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FLRT Are Endogenous Ligands and Regulate Excitatory Synapse Development

Matthew L. O’Sullivan,1,5 Joris de Wit,1,5 Jeffrey N. Savas,2 Davide Comoletti,3 Stefanie Otto-Hitt,1,6 John R. Yates III,2 and Anirvan Ghosh1,4,* 1Neurobiology Section, Division of Biology, University of California San Diego, La Jolla, CA 92093, USA 2Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA 3Child Health Institute of New Jersey and Department of Neuroscience and Cell Biology, UMDNJ/Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA 4CNS Discovery, F. Hoffmann-La Roche, 4070 Basel, Switzerland 5These authors contributed equally to this work 6Present address: Department of Natural Sciences, Carroll College, Helena, MT 59625, USA *Correspondence: [email protected] DOI 10.1016/j.neuron.2012.01.018

SUMMARY much effort has been expended investigating the mechanisms of a- action (Su¨ dhof, 2001), nothing is known about (LPHNs) are a small family of G - the endogenous function of latrophilins in vertebrates. Further coupled receptors known to mediate the massive evidence for the importance of latrophilins in the proper synaptic exocytosis caused by the black widow functioning of neural circuits comes from recent human spider venom a-latrotoxin, but their endogenous genetics studies that have linked LPHN3 mutations to attention ligands and function remain unclear. Mutations in deficit hyperactivity disorder (ADHD), a common and highly LPHN3 are strongly associated with attention deficit heritable developmental psychiatric disorder (Arcos-Burgos et al., 2010; Domene´ et al., 2011; Jain et al., 2011; Ribase´ s hyperactivity disorder, suggesting a role for latrophi- et al., 2011). lins in human cognitive function. Using affinity chro- Here we report the identification of fibronectin leucine-rich matography and mass spectrometry, we identify repeat transmembrane (FLRT) proteins as endogenous ligands the FLRT family of leucine-rich repeat transmem- for latrophilins. There are three FLRT isoforms encoded by brane proteins as endogenous postsynaptic ligands different , Flrt1-3, that each encode single-pass trans- for latrophilins. We demonstrate that the FLRT3 and membrane proteins with ten extracellular leucine-rich repeat LPHN3 ectodomains interact with high affinity in (LRR) domains and a juxtamembrane fibronectin type 3 (FN3) trans and that interference with this interaction using domain (Lacy et al., 1999). FLRTs are expressed in many tissues, soluble recombinant LPHN3, LPHN3 shRNA, or including brain, where the different isoforms have striking cell- FLRT3 shRNA reduces excitatory synapse density type-specific expression patterns in the hippocampus and in cultured neurons. In addition, reducing FLRT3 cortex (Allen Mouse Brain Atlas, 2009). FLRTs have recently been reported to function in axon guidance and cell migration levels with shRNA in vivo decreases afferent input through an interaction with Unc5 proteins (Yamagishi et al., strength and dendritic spine number in dentate 2011), but they have no known role at synapses. granule cells. These observations indicate that We report the identification of FLRT3 and LPHN3 as a synaptic LPHN3 and its ligand FLRT3 play an important role ligand-receptor pair. This interaction is of high affinity, can occur in glutamatergic synapse development. in trans, and is mediated by the extracellular domains of FLRT3 and LPHN3. Moreover, we present evidence that FLRT3 and INTRODUCTION LPHN3 regulate excitatory synapse number in vitro and in vivo. These results demonstrate a role for LPHN3 and its ligand Latrophilins (LPHNs) have long been known to mediate the FLRT3 in the development of synaptic circuits. potent exocytotic effect that the black widow spider venom a-latrotoxin exerts on synaptic terminals (Krasnoperov et al., RESULTS 1997; Lelianova et al., 1997). The latrophilin family consists of three isoforms, LPHN1-3, encoded by different genes, with Identification of FLRTs as Candidate LPHN Ligands Lphn1 and Lphn3 expression largely restricted to the CNS To identify candidate LPHN ligands, we used recombinant ecto- (Ichtchenko et al., 1999; Sugita et al., 1998). All three LPHNs LPHN3-Fc protein (Figure 1A) to identify putative binding have a similar domain organization, consisting of a G protein- proteins from 3-week-old rat synaptosome extracts by affinity coupled receptor (GPCR) subunit and an unusually large chromatography. Proteins bound to ecto-LPHN3-Fc were adhesion-like extracellular N-terminal fragment (NTF) with lec- analyzed by shotgun mass spectrometry (de Wit et al., 2009; tin, olfactomedin, and domains. Though Savas et al., 2011). Of the proteins that bound selectively to

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Figure 1. Identification of FLRT3 as an Endogenous Ligand for LPHN3 (A) Coomassie-stained SDS-PAGE gels showing ecto-LPHN3-Fc (left) and ecto-FLRT3-Fc (right) purified proteins running as single bands. (B and C) Number of distinct tryptic peptides for selected proteins detected by mass spectrometry from ecto-LPHN3-Fc (B) or ecto-FLRT3-Fc (C) affinity purifications. (D) Frequency of detection of all peptides (total spectra count, ‘‘spec #’’) for proteins identified in both LPHN3 and FLRT3 affinity purifications. (E and F) Pull-downs from whole-brain extracts with ecto-FLRT3-Fc protein or control Fc protein. Ecto-FLRT3-Fc precipitates LPHN3 NTF (E), but not NRXN1 (F), from brain. (G– I) Pull-downs on lysate of HEK293 cells trans- fected with FLRT3-myc (G), myc-LRRTM2 (H), or LPHN3-GFP (I) using ecto-Fc or control Fc proteins. Ecto-LPHN3-Fc precipitates FLRT3-myc (G), but not myc-LRRTM2, from HEK cell lysate (H). (I) Ecto-FLRT3-Fc precipitates LPHN3-GFP NTF. (J and K) Binding of soluble ecto-LPHN3-Fc (J) and ecto-FLRT3-Fc (K) proteins to HEK cells expressing FLRT3 or LPHN3. Scale bar represents 10 mm. (L) Direct interaction of FLRT3 and LPHN3 ectodomains. Fc, ecto-LPHN3-Fc, or ecto-NRXN1b(-S4)-Fc was mixed with FLRT3-His, precipitated, and analyzed by western blot. FLRT3-His only binds to ecto-LPHN3-Fc (top). (M) Surface plasmon resonance (SPR)-based measurement of ecto-LPHN3-Fc binding to im- mobilized ecto-FLRT3. Each colored trace represents the binding response for a different concentration of ecto-LPHN3-Fc, from 1 mMto 0.45 nM in 3-fold dilutions, separated by wash and regeneration steps. (N) Concentration-response

function of LPHN3-FLRT3 SPR binding. The KD of the interaction is 14.7 nM.

ecto-LPHN3-Fc, FLRT2 and FLRT3 were among the most abun- When total spectra counts from proteins identified in both dant and were of particular interest due to similarities in domain purifications were compared, LPHN3 and FLRT3 stood out organization to previously identified postsynaptic organizing clearly as the proteins most frequently detected in both purifica- molecules such as the LRRTMs (de Wit et al., 2011), which tions (with each as bait in one condition and prey in the other) were not detected in our purification (Figure 1B). We also identi- (Figure 1D). To support our mass spectrometry results, we veri- fied proteins in the Teneurin family (also named ODZs), which fied the association of FLRT3 with LPHN3 by western blot in have recently been reported as ligands for LPHN1 (Silva et al., similar ecto-Fc pull-down assays on rat brain extract and trans- 2011) (see Figure S1A available online). fected heterologous cell lysate (Figures 1E–1I). Together, these Because FLRT3 was the most abundant FLRT protein findings suggest that FLRTs likely represent endogenous ligands identified in the ecto-LPHN3-Fc pull-down, we carried out for latrophilins. complementary experiments with ecto-FLRT3-Fc to confirm this interaction (Figures 1B and S1A). Affinity chromatography FLRT3 Can Directly Interact with LPHN3 and mass spectrometry using ecto-FLRT3-Fc resulted in the To test whether FLRT3 and LPHN3 can bind to one another in identification of a large number of LPHN1 and LPHN3 peptides, a cellular context, we expressed FLRT3-myc in HEK293 cells with relatively fewer LPHN2 peptides, but not the abundant and applied ecto-LPHN3-Fc or control Fc protein. We observed presynaptic organizing protein NRXN1 (Figure 1C). UNC5B strong binding of ecto-LPHN3-Fc to cells expressing FLRT3- (Figure S1B), a previously reported FLRT3 interactor, was myc, but no binding of Fc (Figure 1J). Ecto-LPHN3-Fc did not also identified, but at much lower abundance (Karaulanov bind to cells expressing myc-LRRTM2 (Figure S1D), showing et al., 2009; So¨ llner and Wright, 2009; Yamagishi et al., 2011). that the LPHN3-FLRT3 interaction is specific. Ecto-LPHN3-Fc

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Figure 2. FLRT3 Is a Postsynaptic Protein Capable of Interacting with LPHN3 In trans (A) In situ hybridization with antisense Flrt3 probe in horizontal sections from P7 (left) and P14 (right) mouse brain. (B) Western blot for FLRT3 and known synaptic proteins PSD95 and synaptophysin (SYP) in fractionated rat brain protein samples. Fractions, from left to right: homogenate, postnuclear supernatant, cytosol, crude membrane, synaptosome, detergent solu- bilized synaptosome, postsynaptic density, and purified postsynaptic density. (C) In dissociated hippocampal neurons, FLRT3-myc (green) local- izes to dendrites in a punctate fashion, partially colocalizes with the postsynaptic marker PSD95 (red), and is closely associated with presynaptic VGluT1 puncta (blue). (D) Hippocampal neurons transfected with LPHN3-GFP were cocultured with HEK cells expressing FLRT3-myc, and transfected axons (arrowheads) were imaged where they contacted transfected HEK cells. LPHN3-GFP, LPHN3 NTF, and FLRT3-myc were all enriched at axon-HEK cell contacts. Scale bars in (C) and (D) represent 10 mm.

FLRT3 Is a Postsynaptic Protein Expressed by Specific Neuronal Populations To identify brain regions where FLRT3 is likely to function, we examined Flrt3 also bound strongly to the other FLRT isoforms, FLRT1 and expression in the developing brain and found that Flrt3 was FLRT2 (Figure S1D), and ecto-LPHN1-Fc bound to all FLRT iso- highly expressed in specific neuronal populations during the first forms as well (Figure S1C). Complementarily, ecto-FLRT3-Fc, 2 postnatal weeks (Figures 2A and S2A). In the hippocampus, the but not control Fc, bound strongly to cells expressing LPHN3- principal cell layers of the dentate gyrus (DG) and CA3 showed GFP (Figure 1K). Ecto-FLRT3-Fc also bound the previously iden- strong signal, whereas Flrt3 expression was not detected in CA1. tified interactors UNC5A, UNC5B, and UNC5C, but did not bind Given its interaction with the extracellular domain of LPHNs, to NRXN1b(+ or ÀS4)-expressing cells (data not shown). We also we hypothesized that FLRT3 might be a postsynaptic protein. confirmed that ecto-LPHN3-Fc, but not ecto-FLRT3-Fc, could We first employed a subcellular fractionation approach to bind to cells expressing teneurin 3, confirming that LPHNs and examine the distribution of FLRT3 across different synaptic frac- teneurins can indeed interact (Figure S1E). Thus, we find that tions (Figure 2B) and found FLRT3 to be enriched in synapto- LPHNs and FLRTs strongly interact, with promiscuity between some and postsynaptic density (PSD) fractions, mirroring the isoforms. distribution of PSD95 and in contrast to synaptophysin, which To eliminate the possibility that an unknown coreceptor is excluded from PSD fractions. Next, because endogenous present in HEK293 cells might mediate the FLRT-LPHN interac- FLRT3 could not be detected by immunofluorescence with tion, we performed a cell-free binding assay using purified currently available antibodies, we expressed FLRT3-myc in proteins. Histidine-tagged FLRT3 and ecto-LPHN3-Fc or control dissociated hippocampal neurons and examined its subcellular Fc proteins were mixed in solution, and the Fc proteins were distribution. FLRT3-myc was found in dendrites in puncta that precipitated with bead-coupled protein A/G and assessed partially colocalized with glutamatergic but not GABAergic by western blot. We found that FLRT3-His coprecipitated with synapses (Figure 2C and S2C). Together, these results suggest ecto-LPHN3-Fc, but not with control Fc or NRXN1b(-S4)-Fc that FLRT3 is a postsynaptic protein of glutamatergic synapses. (Figure 1L), confirming a direct interaction between the ectodo- mains of FLRT3 and LPHN3. To quantitatively characterize LPHN3 Can Interact with FLRT3 In trans the affinity of the FLRT3-LPHN3 interaction, we employed a As a putative trans-synaptic complex, FLRT3 and LPHN3 must surface plasmon resonance (SPR) bioassay to measure specific be able to interact across sites of cell-cell contact. We tested ligand-receptor binding (Figure 1M). Plotting the maximum rela- whether LPHN3 and FLRT3 can interact in trans by overexpress- tive response versus the ecto-LPHN3-Fc concentrations, we ing LPHN3-GFP in dissociated hippocampal neurons and cocul- calculated the dissociation constant (Kd) of the LPHN3-FLRT3 turing them with HEK293 cells expressing FLRT3-myc or interaction to be 14.7 nM (Figure 1N), indicating a high-affinity a control construct. Strong axonal clustering of the GPCR interaction. and NTF fragments of LPHN3, as well as enrichment of

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FLRT3-myc, were observed at sites of contact with FLRT3-myc- aptic GCs reduces the number of excitatory synapses onto those expressing HEK293 cells (Figure 2D). No clustering of LPHN3- neurons. GFP was observed when axons contacted control cells (data If LPHN3 is involved in promoting the number of glutamatergic not shown). The accumulation of FLRT3 at sites of contact with synapses, as suggested by our dominant-negative experiment LPHN3-expressing axons (Figure 2D) demonstrates that FLRT3 with ecto-LPHN3-Fc (Figures 3A–3C), then loss of presynaptic is capable of interacting in trans with axonal LPHN3 and that LPHN3 should also reduce synapse number. To test this predi- the interaction can mediate mutual recruitment or retention. cation, we infected hippocampal cultures with a lentivirus To test whether FLRT3 and LPHN3 are sufficient to induce expressing an shRNA against LPHN3 (shLphn3; Figure S3D) pre- and postsynaptic differentiation, respectively, we cocul- such that the preponderance (>90%) of neurons in the culture tured neurons with HEK293 cells expressing FLRT3 or LPHN3. was transduced and recorded mEPSCs from neurons in We found that FLRT3 was not sufficient to induce clustering of LPHN3 knockdown and control cultures (Figure 3J). Culture- synapsin in axons in this heterologous cell assay (Figures S2D wide knockdown of LPHN3 reduced the frequency of mEPSCs and S2E) but that LPHN3-expressing HEK293 cells did cluster (Figure 3K) without affecting mEPSC amplitude (Figure 3L), sug- PSD95 in contacting dendrites, though less potently than gesting that loss of LPHN3 decreases the number of excitatory NRXN1b(-S4) (Figures S2F and S2G). synapses in these cultures. These experiments in dissociated hippocampal cultures show FLRT3 and LPHN3 Regulate Glutamatergic Synapse that three distinct manipulations designed to perturb LPHN3- Density In Vitro FLRT3 complexes—competition with ecto-LPHN3-Fc (Figures After establishing that the latrophilin ligand FLRT3 is expressed 3A–3C), shRNA knockdown of FLRT3 (Figures 3D–3I), and in hippocampal neurons and localizes to glutamatergic shRNA knockdown of LPHN3 (Figures 3J–3L)—all lead to synapses, we decided to test whether the interaction of endog- a reduction in the number of glutamatergic synapses, strongly enous LPHN3 with its ligand is of functional importance. We suggesting that FLRT3 and LPHN3 serve to positively regulate applied excess soluble ecto-LPHN3-Fc protein to dissociated synapse number. hippocampal cultures to competitively disrupt endogenous LPHN3 complexes and analyzed glutamatergic synapses on Postsynaptic FLRT3 Regulates the Function dentate granule cells (GCs) (identified by nuclear Prox1 immuno- of Glutamatergic Synapses In Vivo reactivity; Williams et al., 2011) by immunofluorescence (Fig- To test whether endogenous FLRT3 contributes to synapse ure 3A). We found that ecto-LPHN3-Fc treatment strongly development in vivo, we first used in utero electroporation to reduced the density of synaptic puncta (Figure 3B) without sparsely label and manipulate DG GCs for anatomical analysis. affecting their size (Figure 3C). These results suggest that In fixed slices from P14 electroporated mice, GFP-filled GC reducing the availability of LPHN3 ligands such as FLRT3 dendrites in the middle molecular layer were imaged on a by competition with ecto-LPHN3-Fc prevents glutamatergic confocal microscope, and dendritic spines were counted (Fig- synapses from developing properly. ure 4A). Knockdown of FLRT3 resulted in a highly significant If ecto-LPHN3-Fc exerts its effect by disrupting the interaction reduction in dendritic protrusion density relative to controls of LPHN3 with FLRT3, loss of postsynaptic FLRT3 might be ex- (Figure 4B). The density of spines on the apical dendrites of elec- pected to result in a similar reduction in synapses. We designed troporated CA1 pyramidal neurons, which do not express Flrt3 a short hairpin RNA (shRNA) to specifically knock down Flrt3 (Figure 2A), did not differ between shFlrt3 and control cells expression (shFlrt3) and verified its efficacy and specificity (Figures 4C and 4D), functionally confirming the specificity of (Figures S3A–S3C). To determine whether endogenous FLRT3 the shRNA used. regulates synapse number, we electroporated postnatal day To determine whether the reduction in GC dendritic spine 0 (P0) hippocampal cultures with shFlrt3 or control plasmids to density reflects a decrease in the strength of synaptic input sparsely knock down FLRT3 and analyzed synapses formed onto these cells, we stereotaxically injected shFlrt3 or control onto GC dendrites by immunofluorescence (Figure 3D). We lentivirus into the hippocampus of P5 rat pups and cut acute observed a large decrease in the density of synapses with slices for electrophysiology between P13 and P16. Infected shFlrt3 (Figure 3E), an effect similar to that observed with ecto- GCs were identified by GFP epifluorescence, and simultaneous LPHN3-Fc treatment. This decrease in synapse number was fully whole-cell voltage-clamp recordings were made from nearby in- rescued by coelectroporation with an shRNA-resistant FLRT3 fected and uninfected cells while perforant path synaptic inputs construct (FLRT3*-myc) (Figures 3E and S3A). Additionally, we were evoked from the middle molecular layer. We observed that saw a small decrease in the area of synaptic puncta following AMPAR-mediated EPSCs onto shFlrt3-infected neurons were FLRT3 knockdown (Figure 3F). To see whether the decrease in strongly reduced in amplitude relative to simultaneously re- synapse density assessed by immunofluorescence corresponds corded uninfected control cells (Figures 4E and 4F). NMDAR- to a decrease in functional synapses, we recorded miniature mediated EPSCs, measured 50 ms after the stimulus at a holding excitatory postsynaptic currents (mEPSCs) from putative GCs potential of +40 mV, were similarly reduced by FLRT3 knock- in similar cultures (Figure 3G). Consistent with the reduction in down (Figures 4G and 4H). This reduction was proportional, synaptic puncta density, we found a marked decrease in mEPSC because the ratio of AMPAR EPSC to NMDAR EPSC for each frequency (Figure 3H) and a slight decrease in the mean ampli- input was not affected by FLRT3 knockdown (Figure 4I), consis- tude of mEPSCs (Figure 3I) after FLRT3 knockdown. These tent with a reduction in number of synapses rather than a selec- experiments together suggest that a loss of FLRT3 in postsyn- tive loss of certain glutamate receptors. GCs infected with a

906 Neuron 73, 903–910, March 8, 2012 ª2012 Elsevier Inc. Neuron FLRTs Are Endogenous Ligands for Latrophilins

Figure 3. FLRT3 and LPHN3 Regulate Glu- tamatergic Synapse Number (A) Competitive disruption of endogenous LPHN interactions with ecto-LPHN3-Fc reduces excit- atory synapse density. Hippocampal cultures were fixed and immunostained for VGluT1 (green), PSD95 (red), and GFP (blue) at 14 days in vitro after 6 days of treatment with 10 mg/ml Fc (left) or ecto-LPHN3-Fc (right). (B) LPHN3-Fc treatment reduces the density of colocalized VGluT1 and PSD95 puncta on dendrites of identified GCs (Fc 1.00 ± 0.05, n = 17; LPHN3-Fc 0.68 ± 0.06, n = 14; p = 0.01). All summary statistics are reported and displayed as mean ± SEM. (C) Synaptic puncta did not significantly differ in size between Fc- and ecto-LPHN3-Fc-treated neurons (Fc 1.00 ± 0.04, n = 17; LPHN3-Fc 0.90 ± 0.08, n = 14; not signifi- cant [NS]). (D) Hippocampal neurons were elec- troporated with GFP control plasmid (GFP), a plasmid encoding shFlrt3 and GFP (shFlrt3), or the shFlrt3 plasmid plus an shFlrt3-resistant FLRT3-myc expression construct (Rescue) and were processed for immunofluorescence as in (A). (E) Synapse density was reduced by shFlrt3 and returned to control levels in the rescue condition (GFP 1.00 ± 0.08, n = 35; shFlrt3 0.66 ± 0.06, n = 33; rescue 0.96 ± 0.09, n = 34; analysis of variance [ANOVA] p < 0.01, significant post hoc compari- sons shown on graph). (F) Synapse area was slightly reduced by shFlrt3 (GFP 1.00 ± 0.05, n = 35; shFlrt3 0.83 ± 0.04, n = 33; rescue 0.98 ± 0.06, n = 34; ANOVA p < 0.05, significant post hoc comparisons shown on graph). (G) Example mEPSC traces recorded from cultured hippo- campal neurons electroporated with GFP (black) or shFlrt3 (red) plasmids. (H) Summary of mEPSC frequencies plotted as cumulative probability distributions of interevent intervals (IEIs) for GFP (black) or shFlrt3 (red) electroporated cells. Inset: quantification of mean mEPSC IEIs. shFlrt3 cells have longer mean IEIs than control neurons (GFP 1028 ± 147 ms, n = 15; shFlrt3 3069 ± 688 ms, n = 15; p < 0.01). (I) Summary of mEPSC amplitude plotted as cumulative probability distributions for GFP (black) or shFlrt3 (red) electroporated cells. Inset: quantification of mean mEPSC amplitude. shFlrt3 electroporated cells show a small decrease in mean mEPSC amplitude (GFP 16.4 ± 1.2 pA, n = 15; shFlrt3 12.8 ± 0.7 pA, n = 15; p < 0.05). (J) mEPSCs were recorded from neurons in hippocampal cultures densely infected with GFP (black) or shLphn3 (red) lentiviruses such that greater than 90% of neurons were transduced. (K) shLphn3 causes a shift toward longer times in the distribution of IEIs (GFP 745 ± 125 ms, n = 19; shLphn3 1254 ± 219 ms, n = 17; p < 0.05). (L) shLphn3 does not affect mEPSC amplitudes (GFP 27.7 ± 2.6 pA, n = 19; shLphn3 28.4 ± 2.3 pA, n = 17; NS). Scale bars in (A) and (D) represent 10 mm. control lentivirus did not differ from uninfected cells by any DISCUSSION of these measures (Figures S4A–S4D; de Wit et al., 2009). When we recorded mEPSCs from infected GCs we found no Latrophilins have garnered much interest because of their role in difference in mEPSC amplitudes and a trend toward reduction a-latrotoxin-stimulated neurotransmitter release, but their in frequency that did not reach significance (Figures S4E–S4G), endogenous functions have until now remained unknown. Here also suggesting that loss of FLRT3 does not affect the strength we report that the single-pass transmembrane proteins of the of single synapses. Furthermore, the paired-pulse ratio of EPSCs FLRT family, FLRT1-3, are endogenous ligands for LPHN1 and evoked at 20 Hz was unaffected (Figure 4J). These results indi- LPHN3. These interactions are mediated by the N-terminal frag- cate that loss of FLRT3 leads to an attenuation of the strength ment of LPHNs and the extracellular domain of FLRTs and are of glutamatergic transmission from the perforant path onto promiscuous among isoforms. The high-affinity interaction GCs and a reduction in the number of GC dendritic spines, further between LPHN3 and FLRT3, together with the postsynaptic supporting a role for FLRT3 in regulating synapse formation enrichment of FLRT3, suggests that LPHN3 and FLRT3 form a onto GCs. trans-synaptic complex (Figure 4K).

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Figure 4. FLRT3 Regulates Perforant Path Synapses onto Dentate Granule Cells In Vivo (A–D) Mouse embryos were electroporated in utero with GFP or shFlrt3 plasmids at E15 and GFP-expressing dendrites imaged at P14–16. (A) Dendrites of electroporated GCs were imaged in the middle molecular layer (MML) of the DG. (B) Flrt3 shRNA significantly reduces the density of dendritic protrusions (GFP 2.01 ± 0.07 protrusions/mm, n = 13; shFlrt3 1.60 ± 0.07 protrusions/mm, n = 10; p < 0.001). All summary statistics are reported as mean ± SEM. (C) Dendrites of electroporated CA1 pyramidal neurons, which do not express Flrt3, were imaged in the stratum radiatum. (D) Flrt3 shRNA does not affect the density of dendritic protrusions in CA1 (GFP 0.87 ± 0.06 protrusions/mm, n = 8; shFlrt3 0.91 ± 0.04 protrusions/mm, n = 8; NS). (E–J) P5 rat pups were stereotaxically injected with shFlrt3 lentivirus, and acute slices were cut at P13–16 for recording. (E) Simultaneous whole-cell voltage-clamp recordings of evoked perforant path AMPAR- mediated EPSCs onto shFlrt3 infected and uninfected DG GCs. Scatterplot shows mean EPSC amplitude from individual experiments. Inset: example average evoked AMPAR-EPSCs recorded simultaneously from shFlrt3 infected (red) and uninfected (black) GCs. (F) shFlrt3 significantly reduces the amplitudeof AMPAR-EPSCs (shFlrt3 101.1 ± 13.4 pA; uninfected 162.8 ± 15.0 pA, n = 15; p < 0.01). (G) Evoked NMDAR-EPSCs were measured simultaneously in shFlrt3 infected (red) and uninfected (black) GCs. Inset: example average evoked NMDAR-EPSCs. The NMDAR-EPSC measurement was taken at the time indicated by the arrow. (H) shFlrt3 significantly reduces the amplitude of NMDAR-mediated EPSCs (shFlrt3 129.6 ± 23.9 pA; uninfected 193.7 ± 16.9 pA, n = 0; p < 0.05). (I) shFlrt3 does not affect the ratio of AMPAR-EPSCs to NMDAR-EPSCs at single inputs (shFlrt3 0.76 ± 0.07; uninfected 0.83 ± 0.09, n = 10; NS). (J) shFlrt3 does not affect the ratio of EPSC amplitudes evoked by pairs of stimuli delivered at 20 Hz (EPSC2/EPSC1) (shFlrt3 1.01 ± 0.07; uninfected 1.04 ± 0.08, n = 13; NS). (K) Schematic of proposed FLRT3-LPHN3 synaptic interaction. FLRT3 is located at postsynaptic sites and interacts trans-synaptically with the NTF of LPHN3 via its extracellular domain. This interaction promotes synaptic development, and its disruption reduces synapse number. Scale bars in (A) and (C) represent 5 mm.

The resemblance of FLRTs to characterized LRR-containing controlling AMPAR recruitment (de Wit et al., 2009; see also synaptic organizers (de Wit et al., 2011) suggested to us that Soler-Llavina et al., 2011). Thus, FLRT-LPHN and LRRTM- the trans-synaptic interaction of LPHN with FLRT might regulate NRXN complexes, along with others, may regulate distinct synaptic development and function. Consistent with this hypoth- aspects of synapses. esis, we observed that three separate manipulations targeting How FLRTs signal postsynaptically is not known, but cis inter- the LPHN3-FLRT3 complex reduce excitatory synapse number actions of FLRTs have been reported in other systems. FLRT3 in cultured neurons (Figure 3). We further show that loss of interacts with FGFRs and can regulate FGF signaling (Bo¨ ttcher FLRT3 in vivo by lentivirus-mediated shRNA knockdown et al., 2004; Wheldon et al., 2010) and may also be capable of reduces the strength of evoked perforant path synaptic inputs modulating cadherin- and protocadherin-mediated cell adhe- onto dentate GCs and the number of dendritic spines. Our sion by signaling intracellularly through the small GTPase Rnd1 results suggest that FLRT3 may primarily regulate synapse (Chen et al., 2009; Karaulanov et al., 2009). Both FGF signaling number, whereas LRRTM2 may regulate synapse function by (Umemori et al., 2004; Terauchi et al., 2010) and cadherin

908 Neuron 73, 903–910, March 8, 2012 ª2012 Elsevier Inc. Neuron FLRTs Are Endogenous Ligands for Latrophilins

adhesion (Takeichi, 2007; Williams et al., 2011) are known to synaptic ligands at different types of synapses (Williams et al., influence synapse development, making them two possible 2010). Understanding how the brain assembles specific types effectors for the postsynaptic action of FLRT3. of synapses between the correct partner cells will require con- Further work is required to clarify the immediate presynaptic sideration of multiple parallel trans-synaptic signaling com- molecular and cell-biological consequences of FLRT binding to plexes, and the latrophilin-FLRT complex is poised to be an LPHN. Binding may activate downstream signaling or serve important unit in accomplishing this task. Given the genetic primarily to stabilize LPHN at appropriate presynaptic sites. association of LPHN3 mutations (Arcos-Burgos et al., 2010; Ar- Ligand binding to LPHNs may result in Ca2+ elevations through cos-Burgos et al., 2010; Domene´ et al., 2011; Jain et al., 2011; G protein signaling and IP3-mediated calcium release from the Martinez et al., 2011; Ribase´ s et al., 2011) and FLRT3 copy endoplasmic reticulum (ER), as has been shown to occur in number variations (Lionel et al., 2011) with ADHD, further charac- response to a-LTX binding (Davletov et al., 1998; Ichtchenko terization of the FLRT3-LPHN3 complex may lead to a better et al., 1998). That we observe FLRT-induced accumulation of understanding of the pathology and etiology of this disorder. both the LPHN3 NTF and GPCR domains (Figure 2D) may also provide a hint of mechanism. Latrophilins are constitutively EXPERIMENTAL PROCEDURES cleaved into two subunits (Krasnoperov et al., 2002; Silva et al., 2009), and it has been suggested that the association Please see the Supplemental Experimental Procedures. between subunits may be dynamically regulated by ligand binding to modulate latrophilin G protein signaling. This raises SUPPLEMENTAL INFORMATION the possibility that FLRT binding to the LPHN NTF may lead to Supplemental Information includes four figures and Supplemental Experi- reassociation of the LPHN subunits and engender subsequent mental Procedures and can be found with this article online at doi:10.1016/ G protein signaling. Whether binding of FLRTs and teneurins to j.neuron.2012.01.018. LPHNs induces similar signaling or has different functional consequences remains to be explored. ACKNOWLEDGMENTS Interestingly, FLRTs have also recently been shown to function in axon guidance during embryonic development by interacting We thank Katie Tiglio, Joseph Antonios, Christine Wu, and Tim Young for assistance with virus injections, virus and recombinant protein production, with axonal Unc5 proteins (Yamagishi et al., 2011). This function and antibody testing. This work was supported by the Brain and Behavior is potentially non-cell-autonomous, given that it is proposed to Research Foundation (formerly NARSAD) (J.d.W.), Autism Speaks grant depend upon proteolytically cleaved, soluble FLRT ectodomains 2617 (D.C.), NIH fellowship F32AG039127 (J.N.S.), and NIH grants acting as diffusible cues. Our manipulations of FLRT3 in vivo NS067216 (A.G.), NS064124 (A.G.), P41 RR011823 (J.R.Y.), and R01 were sparse and, for viral experiments, began at a developmental MH067880 (J.R.Y.). stage at which axon guidance was complete, suggesting that the effects we see of FLRT3 on synapses are cell autonomous and Accepted: January 25, 2012 Published: March 7, 2012 are not the result of axon guidance defects. Thus, an early, non-cell-autonomous FLRT-Unc5 interaction may mediate REFERENCES axon guidance, and a later, cell-autonomous FLRT-LPHN inter- action may regulate synaptic maturation and function. This dual Allen Mouse Brain Atlas. (2009). Allen Institute for Brain Science. (http:// function is reminiscent of the manner in which semaphorins mouse.brain-map.org). (Pasterkamp and Giger, 2009) and Ephs/Ephrins (Klein, 2009) Arcos-Burgos, M., Jain, M., Acosta, M.T., Shively, S., Stanescu, H., Wallis, D., function in both axon guidance and synaptogenesis. 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