDKO synapses. In Figure 2B-D (21 experiments obtained from 3

256 independent cultures), n = 841 for internal control synapses, n = 854 for Nlgn1

257 synapses, n = 700 for internal DAPT synapses, n = 684 for DAPT Nlgn1

258 synapses. In Figure 2F-H (10 experiments obtained from 2 independent

259 cultures) n = 383 for internal control synapses, n = 387 for Nlgn1 synapses, n =

260 332 for internal PS1 D385A synapses, n = 348 for PS1 D385A Nlgn1 synapses.

261 In Figure 7C (23 experiments from 3 independent cultures) n = 210 for control

262 synapses, n = 196 for HA-Nrxn-CTF'PDZ synapses, n = 196 for HA-Nrxn-CTF

263 synapses. In Figure 8B (6 experiments from 2 independent cultures) n = 55 for

264 internal control synapses, n = 133 for Nlgn1 synapses, n = 45 for internal ΔCre

265 synapses, n = 61 for Nlgn1 ΔCre synapses, n = 64 for internal PS cDKO

266 synapses, n = 142 for Nlgn1 PS cDKO synapses. In Figure 8D (13 experiments

267 from 2 independent cultures) n = 133 for control synapses, n = 135 for HA-Nrxn-

268 CTF'PDZ synapses, n = 97 for HA-Nrxn-CTF synapses. In Figure 9D (10

269 experiments from 2 independent cultures) n = 91 for control synapses, n = 267

270 for HA-Nrxn1-beta synapses, n = 213 for HA-Nrxn1-beta Δ368-393 synapses.

271 Statistical analyses were performed with GraphPad Prism software

272 RRID:SCR_002798). For comparison of two groups, the two-tailed Student's t

273 test or the Mann-Whitney U-test were applied. Differences in means across

274 multiple groups were analyzed using one-way ANOVA. When the ANOVA

275 analysis revealed significant differences, the post hoc Bonferroni's test was

276 used for pairwise comparisons. When parametric assumptions were not met,

277 Kruskal-Wallis followed by the post hoc Dunn’s test was used. All data are

278 presented as mean ± SEM.

279

280 Results

281 PS function is essential for presynaptic release at glutamatergic terminals

282 mediated by Nrxn-Nlgn1

283 Following the shedding of the extracellular domain of Nrxn isoforms by

284 metalloproteases, Nrxn-CTF is subsequently processed by PS/gamma-

285 secretase at glutamatergic terminals (Saura et al., 2011). However, the

286 functional consequences of the proteolytic processing of Nrxns at glutamatergic

287 synapses are not known. Specifically, as the cytoplasmic tail of Nrxns regulates

288 neurotransmitter release (Futai et al., 2007; Rabaneda et al., 2014; Aoto et al.,

289 2015), we speculated that accumulation of Nrxn-CTF due to impaired PS

290 function might interfere with synaptic vesicle release. For that, we developed an

291 approach that allows for the analysis of presynaptic release at glutamatergic

292 terminals mediated by the Nrxn-Nlgn1 complex in control and PS cDKO

293 hippocampal neurons (Fig. 1A). PS cDKO neurons were generated by Cre-

294 dependent ablation of PS1 in fPS1/fPS1; PS2 -/- hippocampal cultures. As

295 expected, Nrxn-CTF bands recognized by a cyto-neurexin antibody

296 accumulated in PS cDKO neurons, but not in cultures infected with an inactive

297 'Cre, by a PS-dependent proteolytic processing (Fig.1B). Control and PS cDKO

298 hippocampal cultures were infected at 3 days in vitro (DIV) with lentivirus

299 expressing SypHy, a pH-sensitive probe that measures exo/endocytosis of

300 synaptic vesicles (Granseth et al., 2006). Following infection, the cultures were

301 co-transfected at 11-12 DIV with DsRed and a N-terminal HA-tagged Nlgn1.

302 Thus, Nlgn1 co-expressed in DsRed-dendrites binds to endogenous Nrxns in

303 contacting axons and the activity of the resulting glutamatergic terminals

304 containing SypHy can be analyzed upon stimulation (Fig. 1C).

305 Previous reports using electrophysiological recordings in hippocampal

306 slices have shown that Nlgn1 increases release probability by a retrograde

307 mechanism (Futai et al., 2007). Because release probability positively correlates

308 with the size of the readily releasable pool (RRP) of synaptic vesicles, which

309 corresponds to the number of docked vesicles at presynaptic active zones

310 (Murthy and Stevens, 1999; Murthy et al., 2001; Schikorski and Stevens, 2001),

311 we measured the RRP with a stimulus train of 40 action potentials (AP) at 20

312 Hz, followed by 300 AP at the same frequency to mobilize the recycling pool

313 (RP). Presynaptic response at Nlgn1 synapses was compared in each

314 experimental condition with the response of internal synapses, which were

315 identified in the same fields as SypHy-positive puncta not contacting

316 Nlgn1/DsRed transfected dendrites. Interestingly, postsynaptic expression of

317 Nlgn1 in control cultures induced a a 3-fold increase in presynaptic response at

318 40 and 300 AP compared with neighboring internal synapses ('F ratio for

319 Nlgn1/internal synapses at 40 AP: 2.96; 'F ratio for Nlgn1/internal synapses at

320 300 AP: 3.54) (Fig. 1D,E,G). These data indicated that Nlgn1 proportionally

321 increases the size of the RRP and the RP at contacting terminals. Remarkably,

322 the positive effect of Nlgn1 increasing presynaptic release at 40 and 300 AP

323 was strongly reduced in PS cDKO neurons (normalized 'F at 40 AP in Nlgn1

324 synapses: 2.963 r 0.074; normalized 'F at 40 AP in Nlgn1 PS cDKO synapses:

325 1.658 r 0.055, p<0.0001, Kruskal-Wallis followed by the post hoc Dunn’s test;

326 normalized 'F at 300 AP in Nlgn1 synapses: 10.535 r 0.272; normalized 'F at

327 300 AP in Nlgn1 PS cDKO synapses: 4.183 r 0.141, p<0.0001, Kruskal-Wallis

328 followed by the post hoc Dunn’s test) (Fig. 1D,E,G). However, presynaptic

329 release at internal synapses were not affected at 40 AP or slightly reduced at

330 300 AP in PS cDKO neurons (normalized 'F at 40 AP in internal control

331 synapses: 1.0 r 0.025; normalized 'F at 40 AP in internal PS cDKO synapses:

332 1.009 r 0.047, p>0.999, Kruskal-Wallis followed by the post hoc Dunn’s test;

333 normalized 'F at 300 AP in internal control synapses: 2.975 r 0.085;

334 normalized 'F at 300 AP in internal PS cDKO synapses: 2.509 r 0.110,

335 p=0.048, Kruskal-Wallis followed by the post hoc Dunn’s test) (Fig. 1D,F,G).

336 The higher impact of the loss of PS function in Nlgn1 synapses was observed

337 even when presynaptic responses of similar magnitude were compared (40 AP

338 in Nlgn1 synapses vs 300 AP in internal synapses, Fig. 1). These findings

339 suggest a higher susceptibility of Nlgn1 synapses to the loss of PS function.

340 Moreover, neither Nlgn1 synapses nor internal synapses were affected in

341 neurons infected with inactive 'Cre, used as a negative control (Fig. 1D-G).

342 These data indicate that Nrxn-Nlgn1 trans-synaptic signaling locally increases

343 the size of the RRP by a mechanism that is affected by the loss of PS function.

344

345 PS regulates presynaptic release by a proteolytic mechanism

346 PS genes have been proposed to regulate cellular signaling pathways by

347 gamma-secretase dependent and independent mechanisms (Tu et al., 2006;

348 Hass et al., 2009). To analyze whether PS regulates presynaptic release at

349 Nlgn1 synapses by a proteolytic activity or by an alternative mechanism, we

350 inhibited PS/gamma-secretase proteolytic activity, but not PS expression, using

351 the gamma-secretase inhibitor DAPT (1 PM) or expressing the dominant-

352 negative mutant PS1 D385A, two approaches that induce the accumulation of

353 Nrxn-CTF in hippocampal neurons (Saura et al., 2011). Interestingly, we found

354 that impairment of PS/gamma-secretase activity in hippocampal neurons

355 strongly reduced presynaptic release at 300 AP in Nlgn1 synapses in both,

356 cultures treated with DAPT (normalized 'F in Nlgn1 synapses: 3.472 r 0.054;

357 normalized 'F in DAPT Nlgn1 synapses: 2.350 r 0.050, p<0.0001, Kruskal-

358 Wallis followed by the post hoc Dunn’s test) and cultures expressing PS1

359 D385A (normalized 'F in Nlgn1 synapses: 3.159 r 0.082; normalized 'F in PS1

360 D385A Nlgn1 synapses: 1.585 r 0.057, p<0.0001, Kruskal-Wallis followed by

361 the post hoc Dunn’s test) (Fig. 2). Notably, presynaptic release at internal

362 synapses was not affected by DAPT treatment (normalized 'F in internal

363 synapses of control neurons: 1.0 r 0.023; normalized 'F in internal synapses of

364 DAPT neurons: 0.98 r 0.025, p>0.999, Kruskal-Wallis followed by the post hoc

365 Dunn’s test) or slightly reduced by the expression of PS1 D385A (normalized

366 'F in internal synapses of control neurons: 1.0 r 0.030; normalized 'F in

367 internal synapses of PS1 D385A neurons: 0.825 r 0.027, p=0.0072, Kruskal-

368 Wallis followed by the post hoc Dunn’s test) (Fig. 2). These data obtained with

369 genetic or chemical inhibitors of PS/gamma-secretase activity recapitulated the

370 effects observed in PS cDKO neurons, suggesting that loss of PS proteolytic

371 activity at Nrxn-Nlgn1 synapses accounts for the inhibitory effect on presynaptic

372 release.

373

374 PS function is dispensable for the recruitment of synaptic vesicles

375 induced by Nlgn1

376 Expression of Nlgn1 increases the recruitment of synaptic vesicles and the

377 density of presynaptic terminals in contacting axons (Scheiffele et al., 2000;

378 Dean et al., 2003). Therefore, we asked if PS activity is also required for the

379 synaptogenic function of Nlgn1 or rather loss of PS function specifically inhibits

380 presynaptic release. Quantitative analysis of SypHy, used as a synaptic vesicle

381 marker, by confocal microscopy showed an over 2-fold increase in SypHy

382 intensity at Nlgn1 synapses compared to internal synapses in control neurons

383 (normalized SyPhy intensity in internal synapses: 1.0 r 0.007; normalized

384 SyPhy intensity in Nlgn1 synapses: 2.194 r 0.027, p<0.0001, Mann-Whitney U-

385 test) (Fig. 3). However, inhibition of PS function did not affect the area or the

386 intensity of presynaptic SypHy recruited at Nlgn1 synapses in none of the three

387 experimental approaches studied, namely PS cDKO hippocampal neurons (Fig.

388 3A-C) and hippocampal cultures treated with DAPT or expressing PS1 D385A

389 (Fig. 3D-I). In agreement with these data, the levels of the endogenous synaptic

390 vesicle marker VGluT1 recruited by Nlgn1 were not affected in PS cDKO

391 neurons or in neurons treated with DAPT (data not shown). Moreover, the levels

392 of transfected Nlgn1 in PS defective neurons were not different from control

393 neurons in any of the different approaches (Fig. 3C,F,I). These data further

394 support that the inhibition in presynaptic release caused by inactivation of PS is

395 not an indirect consequence of reduced synaptic vesicle recruitment or Nlgn1

396 expression, pointing at a presynaptic mechanism. Moreover, the

397 immunofluorescence signal obtained with a cyto-neurexin antibody was

398 increased at Nlgn1 synapses of PS cDKO neurons compared with Nlgn1

399 synapses of control cultures (Fig. 3J,K). These data suggested local

400 accumulation of Nrxn-CTF at glutamatergic terminals of PS defective neurons,

401 in agreement with previous results (Saura et al., 2011). The levels of CASK, a

402 synaptic scaffolding protein that can interact with a PDZ-binding domain in the

403 cytoplasmic tail of Nrxns (Butz et al., 1998), were significantly increased at

404 Nlgn1 synapses of PS cDKO neurons (Fig. 3J,L). These results indicate that the

405 proteolytic activity of the PS/gamma-secretase complex plays a selective role in

406 the release of synaptic vesicles, which can be dissected apart from the

407 recruitment of synaptic vesicles and the synapse-formation activity induced by

408 Nlgn1.

409

410 Activity-dependent processing of Nrxns

411 The previous results suggested the generation of a PS/gamma-secretase

412 substrate that abnormally accumulates at Nrxn-Nlgn1 synapses with impaired

413 PS activity and ultimately mediates the inhibition in presynaptic release. The

414 accumulation of Nrxn-CTF at glutamatergic terminals makes it a strong

415 candidate to mediate the inhibitory effect of PS in neurotransmitter release. It

416 has been previously reported that depolarization with KCl or incubation with

417 glutamate induces the generation of Nrxn-CTF in cultured neurons (Bot et al.,

418 2011). To further analyze the mechanism responsible for the activity-dependent

419 proteolytic processing of Nrxns, we depolarized hippocampal cultures with 50

420 mM KCl for 30 minutes and evaluated the levels of Nrxn-CTF by western-blot.

421 We confirmed that stimulation with KCl resulted in over 2-fold increase in Nrxn-

422 CTF levels compared to non-stimulated cultures (Fig. 4). Inhibition of the

423 PS/gamma-secretase complex with DAPT (1 PM) further increased the levels of

424 Nrxn-CTF in cultures stimulated with KCl, indicating activity of the PS/gamma-

425 secretase complex during synaptic stimulation. Importantly, we found that

426 incubation with the broad metalloprotease inhibitor GM6001 (2.5 PM) abolished

427 the KCl-induced accumulation of Nrxn-CTF (Fig. 4). These data suggest that

428 the cleavage of Nrxn ectodomain by metalloproteases initiates the activity-

429 dependent production of Nrxn-CTF, which is subsequently processed by PS.

430 We next tested the effect of several antagonists in the activity-dependent

431 generation of Nrxn-CTF. Inhibition of sarco-ER calcium ATPase (SERCA)

432 activity with thapsigargin (4 PM), which results in depletion of calcium stores at

433 the ER, or blockage of voltage-dependent calcium-channels with Z-agatoxin-

434 IVA (100 nM) did not affect the activity-dependent accumulation of Nrxn-CTF

435 (Fig. 4). However, blocking NMDA and AMPA receptors with APV (50 PM) and

436 CNQX (10 PM) abolished the accumulation of Nrxn-CTF induced by activity

437 (Fig. 4). These experiments indicate that NMDA/AMPA receptor signaling is

438 required to exscind the ectodomain of Nrxns leading to the generation of Nrxn-

439 CTF, which is cleared form glutamatergic terminals by a PS/gamma-secretase

440 activity.

441

442 Identification of the cleavage site that generates Nrxn-CTF

443 To identify the residues responsible for the generation of Nrxn-CTF, we

444 produced deletions in the juxtamembrane region of human HA-Nrxn1-beta, a

445 known Nlgn1 presynaptic receptor, and analyzed the generation of the

446 ectodomain and Nrxn-CTF fragments (Fig. 5A). Immunoprecipitates of

447 conditioned media from transfected HEK293T cells showed that increasing the

448 size of deletions within residues 368-385 produced the release of HA-Nrxn1-

449 beta ectodomains of decreasing size, suggesting that the localization of the

450 cleavage site was further downstream (Fig. 5B). Furthermore, a larger deletion

451 comprising residues 368-393 inhibited the release of HA-Nrxn1-beta

452 ectodomain and increased the levels of the mature protein in cell lysates (Fig.

453 5B). In agreement with a sequential proteolytic processing, the generation of

454 Nrxn-CTF, both basal and accumulated in presence of DAPT, was strongly

455 inhibited when residues 368-393 were deleted (Fig. 5C). These data revealed a

456 major shedding site within residues 386-393 of Nrxn1-beta responsible for the

457 release of the ectodomain and the subsequent generation of Nrxn-CTF.

458 Intriguingly, deletion of residues 368-385 increased the levels of HA-Nrxn1-beta

459 ectodomain in the media, suggesting that residues adjacent to the shedding site

460 negatively regulate Nrxn1-beta shedding (Fig. 5B). Sequence alignment

461 showed high homology of the predicted Nrxn-CTFs originating from the different

462 Nrxn genes (Fig. 5D).

463 Next, we generated an HA-Nrxn-CTF expression vector encoding the last

464 85 C-terminal residues of Nrxn1 starting at V388 and tagged at the mature N-

465 terminus with an HA epitope (Fig. 6A). In parallel, we also generated HA-Nrxn-

466 CTF'PDZ, a mutant lacking the last three C-terminal residues essential in intact

467 Nrxns for the binding to PDZ-synaptic proteins (Butz et al., 1998; Grootjans et

468 al., 2000) (Fig. 6A). In western-blot experiments of transfected HEK293T cells,

469 the levels of HA-Nrxn-CTF and HA-Nrxn-CTF'PDZ proteins increased in the

470 presence of DAPT, as expected for a PS substrate (Fig. 6B). To confirm the

471 proteolytic processing in neurons, we expressed HA-Nrxn-CTF proteins in

472 hippocampal neurons and analyzed the expression in the presence of DAPT by

473 western-blot using anti-HA and cyto-neurexin antibodies. HA-Nrxn-CTF showed

474 a similar mobility than Nrxn-CTF originating from endogenous Nrxns or from the

475 expression of Nrxn1-beta in the same cultures (Fig. 6C). Moreover, the finding

476 that HA-Nrxn-CTF was readily detectable in untreated neurons and that its

477 levels further increased upon inhibition of PS activity, indicated accumulation of

478 HA-Nrxn-CTF in untreated neurons with otherwise normal PS activity. HA-Nrxn-

479 CTF, but not HA-Nrxn-CTF'PDZ, co-immunoprecipitated with CASK in

480 heterologous and neuronal cells (Fig. 6 D,E), indicating that the PDZ-binding

481 domain of Nrxn-CTF can mediate protein-protein interactions. These data

482 demonstrated that HA-Nrxn-CTF is a PS substrate comparable to endogenous

483 Nrxn-CTF and provided a molecular tool to evaluate the functional

484 consequences of the proteolytic activity of PS on a single substrate, Nrxns.

485

486 Nrxn-CTF inhibits presynaptic release

487 The effect of the accumulation of HA-Nrxn-CTF on presynaptic release was

488 analyzed in hippocampal neurons. We chose transfection experiments to

489 achieve expression in sparse neurons, so that the effect of HA-Nrxn-CTF on

490 presynaptic release can be readily attributable to the presynaptic neuron.

491 Moreover, within the transfected axons HA-Nrxn-CTF showed localization in

492 synaptic puncta (Fig. 7A), thereby resembling the presynaptic distribution of

493 endogenous Nrxn-CTF. Remarkably, presynaptic expression of HA-Nrxn-CTF

494 strongly inhibited evoked release at 40 and 300 AP, whereas the inhibitory

495 effect was attenuated in neurons expressing HA-Nrxn-CTF'PDZ despite similar

496 localization and expression levels (normalized 'F at 40 AP: control 1.0 r 0.060;

497 HA-Nrx-CTF 0.411 r 0.023, p<0.0001, Kruskal-Wallis followed by the post hoc

498 Dunn’s test; HA-Nrxn-CTF'PDZ 0.697 r 0.041, p=0.0008, Kruskal-Wallis

499 followed by the post hoc Dunn’s test; normalized 'F at 300 AP: control 3.580 r

500 0.178; HA-Nrxn-CTF 0.980 r 0.059, p<0.0001, Kruskal-Wallis followed by the

501 post hoc Dunn’s test; HA-Nrxn-CTF'PDZ 1.949 r 0.085, p<0.0001, Kruskal-

502 Wallis followed by the post hoc Dunn’s test) (Fig. 7B,C). CASK levels increased

503 in axons transfected with HA-Nrxn-CTF compared with control neurons and

504 reached intermediate levels in axons expressing HA-Nrxn-CTF'PDZ (Fig.

505 7D,E). These results indicated that presynaptic accumulation of Nrxn-CTF by

506 itself recapitulates the deficits in presynaptic release caused by the loss of PS

507 activity.

508

509 Nrxn-CTF mimics the inhibitory effect of PS on presynaptic calcium levels

510 Synaptic vesicle release depends on the levels of calcium in the presynaptic

511 active zone. To gain insight into the mechanism by which Nrxn-CTF impairs

512 neurotransmitter release, we measured presynaptic calcium using SyGCaMP3,

513 a fusion of the genetically encoded calcium indicator CaMP3 to the synaptic

514 vesicle resident protein synaptophysin (Dreosti et al., 2009; Tian et al., 2009).

515 Calcium imaging was performed in two experimental conditions that accumulate

516 Nrxn-CTF at presynaptic terminals, glutamatergic terminals induced by Nlgn1 in

517 PS cDKO neurons and hippocampal neurons transfected with exogenous HA-

518 Nrxn-CTF (Fig. 8). In each condition, presynaptic response was evoked with 1,

519 3 and 5 AP, a stimulus range within the linear response reported for SyGCaMP

520 probes (Tian et al., 2009; Li et al., 2011; Akerboom et al., 2012). Stimulation of

521 Nrxn signaling with postsynaptic Nlgn1 in control cultures locally increased

522 evoked presynaptic calcium signal compared with untransfected synapses (Fig.

523 8A,B). Importantly, genetic inactivation of PS genes in PS cDKO neurons

524 decreased evoked calcium levels at Nlgn1 synapses, leaving internal synapses

525 contacting untransfected neurons unaffected (Fig. 8A,B). The peak SyGCaMP3

526 amplitude per AP, a measure of the average calcium signal evoked by a single

527 AP, was increased at Nlgn1 synapses of control cultures compared to internal

528 synapses (1.145 r 0.047 and 1.765 r 0.101 for internal and Nlgn1 synapses of

529 control cultures; 1.060 r 0.038 and 1.695 r 0.106 for internal and Nlgn1

530 synapses of ΔCre cultures), whereas ablation of PS genes decreased

531 SyGCaMP3 amplitude per AP at Nlgn1 synapses (1.058 r 0.017 and 1.288 r

532 0.038 for internal and Nlgn1 synapses of PS cDKO synapses). Thus, the

533 augmented presynaptic release induced by Nlgn1 was found to be associated

534 to an increase in calcium levels at presynaptic terminals. Conversely, inhibition

535 of PS function strongly decreased presynaptic calcium levels and mobilization

536 of synaptic vesicles at Nlgn1 synapses.

537 Then, we analyzed if Nrxn-CTF mediates the inhibitory response in presynaptic

538 calcium flux induced by PS inactivation. Indeed, transfection of HA-Nrxn-CTF in

539 hippocampal neurons inhibited evoked calcium levels at presynaptic terminals,

540 whereas expression of HA-Nrxn-CTF'PDZ showed an attenuated reduction in

541 presynaptic calcium (Fig 8C,D). The peak SyGCaMP3 signal per AP was 1.052

542 r 0.023 for control synapses, 0.380 r 0.017 for HA-Nrxn-CTF synapses, and

543 0.707 r 0.024 for HA-Nrxn-CTF'PDZ synapses. Together, these findings

544 indicate that accumulation of Nrxn-CTF at presynaptic terminals accounts for

545 the inhibitory effect of loss of PS function by a mechanism that reduces

546 presynaptic calcium levels and, consequently, synaptic vesicle release.

547

548 Inhibition of ectodomain shedding in Nrxn1-beta increases presynaptic

549 release

550 The presynaptic effect of overexpressed postsynaptic Nlgn1 relies on the trans-

551 synaptic recruitment of Nrxn-beta isoforms. In this scenario, increasing Nrxn-

552 beta levels at the axonal surface should mimic the effect of overexpressed

553 Nlgn1 at the postsynapse. However, it has been shown that the surface/total

554 ratio of Nrxn-beta proteins at synapses is tightly controlled (Fu and Huang,

555 2010), precluding an effect of their overexpression. Our data showing that

556 blocking ectodomain shedding increases Nrxn1-beta levels in lysates of

557 HEK293T cells (Fig. 5) suggest the intriguing possibility that the synaptic levels

558 of Nrxn-beta proteins are regulated by proteolysis of the extracellular domain.

559 Interestingly, we found that expression of mutant HA-Nrxn1-beta '368-393

560 resistant to ectodomain shedding increased Nrxn1-beta surface levels in axons

561 of hippocampal neurons (Fig. 9). As opposed to the lack of effect of HA-Nrxn1-

562 beta, expression of HA-Nrxn1-beta '368-393 proportionally increased the

563 release of synaptic vesicles at 40 and 300 AP (Fig. 9). Thus, increasing

564 postsynaptic Nlgn1 levels or blocking ectodomain shedding of axonal Nrxn-beta

565 isoforms increase synaptic levels of Nrxn-beta and stimulates presynaptic

566 release.

567

568 Discussion

569 Although PS mediates the proteolytic processing of Nrxns, it has been unclear

570 whether the synaptic clearance of Nrxn-CTF by PS is important for synaptic

571 physiology. In this study, we performed experimental approaches based on

572 fluorescent synaptic probes to analyze the effect of the inhibition of Nrxns

573 processing by PS. Our results indicate that normal PS function is required to

574 maintain neurotransmitter release and presynaptic calcium levels at

575 glutamatergic synapses induced by Nrxn-Nlgn1 complex. However, PS function

576 is dispensable for the recruitment of synaptic vesicles and the induction of

577 glutamatergic synapses mediated by Nlgn1. Moreover, presynaptic expression

578 of exogenous Nrxn-CTF in hippocampal neurons decreases synaptic vesicle

579 release and presynaptic calcium levels, thereby recapitulating the effect caused

580 by inactivation of PS activity. The inhibitory effect of Nrxn-CTF is attenuated

581 with the deletion of the last three C-terminal residues, which form a functional

582 PDZ-binding domain in Nrxn-CTF. Taken together, our data indicate that

583 accumulation of Nrxn-CTF due to impaired PS activity is deleterious to the

584 function of glutamatergic synapses.

585 Due to the role of Nrxns in presynaptic function, the accumulation of

586 Nrxn-CTF at glutamatergic terminals favored a potential impact of Nrxn

587 processing in the synaptic phenotype associated with the loss of PS function.

588 However, the challenges in assigning synaptic phenotypes to specific PS

589 substrates and the complexity in analyzing presynaptic function have precluded

590 answering these questions. In this study, we analyzed the functional

591 implications of the proteolytic processing of Nrxns at glutamatergic terminals.

592 Electrophysiological recording of Nlgn1-expressing neurons supported the

593 notion of a trans-synaptic mechanism that increases release probability at

594 glutamatergic synapses (Futai et al., 2007). Surprisingly, expression of Nrxn1-

595 beta failed to increase neurotransmitter release, while expression of a Nrxn1-

596 beta deletion mutant lacking the cytoplasmic tail decreases neurotransmitter

597 release while preserving trans-synaptic interaction with Nlgn1 (Futai et al.,

598 2007; Wittenmayer et al., 2009; Rabaneda et al., 2014). These data suggested

599 that the function of Nrxn-beta isoforms at glutamatergic terminals is tightly

600 regulated by postsynaptic binding partners. Using synaptic probes to directly

601 visualize presynaptic release, we found that Nlgn1 enhances the size of the

602 different pools of synaptic vesicles at single terminals. Moreover, calcium-

603 imaging experiments showed that expression of Nlgn1 increases AP-evoked

604 calcium levels at contacting terminals. Because the probability of

605 neurotransmitter release correlates with the size of the RRP (Murthy and

606 Stevens, 1999), our data are consistent with a role of Nlgn1 as a rate-limiting

607 factor that regulates the number of functional release sites at contacting

608 terminals by stimulating trans-synaptic signaling through Nrxn-beta isoforms. In

609 this regard, blocking ectodomain shedding in HA-Nrxn1-beta increases

610 presynaptic release and the levels of the protein at the axonal membrane.

611 Therefore, the increase in presynaptic release sites mediated by Nrxn-beta can

612 at least partly explain the positive effect of Nlgn1 in neurotransmitter release

613 probability.

614 Importantly, we found that ablation of PS genes in PS cDKO neurons or

615 inactivation of PS activity with genetic and chemical approaches reduced the

616 activity-dependent mobilization of synaptic vesicles and decreased calcium

617 levels at Nlgn1 synapses. Although Nrxn-Nlgn1 signaling increases the number

618 of glutamatergic synapses (Dean et al., 2003; Boucard et al., 2005; Chih et al.,

619 2006), the increase in density and intensity of synaptic vesicle markers induced

620 by Nlgn1 was not altered in neurons lacking PS activity, suggesting a selective

621 role of PS in the release of synaptic vesicles downstream of Nrxn activation.

622 The most plausible explanation for these results is the generation of a PS

623 substrate at Nlgn1-induced presynaptic terminals whose local accumulation

624 renders Nlgn1 synapses susceptible to the loss of PS function. It is noteworthy

625 to mention that while Nrxn-CTF is generated in neurons with impaired PS

626 activity (Saura et al., 2011; this study), presynaptic release in synapses not

627 contacting Nlgn1 was only slightly reduced at 300 AP by the loss of PS activity.

628 Importantly, the immunoreactivity for a cyto-neurexin antibody was found

629 increased at Nlgn1 synapses of PS cDKO cultures, expanding our previous

630 data using PS1 dominant-negative mutants (Saura et al., 2011). Our data favor

631 two potential non-excluding mechanisms for the local generation of Nrxn-CTF at

632 glutamatergic terminals: i) increase Nrxn availability through adhesion to

633 postsynaptic Nlgn1; ii) initiation of the proteolytic processing by an activity-

634 dependent mechanism mediated by NMDA/AMPA receptor signaling. In this

635 regard, it has been reported that Nlng1 increases the recruitment of NMDA

636 receptors (Budreck et al., 2013), which in turn can become activated by

637 glutamate released from the apposing terminal. Thus, the higher susceptibility

638 of Nrxn-Nlgn1 synapses to the loss of PS function correlates with increased

639 concentration of Nrxn-CTF at presynaptic terminals.

640 The accumulation of Nrxn-CTF is governed by the balance of the

641 cleavage at the extracellular domain and the clearance of the resulting Nrxn-

642 CTF by PS. Using deletion mutants in the extracellular domain of human Nrxn1-

643 beta, we identified a short region in the juxtamembrane domain comprising

644 residues 386-393 responsible for the release of the ectodomain and the

645 generation of Nrxn-CTF. The predicted Nrxn-CTF contains the transmembrane

646 and cytoplasmic domains of Nrxns and is conserved among Nrxn isoforms.

647 Moreover, we demonstrated that inhibition of PS activity in heterologous and

648 neuronal cells increases the levels of an HA-tagged Nrxn-CTF construct

649 containing the last 85 residues of human Nrxn1, further indicating that Nrxn1 C-

650 terminal fragments starting at V388 function as a PS substrate, as recently

651 shown for Nrxn3 (Borcel et al., 2016). One important consequence of the

652 identification of the cleavage site that generates Nrxn-CTF is that it enabled the

653 functional study of the proteolytic processing of Nrxns by PS, without broadly

654 affecting PS activity towards other neuronal substrates. A key finding of our

655 study is that presynaptic accumulation of exogenous Nrxn-CTF strongly

656 decreases evoked calcium influx at axon terminals and consequently synaptic

657 vesicle release, thereby mimicking the inhibitory effect of the loss of PS

658 function. The inhibition in presynaptic function induced by Nrxn-CTF is mediated

659 by a synaptic mechanism that depends on the integrity of the cytoplasmic tail,

660 as shown by the attenuated effect of a deletion mutant that lacks the PDZ-

661 binding domain of Nrxn-CTF. The increased CASK levels indicate that

662 accumulation of ligands that interact with Nrxn-CTF might interfere with

663 presynaptic signaling occurring at contact sites downstream of the cytoplasmic

664 tail of Nrxns, thereby uncoupling presynaptic release from trans-synaptic

665 adhesion. Regions other than the PDZ-binding domain could be contributing to

666 the intermediate CASK levels accumulated by the Nrxn-CTF'PDZ mutant. In

667 this regard, the cytoplasmic tail of Nrxn1 contains a conserved juxtamembrane

668 domain that indirectly binds to CASK through neuronal Protein 4.1N (Biederer

669 and Sudhof, 2001). Whether CASK levels are increased by this or by an

670 alternative mechanism remains to be investigated, however it is interesting to

671 note that the inhibition in presynaptic release caused by Nrxn-CTF and Nrxn-

672 CTF'PDZ constructs correlates with the levels of accumulated CASK.

673 Collectively, our data indicate that stimulation of Nrxn-Nlgn1 trans-

674 synaptic signaling increases the number of functional release sites at contacting

675 presynaptic terminals. Synaptic activity initiates the proteolytic shedding of the

676 extracellular domain of Nrxns by a NMDA/AMPA receptor-dependent signaling,

677 which results in the generation of Nrxn-CTF. Interestingly, Nrxn1-beta

678 ectodomain accelerates the activity-dependent shedding of Nlgn1, which in turn

679 can modulate the retrograde pathway that controls presynaptic release

680 probability (Peixoto et al., 2012; Suzuki et al., 2012). Therefore, the Nrxn-Nlgn1

681 signaling pathway is limited by the coordinated activity-dependent processing of

682 their members. Nrxn-CTF produced at glutamatergic terminals is further

683 processed by PS/gamma-secretase complex, leading to its synaptic clearance.

684 Loss of PS activity with chemical inhibitors or by genetic inactivation

685 accumulates Nrxn-CTF at presynaptic terminals, which selectively reduces

686 evoked presynaptic calcium flux and, as a consequence, exocytosis of synaptic

687 vesicles. Therefore, accumulation of Nrxn-CTF explains the inhibitory effects

688 caused by loss of PS function at glutamatergic synapses. In this regard, it has

689 been proposed that FAD-linked PS mutations produce a partial loss of function

690 mechanism, reducing the proteolytic activity of PS/gamma-secretase complex

691 toward its substrates (Shen and Kelleher, 2007; Sun et al., 2017), including

692 Nrxns (Bot et al., 2011; Saura et al., 2011). Importantly, genetic inactivation of

693 PS genes in excitatory neurons results in deficits in glutamatergic release by a

694 presynaptic mechanism, followed by age-dependent neurodegeneration (Saura

695 et al., 2004; Zhang et al., 2009; Zhang et al., 2010). Remarkably, the

696 accumulation of Nrxn-CTF at glutamatergic terminals of PS cDKO mice occurs

697 at an early developmental time that coincides with deficits in glutamatergic

698 transmission, but precedes neurodegeneration (Saura et al., 2011). Whereas

699 the consequences of impaired PS activity toward specific substrates remain to

700 be fully clarified, our data suggest that presynaptic accumulation of Nrxn-CTF

701 can contribute to the synaptic and neuronal deficits associated with the loss of

702 PS function in FAD.

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894

895

896 Figure Legends

897 898 Figure 1. Inhibition of presynaptic release at Nlgn1 synapses of PS cDKO

899 neurons. A, Experimental design. fPS1/fPS1; PS2 -/- hippocampal neurons

900 were infected with lentivirus expressing SypHy alone or double-infected with

901 'Cre or Cre, followed by co-transfection of HA-tagged Nlgn1 and DsRed. The

902 same population of axons forms synaptic puncta in Nlgn1 expressing dendrites

903 (filled arrowheads) and in non-expressing Nlgn1 synapses (open arrowheads),

904 which represent an internal control. B, Expression of Nrxn-CTF in PS cDKO

905 hippocampal neurons. Lysates from fPS1/fPS1; PS2 -/- cultures (control) and

906 from cultures infected with 'Cre, Cre, or double-infected with Cre and PS1 or

907 PS1 D385A, were incubated with the indicated antibodies. The cyto-neurexin

908 antibody recognizes the cytoplasmic tail in intact Nrxns and in Nrxn-CTF

909 proteins. C, Co-recruitment of SypHy and VGluT1 by Nlgn1 in transfected

910 neurons. D, Images showing SypHy fluorescence from hippocampal cultures

911 expressing SypHy alone (control) or together with 'Cre or Cre (PS cDKO

912 neurons). SypHy response was elicited with 40 and 300 AP, as indicated. Note

913 that the increased SypHy response in Nlgn1 expressing dendrites was

914 decreased in PS cDKO neurons. E-G Kinetics (E,F) and peak amplitudes (G) of

915 normalized SypHy fluorescence in Nlgn1-induced (E) and internal synapses (F).

916 Data were normalized to the maximum SypHy fluorescence value in control

917 cultures (E,F), or to the peak SypHy fluorescence value elicited with 40 AP at

918 internal synapses of control cultures (G) (normalized 'F at 40 AP: internal

919 control synapses 1.0 r 0.025; internal 'Cre synapses 1.032 r 0.038; internal

920 PS cDKO synapses 1.009 r 0.047; Nlgn1 synapses 2.963 r 0.074; Nlgn1 'Cre

921 synapses 3.036r 0.088; Nlgn1 PS cDKO synapses 1.658 r 0.055; normalized

922 'F at 300 AP: internal control synapses 2.975 r 0.085; internal 'Cre synapses

923 3.048 r 0.130; internal PS cDKO synapses: 2.509 r 0.110; Nlgn1 synapses

924 10.535 r 0.272, Nlgn1 'Cre synapses 9.856r 0.320, Nlgn1 PS cDKO synapses

925 4.183 r 0.141, #p<0.05, ***p<0.001, Kruskal-Wallis followed by the post hoc

926 Dunn’s test). Scale bars 5 Pm. All error bars are SEM.

927

928 Figure 2. Inhibition of PS proteolytic activity reduces presynaptic release. A,

929 Images showing SypHy response at 300 AP of hippocampal neurons incubated

930 with DAPT for 12 hours after co-transfection of DsRed and Nlgn1. B, C, Time

931 kinetics of Nlgn1 (B) and internal synapses (C) in the presence of DAPT. D,

932 Peak amplitudes of SypHy fluorescence in internal and Nlgn1-induced

933 synapses in the presence of DAPT (normalized 'F: internal control synapses

934 1.0 r 0.023; internal DAPT synapses 0.98 r 0.025; Nlgn1 synapses 3.472 r

935 0.054; DAPT Nlgn1 synapses 2.350 r 0.050, ***p<0.001, Kruskal-Wallis

936 followed by the post hoc Dunn’s test). E, Changes in SypHy fluorescence

937 evoked by 300 AP in hippocampal cultures expressing PS1 D385A.

938 Hippocampal neurons were double-infected with lentivirus expressing PS1

939 D385A and SypHy before transfection of DsRed and Nlgn1. F, G, Time kinetics

940 of Nlgn1 (F) and internal synapses (G) of cultures expressing PS1 D385A. H,

941 Peak amplitudes of SypHy response in internal and Nlgn1-induced synapses

942 expressing PS1 D385A (normalized 'F: internal control synapses 1.0 r 0.030;

943 internal PS1 D385A synapses 0.825 r 0.027; Nlgn1 synapses 3.159 r 0.082;

944 PS1 D385A Nlgn1 synapses 1.585 r 0.057, #p<0.01, ***p<0.0001, Kruskal-

945 Wallis followed by the post hoc Dunn’s test). Data were normalized to the

946 maximum SypHy fluorescence value of control cultures (B, C, F, G), or to the

947 peak SypHy fluorescence of internal control synapses (D, H). Scale bars 5 Pm.

948 All error bars are SEM.

949

950 Figure 3. Distribution of synaptic markers at synapses induced by Nlgn1 in

951 hippocampal neurons with impaired PS function. A-C, Immunofluorescence

952 stainings and quantifications of the synaptic-vesicle resident marker SypHy and

953 Nlgn1 in PS cDKO hippocampal neurons (normalized SypHy area: control 1.0 r

954 0.105; control 'Cre 1.103 r 0.076; PS cDKO 0.992 r 0.073, p>0.05, one-way

955 ANOVA followed by the post hoc Bonferroni’s test; normalized SypHy intensity:

956 control 1.0 r 0.025; control 'Cre 0.986 r 0.043; PS cDKO 0.921 r 0.050,

957 p>0.05, Kruskal-Wallis followed by the post hoc Dunn’s test; normalized Nlgn1

958 intensity: control 1.0 r 0.095; control 'Cre 0.902 r 0.060; PS cDKO 1.004 r

959 0.041, p>0.05, Kruskal-Wallis followed by the post hoc Dunn’s test). Nlgn1 was

960 detected with an HA antibody. Only Nlgn1 intensity was analyzed due to the

961 high dendritic area covered by overexpressed Nlgn1. D-F, Immunofluorescence

962 stainings of SypHy and Nlgn1 and quantifications in hippocampal neurons

963 incubated with DAPT (normalized SypHy area: control 1.0 r 0.035; DAPT 0.896

964 r 0.044, p=0.076, Student’s t test; normalized SypHy intensity: control 1.0 r

965 0.017; DAPT 1.004 r 0.022, p=0.878, Student’s t test; normalized Nlgn1

966 intensity: control 1.0 r 0.040; DAPT 1.046 r 0.034, p=0.402. Mann-Whitney U-

967 test). (G-I) Immunofluorescence signal of SypHy and Nlgn1 and quantifications

968 in hippocampal neurons expressing PS1 D385A (normalized SypHy area:

969 control 1.0 r 0.076; PS1 D385A 0.825 r 0.058, p=0.075, Student’s t test;

970 normalized SypHy intensity: control 1.0 r 0.011; PS1 D385A 0.966 r 0.019,

971 p=0.133, Student’s t test; normalized Nlgn1 intensity: control 1.0 r 0.034; PS1

972 D385A 1.029 r 0.031, p=0.538, Student’s t test). J, Immunofluorescence

973 experiments with CASK and cyto-neurexin antibodies showing increased

974 signals at Nlgn1 synapses of PS cDKO neurons. (K,L) Graphs showing

975 quantifications of the relative area and mean intensity of cyto-neurexin (K) and

976 CASK (L) fluorescent signals at Nlgn1 synapses of control ('Cre) and PS cDKO

977 neurons (normalized Nrxn area: control 'Cre 1.0 r 0.132; PS cDKO 1.716 r

978 0.153, p=0.001, Student’s t test; normalized Nrxn intensity: control 'Cre 1.0 r

979 0.015; PS cDKO, 1.061 r 0.022, p=0.029, Mann-Whitney U-test; normalized

980 CASK area: control 'Cre 1.0 r 0.109; PS cDKO 1.869 r 0.178, p=0.0002,

981 Mann-Whitney U-test; normalized CASK intensity: control 'Cre 1.0 r 0.021; PS

982 cDKO 1.094 r 0.031, p=0.033, Mann-Whitney U-test). Data from 15-25

983 experiments obtained form 2-3 independent cultures. Scale bars 5 Pm. *p<0.05,

984 ***p<0.001. All error bars are SEM.

985

986 Figure 4. Activity-dependent proteolytic processing of Nrxns. A, Western-blot

987 experiments of hippocampal lysates incubated with 50 mM KCl for 30 minutes

988 and treated in the presence of the indicated inhibitors. Nrxns were detected with

989 a cyto-neurexin antibody, as indicated. B, Graph shows quantification of

990 normalized data (control 1.0 ± 0.127; DAPT 1.739 ± 0.051; KCl 2.077 ± 0.123;

991 KCl+DAPT 2.576 ± 0.130; KCl+DAPT+APV+CNQX 1.234 ± 0.030;

992 KCl+DAPT+Z-agatoxin-IVA 2.103 ± 0.273; KCl+DAPT+thapsigargin 2.280 ±

993 0.187; KCl+DAPT+GM6001 1.419 ± 0.179, n=8, *p<0.05, ***p<0.001, one-way

994 ANOVA followed by the post hoc Bonferroni’s test). All error bars are SEM.

995

996 Figure 5. Proteolytic processing of Nrxn1-beta deletion mutants. A, Protein

997 sequences of deletion mutants generated within the juxtamembrane region of

998 human Nrxn1-beta. SP, signal peptide; LNS, laminin/neurexin/sex-hormone

999 binding protein domain; TM, transmembrane region. Wild-type protein and each

1000 deletion mutant were labeled with a N-terminal HA-tag. B, Western-blot

1001 experiments with an anti-HA antibody of cell lysates or HA-immunoprecipitates

1002 from conditioned media of HEK293T cells transfected with the indicated

1003 construct (cell lysates: WT 1 r 0.132; '368-372 1.184 r 0.130; '368-378 1.173

1004 r 0.152; '368-385 1.234 r 0.123; '368-393 1.789 r 0.171, **p<0.01, Kruskal-

1005 Wallis followed by the post hoc Dunn’s test; HA-immunoprecipitates: WT 1 r

1006 0.036; '368-372 1.240 r 0.050; '368-378 1.122 r 0.081; '368-385 1.662 r

1007 0.108, '368-393 0.232 r 0.006, n=5, **p<0.01, ***p<0.001, Kruskal-Wallis

1008 followed by the post hoc Dunn’s test). A cell lysate expressing HA-Nrxn1-beta

1009 was included to show the lower molecular weight of the immunoprecipitated

1010 ectodomains. C, Detection of Nrxn-CTF in western-blot experiments of

1011 transfected HEK293T cells using cyto-neurexin antibodies (normalized Nrxn FL/

1012 actin levels: WT 1 r 0.086; '368-378 0.998 r 0.073; '368-385 0.830 r 0.064;

1013 '368-393 1.449 r 0.132, WT+DAPT 0.978 r 0.089; '368-378+DAPT 0.926 r

1014 0.076; '368-385+DAPT 0.812 r 0.066; '368-393+DAPT 1.492 r 0.160, n=5,

1015 **p<0.01, Kruskal-Wallis followed by the post hoc Dunn’s test) (normalized

1016 Nrxn-CTF/Nrxn levels: WT 1 r 0.133; '368-378 1.494 r 0.186; '368-385 1.423

1017 r 0.187; '368-393 0.118 r 0.013; WT+DAPT 4.428 r 0.787; '368-378+DAPT

1018 4.753 r 0.962; '368-385+DAPT 4.354 r 0.860; '368-393+DAPT 0.194 r 0.026,

1019 n=5, ***p<0.001, Kruskal-Wallis followed by the post hoc Dunn’s test). D,

1020 Sequence alignment of human Nrxn1-3 proteins showing the high conservation

1021 of the shedding site and the predicted Nrxn-CTFs. Dots indicate identical amino

1022 acids. V388 is indicated with an arrow.

1023

1024 Figure 6. Processing of HA-Nrx-CTF proteins by PS. A, Schematic drawings

1025 showing HA-Nrxn-CTF and HA-Nrxn-CTF'PDZ constructs compared to Nrxn1-

1026 beta. PDZ labels the last three residues that form a PDZ-binding domain. B,

1027 Western-blot of transfected HEK293T cells in the presence of DAPT. C, Lysates

1028 of hippocampal neurons infected with HA-Nrxn1-beta, HA-Nrxn-CTF or HA-

1029 Nrxn-CTF'PDZ and incubated with DAPT were immunoblotted with HA and

1030 cyto-neurexin antibodies, as indicated. The decreased mobility observed for

1031 HA-Nrxn-CTF compared to Nrxn-CTF is likely due to the insertion of the HA tag.

1032 D, Lysates and HA-immunoprecipitates of HEK293T cells co-expressing CASK

1033 and empty vector, HA-Nrxn-CTF or HA-Nrxn-CTF'PDZ. HA-Nrxn1-beta was

1034 co-expressed as a positive control. The isolated HA-complexes were

1035 immunoblotted with CASK and HA antibodies, as indicated. E, Hippocampal

1036 neurons expressing HA-Nrxn-CTF, HA-Nrxn-CTF'PDZ or HA-Nrxn1-beta by

1037 lentiviral infections were immunoprecipitated with HA antibodies and the

1038 presence of CASK and HA proteins analyzed by western-blot.

1039

1040 Figure 7. Expression of Nrxn-CTF inhibits presynaptic release. A, HA-Nrx-CTF

1041 or HA-Nrxn-CTF'PDZ were co-transfected with SypHy in hippocampal neurons

1042 and stained with HA antibody. Presynaptic terminals expressing SypHy were

1043 detected with GFP fluorescence. B, Images showing SypHy response at 40 and

1044 300 AP in axons expressing HA-Nrxn-CTF and HA-Nrxn-CTF'PDZ. C, Peak

1045 amplitudes of SypHy fluorescence in axons transfected with HA-Nrxn-CTF and

1046 HA-Nrxn-CTF'PDZ compared to control neurons (control synapses at 40 AP

1047 1.0 r 0.060; HA-Nrxn-CTF synapses at 40 AP 0.411 r 0.023; HA-Nrxn-

1048 CTF'PDZ synapses at 40 AP 0.697 r 0.041; control synapses at 300 AP 3.580

1049 r 0.178; HA-Nrxn-CTF synapses at 300 AP 0.980 r 0.059; HA-Nrxn-CTF'PDZ

1050 synapses at 300 AP 1.949 r 0.085, ***p<0.001, Kruskal-Wallis followed by the

1051 post hoc Dunn’s test). D, Immunofluorescence experiments with HA and CASK

1052 antibodies showing CASK accumulation at HA-Nrxn-CTF expressing axons. E,

1053 Quantification of CASK staining (normalized CASK area in transfected axons:

1054 GFP 1.0 ± 0.079; HA-Nrxn-CTF 2.636 ± 0.107; HA-Nrxn-CTF'PDZ 1.717 ±

1055 0.113; ***p<0.001, one-way ANOVA followed by the post hoc Bonferroni’s test.

1056 Normalized CASK intensity in transfected axons: GFP 1.0 ± 0.010; HA-Nrxn-

1057 CTF 1.365 ± 0.028, HA-Nrxn-CTF'PDZ 1.081 ± 0.015, *p<0.05, ***p<0.001,

1058 one-way ANOVA followed by the post hoc Bonferroni’s test. Data from 26-31

1059 images from 2 independent experiments from different cultures. Scale bars 5

1060 μm. All error bars are SEM.

1061

1062 Figure 8. Impaired calcium flux at presynaptic terminals accumulating Nrxn-

1063 CTF. A, Cultures from PS1/fPS1; PS2 -/- hippocampal neurons were co-

1064 infected with lentivirus expressing SyGCaMP3 alone or together with lentivirus

1065 expressing 'Cre or Cre (PS cDKO), before co-transfection of DsRed and Nlgn1.

1066 Images show peak SyGCaMP3 fluorescence of hippocampal cultures

1067 stimulated with 1, 3 and 5 AP. B, Peak amplitude ('F) of normalized

1068 SyGCaMP3 signal plotted as a bar graph (left) or as a function of the number of

1069 APs elicited (right). In each condition, quantitative analyses were performed in

1070 Nlgn1 and internal synapses not contacting Nlgn1 (internal synapses at 1 AP:

1071 control 1.0 r 0.080; control 'Cre 1.137 r 0.061; PS cDKO 1.049 r 0.063;

1072 internal synapses at 3 AP: control 3.768 r 0.449; control 'Cre 3.438 r 0.215;

1073 PS cDKO 3.299 r 0.220; internal synapses at 5 AP: control 5.557 r 0.513;

1074 control 'Cre 5.129 r 0.264; PS cDKO 5.220 r 0.263; Nlgn1 synapses at 1 AP:

1075 control 1.456 r 0.069; control 'Cre 1.656 r 0.010; PS cDKO 1.238 r 0.048;

1076 Nlgn1 synapses at 3 AP: control 5.994 r 0.430; control 'Cre 5.849 r 0.425; PS

1077 cDKO 4.142 r 0.201; Nlgn1 synapses at 5 AP: control, 8.465 r 0.519; control

1078 'Cre 8.024 r 0.547; PS cDKO 6.283 r 0.300, **p<0.01, ***p<0.001, Kruskal-

1079 Wallis followed by the post hoc Dunn’s test). Slopes: internal control synapses

1080 1.145 r 0.047; internal ΔCre synapses 1.060 r 0.038; internal PS cDKO

1081 synapses 1.058 r 0.017; Nlgn1 synapses 1.765 r 0.101, Nlgn1 ΔCre synapses

1082 1.695 r 0.106; Nlgn1 PS cDKO synapses 1.288 r 0.038, **p<0.01, ***p<0.001,

1083 one-way ANOVA followed by the post hoc Bonferroni’s test. C, Images showing

1084 peak SyGCaMP3 fluorescence of hippocampal neurons co-transfected with

1085 SyGCaMP3 alone (control) or with HA-Nrxn-CTF or HA-Nrxn-CTF'PDZ. D,

1086 Peak signal ('F) of normalized SyGCaMP3 fluorescence plotted as in B (control

1087 synapses at 1 AP 1.0 r 0.082; HA-Nrxn-CTF synapses at 1 AP 0.274 r 0.017;

1088 HA-Nrxn-CTF'PDZ synapses at 1 AP 0.556 r 0.032; control synapses at 3 AP

1089 2.997 r 0.246; HA-Nrxn-CTF synapses at 3 AP 1.235 r 0.115; HA-Nrxn-

1090 CTF'PDZ synapses at 3 AP 2.252 r 0.093; control synapses at 5 AP 5.368 r

1091 0.387; HA-Nrxn-CTF synapses at 5 AP 1.869 r 0.148; HA-Nrxn-CTF'PDZ

1092 synapses at 5 AP 3.490 r 0.139, *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis

1093 followed by the post hoc Dunn’s test). Slopes: control 1.052 r 0.023; HA-Nrxn-

1094 CTF synapses 0.380 r 0.017; HA-Nrxn-CTF'PDZ synapses 0.707 r 0.024,

1095 ***p<0.001 one-way ANOVA followed by the post hoc Bonferroni’s test. Scale

1096 bars 5 Pm. All error bars are SEM.

1097

1098 Figure 9. Effect of the inhibition of ectodomain shedding of Nrxn1-beta in

1099 axonal surface levels and presynaptic release. A, Immunofluorescence using

1100 HA and GFP antibodies in unpermeabilized neurons co-transfected with GFP

1101 and HA-Nrxn1-beta mutants. B, Normalized HA-Nrxn1-beta area in transfected

1102 axons (Wild-type 1.0 ± 0.028; Δ368-372 0.939 ± 0.040; Δ368-378 1.027 ±

1103 0.050; Δ368-385 0.817±0.036; Δ368-393 1.474 ± 0.058, *p<0.05, ***p<0.001,

1104 one-way ANOVA followed by the post hoc Bonferroni’s test). C, Normalized HA-

1105 Nrxn1-beta intensity in transfected axons (Wild-type 1.0 ± 0.022; Δ368-372

1106 0.977 ± 0.021; Δ368-378 1.015 ± 0.027; Δ368-385 0.959 ± 0.023; Δ368-393

1107 1.190 ± 0.022, ***p<0.001, Kruskal-Wallis with Dunn’s multiple comparisons

1108 test). Data from 15-18 images from 2 independent experiments from different

1109 cultures. D, Images showing SypHy fluorescence and quantifications of

1110 hippocampal cultures expressing SypHy alone (control) or together with wild-

1111 type HA-Nrxn1-beta or mutant HA-Nrxn1-beta Δ368-393 (control synapses at

1112 40 AP 1.0 r 0.064; HA-Nrxn1-beta synapses at 40 AP 0.932 r 0.039; HA-

1113 Nrxn1-beta Δ368-393 synapses at 40 AP 1.677 r 1.100; control synapses at

1114 300 AP 2.207 r 0.137; HA-Nrxn1-beta synapses at 300 AP 2.383 r 0.102; HA-

1115 Nrxn1-beta Δ368-393 synapses at 300 AP 3.850 r 0.182, ***p<0.001, Kruskal-

1116 Wallis followed by the post hoc Dunn’s test). Scale bar 5 μm. All error bars are

1117 SEM.