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. Author manuscript; available in PMC: 2014 Jun 12.
Published in final edited form as: Cell Rep. 2013 Dec 5;5(5):1330–1341. doi: 10.1016/j.celrep.2013.11.008

High-throughput Genetic Screen for Synaptogenic Factors: Identification of LRP6 as Critical for Excitatory Synapse Development

Kamal Sharma 1, Se-Young Choi 2, Yong Zhang 3, Thomas JF Nieland 4, Shunyou Long 5, Min Li 6, Richard Huganir 7,*
PMCID: PMC3924421  NIHMSID: NIHMS551219  PMID: 24316074

SUMMARY

Genetic screens in invertebrates have discovered many synaptogenic genes and pathways. However, similar genetic studies have not been possible in mammals. We have optimized an automated high-throughput platform that employs automated liquid handling and imaging of primary mammalian neurons. Using this platform we have screened 3200 shRNAs targeting 800 proteins. One of the hits identified was LRP6, a co-receptor for canonical Wnt ligands. LRP6 regulates excitatory synaptogenesis, and is selectively localized to excitatory synapses. In vivo knockdown of LRP6 leads to a reduction in the number of functional synapses. Moreover, we show that the canonical Wnt ligand, Wnt8A, promotes synaptogenesis via LRP6. These results provide a proof of principle for using a high content approach to screen for synaptogenic factors in the mammalian nervous system, and identify and characterize a Wnt ligand receptor complex that is critical for development of functional synapses in vivo.

INTRODUCTION

Synaptogenesis is a highly specialized and complex phenomenon that involves precise target recognition by the pre and postsynaptic machinery (McAllister, 2007; Okabe, 2012; Shen and Scheiffele, 2010; Siddiqui and Craig, 2011). Secreted ligands and cell surface molecules play key roles in the identification of the target and also initiate assembly of synaptic modules. Once a growth cone arrives in close proximity of its target site on the dendrite, a series of events act in an orchestrated fashion that includes assembly of macro molecular complexes required for signaling, adhesion and neurotransmission (Chih et al., 2005; Dalva et al., 2000; Garner et al., 2000; Graf et al., 2004; Penzes et al., 2003; Scheiffele et al., 2000; Shen and Scheiffele, 2010; Williams et al., 2010). A rich repertoire of cell adhesion and signaling molecules allows for the construction of synapses with diverse structural and functional identities (O’Rourke et al., 2012). Molecular pathways that can selectively regulate the development of excitatory or inhibitory synapses fulfill the need for maintaining a dynamic balance between excitation and inhibition to ensure proper functioning of neuronal networks. Thus, it is of the utmost importance to identify the building blocks that define a synapse subtype. Moreover, the fact that many neurological disorders have been attributed to morphological defects at the synaptic level (Melom and Littleton, 2011; Mitchell, 2011) make it important to discover the genes that regulate the development and maturation of a wide array of synapse subtypes. However, despite a significant effort put into understanding synaptogenesis at the molecular level, master molecule(s) have remained elusive. In invertebrates many genome-wide genetic screens have been carried out that led to the discovery of a handful of candidates involved in synapse development and maturation (Aberle et al., 2002; Featherstone et al., 2000; Kurusu et al., 2008; Schaefer et al., 2000). In the mammalian CNS, however, such screens have not been performed at a scale comparable to what has been possible in invertebrates. Biochemical approaches have proven useful in discovering some important genes involved in synaptogenesis at the neuromuscular junction such as Agrin and FGF-2, or Thrombospondin in retinal ganglion neurons (Allen et al., 2012; Christopherson et al., 2005; Nitkin et al., 1987; Peng et al., 1991) but such efforts have not had much success for synapses in the central nervous system. In recent years a strategy of pooling and deconvoluting siRNA (Paradis et al., 2007) or ORF-cDNA clones has led to the discovery of novel synaptogenic proteins in the mammalian CNS (Linhoff et al., 2009; Takahashi et al., 2011).

Here we adapted a high content approach to develop a platform that can be employed in high throughput screening for synaptogenic molecules using primary neuronal cultures. Employing automated high content liquid handling and high throughput image acquisition to screen several thousand shRNAs we identified several factors, including LRP6, a co receptor for Wnt ligands and signaling hub in canonical Wnt signaling (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000), as a potential synaptogenic factor. Although the importance of canonical Wnt signaling in the development of the neuromuscular junction in Drosophila melanogaster is well established (Koles and Budnik, 2012; Miech et al., 2008; Packard et al., 2002), the localization and function of any of the Wnt receptors at mammalian central synapses in vivo is unknown. Since Wnt receptors determine the nature of Wnt signaling triggered by a complex family of Wnt ligands (van Amerongen et al., 2008), knowledge of localization and function of Wnt receptors is critical. Hence, we carried out a detailed characterization of the role of LRP6 in central synapse formation. We show that LRP6 is exclusively localized to excitatory postsynaptic densities (PSDs) and is critical for synapse formation in vitro. In addition we found that Wnt8A, a Wnt ligand that is predominantly expressed in forebrain, and is known to interact with LRP6, promotes excitatory synaptogenesis in vitro. Furthermore we confirm that LRP6 is critical for the formation of functional excitatory synapses in vivo.

RESULTS

Optimization of shRNA High Content Handling for Screening

To develop the high-throughput shRNA screen we optimized automated culturing and immunolabeling using automated high content liquid handling. A flow chart to outline the strategy is shown in Figure 1A where neurons were handled using a multidrop and robotic arm assisted 96-well plate washing system. Once we optimized the cell density and the steps in robotic handling and image acquisition, we assessed the physiological health of neurons in a depolarization induced CREB phosphorylation assay (Sheng et al., 1990). Neurons plated and handled in a high content fashion were depolarized at DIV14 with 50 mM KCl and probed with CREB and phospho-CREB antibodies. We found that neurons under such conditions robustly responded to depolarization as evident by significant increase in phospho-CREB signal in the nucleus indicating good physiological health (Figure 1B). Next, we tested the method for specificity and efficiency of immunolabeling of synaptic and somatodendritic markers. As shown in Figure 1C immunolabeling with MAP-2, PSD95 and Gephyrin, well-characterized markers for somatodendritic regions, excitatory synapses and inhibitory synapses respectively, confirmed the morphological integrity of neurons as well as the specificity of acquired signals in automated handling and imaging.

Figure 1. Strategy for high content handling of hippocampal neurons.

Figure 1

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A) Outline of high content methodology. B) Neurons are physiologically responsive to changes in activity in the network as evident by enhanced phosphorylation of CREB in response to KCl induced depolarization. Each dot in pCREB/CREB signal plot represents signal intensity from Phospho CREB and total CREB antibodies. Panels where neurons were depolarized with KCl show a significant increase in number of cells with higher pCREB signal intensity. C) Immunolabeling performed in an automated fashion is efficient and specific as shown by differential labeling with MAP-2, PSD95 and Gephyrin. D) One of the hits identified in the loss of function genetic screen is LRP6. Right panel shows a representative region of interest (ROI) from the well that received shRNA lentivirus against LRP6, and left panel shows ROI from control well. shRNA-LRP6 led to reduction in PSD95 clustering in comparison to control. E) Quantification of a well that received shRNA-LRP6 shows a significant reduction in PSD95 to Gephyrin puncta ratio. (n=30 ROIs from each well, P < .05, t test). F) shRNA-LRP6 that caused reduction in PSD95 clusters reduces levels of endogenous LRP6 protein significantly in neurons. See also Figure S1 and S2.

With an optimized platform to carry out an assay using primary hippocampal neurons we initiated a pilot screen to employ shRNA-based screening to search for synaptogenic factors in the mammalian CNS. The Sigma MISSION shRNA library available at the time of our initial screen offered 4–5 shRNAs for each target gene to enhance the possibility of efficient knockdown of a given target. However, a major limitation of the MISSION shRNA library is that it did not have a reporter gene appropriate for assessing infection efficiency. The library employs a Puromycin selection marker that works on the principle of conferring resistance to infected neurons against the protein synthesis inhibitor Puromycin. For non-neuronal cells and for young neurons that are not extensively connected in a network, Puromycin works suitably but once neurons are mature activity in the network becomes critical for neuronal survival. In such a scenario death of even 50% of uninfected neurons will have adverse effects on the health of infected neurons despite Puromycin resistance. This makes Puromycin an unsuitable selection marker for mature neuronal networks. However, by assessing the effect of Puromycin on younger neurons we found that majority of the infected wells, when treated with Puromycin, showed survival rate of more than 75% (Suppl. Figure 1) suggesting a high probability of infection. We also tested a few sample shRNA clones to assess the potency of shRNA and efficiency of knockdown. As shown in an example in supplementary figure 2, we observed a significant knockdown of Gephyrin by 2 out of 5 shRNA clones.

Next, we selected 800 transmembrane and secreted candidate proteins with 4 shRNAs against each (i.e. 3200 shRNA clones), and designed a high-throughput screen to look for synaptogenic factors. Neurons were infected with lentiviral particles at DIV4, and fixed and immunostained at DIV13 using the high content handling methodology optimized earlier. Since there is currently no appropriate efficient automated image analysis tool that can carry out reliable quantitative analysis we shortlisted the hits by manual browsing of the wells. The wells that had a noticeably changed pattern for PSD95/Gephyrin staining were quantified in a low-throughput manner. Potential shRNAs hits were sub-cloned into the pSuper plasmid that allows more efficient expression of shRNA. This was followed by secondary screening to address whether the shRNA clone can knockdown its endogenous target, and to assess the reproducibility of the primary screen.

To address first question we electroporated shRNA into dissociated hippocampal neurons and 5 days post-electroporation and plating, lysed the neurons and probed for putative target protein. To address the second question we transfected hippocampal neurons with shRNA in pSuper plasmid backbone along with GFP at DIV7, and analyzed synapse number at DIV14. shRNAs that showed an effect on puncta number, and knocked down their putative target protein were considered as positive hits. We describe here LRP6 as one of the hits that was discovered following this strategy and validated its role in development of functional excitatory synapses (Figure 1 and 2).

Figure 2. LRP6 is required selectively for excitatory synaptogenesis.

Figure 2

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A) (i–iv) Loss of LRP6 leads to reduction in excitatory synapses as evident by reduced number of PSD95 and vGluT1 puncta (n = 20 each). Human LRP6 (hLRP6) that has sequence mismatch with shRNA against mouse/rat LRP6 can rescue the phenotype from effect of shRNA. (v) Loss of LRP6 causes reduction in spine density that can be rescued by hLRP6 (n = 20 each). B) Knockdown of LRP6 does not affect formation of inhibitory synapses, as there was no change in the number of Gephyrin and vGAT puncta in neurons that were transfected with shRNA-LRP6 (n=12). (P < .05, t test). Merged frames show GFP (Green), PSD95 (Red), vGluT1 (Blue) in panel A, and GFP (Green), Gephyrin (Red) and vGAT (Blue) in panel B.

LRP6 Regulates Excitatory Synapse Development

In the first round of the screen, wells infected with shRNA-lentiviral particles targeting LRP6 (shRNA-LRP6) showed a significant reduction in the number of PSD95 puncta but no change in Gephyrin puncta (Figure 1 D, E). shRNA-LRP6 significantly and specifically reduced endogenous LRP6 levels but had no effect on total PSD95 levels in hippocampal neurons. Next, we tested if knockdown of endogenous LRP6 lead to a reduction in the number of morphological synapses labeled with pre and postsynaptic markers. We expressed shRNA-LRP6 in dissociated neurons at DIV7 and analyzed the number of excitatory and inhibitory synapses at DIV14 using PSD95/VGluT1 and Gephyrin/VGAT immunostaining respectively. Knockdown of LRP6 selectively reduced the number of excitatory synapses (Figure 2A i-iv), and did not have any effect on inhibitory synapse number (Figure 2B). Reduction in excitatory synapse number was also reflected by a loss of spines as a result of LRP6 knockdown (Figure 2A v). The effect caused by the shRNA could be rescued by shRNA-resistant human LRP6. In summary we confirmed LRP6 as a bona fide hit in dissociated neuronal cultures that plays an important specific role in excitatory synapse development.

LRP6 is Selectively Localized to Excitatory Synapses

To investigate the subcellular localization of endogenous LRP6 we immunostained dissociated neurons using LRP6 antibodies in conjunction with synaptic markers. First, we tested the specificity of antibodies in the neurons that were transiently transfected with shRNA-LRP6. We saw a significant reduction in LRP6 immunolabeling in neurons transfected with shRNA-LRP6 in comparison to neighboring untransfected neurons (Suppl. Figure 3A). Specificity of the antibodies was also evident in western blot of total protein lysate from neurons electroporated with shRNA-LRP6 (Suppl. Figure 3B). Next, in immunolocalization studies LRP6 showed a punctate pattern with enrichment in spine heads. LRP6 was selectively colocalized with PSD95 and vGluT1 at excitatory synapses. (Figure 3A). In contrast, LRP6 did not show significant colocalization with inhibitory postsynaptic marker Gephyrin (Figure 3B,C). To confirm the synaptic localization with an alternative biochemical method we prepared postsynaptic density fractions and probed the samples with LRP6 antibodies. Supporting the immunostaining data LRP6 showed a pattern similar to that of PSD95 and GluA1 (Figure 3D), and was found to be component of PSD fractions I, II and III. Taken together these results suggest that LRP6 is predominantly localized at excitatory synapses.

Figure 3. LRP6 is localized at excitatory synapses in mature neurons.

Figure 3

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A) Immunolabeling of endogenous LRP6, PSD95 and presynaptic marker vGluT1 reveals that LRP6 is present at excitatory synapses. B) LRP6 does not show any significant level of colocalization with Gephyrin, a postsynaptic marker for inhibitory synapses. Merged frames show LRP6 (Red), PSD95 (Green), and vGluT1/Gephyrin (Blue). C) Quantification of overlap between LRP6 and PSD95 clusters in comparison to LRP6 and Gephyrin clusters reveals significant colocalization between LRP6 and PSD95 but not between LRP6 and Gephyrin. (n= 15 each, P < .001, t test) D) In biochemical fractions of postsynaptic densities from mouse brain endogenous LRP6 is detected in synaptosomal fractions including postsynaptic density fractions 1, 2 and 3 synaptic fractions. Synaptophysin (Synapto.) was used as a presynaptic marker for PSD samples. See also Figure S3.

Wnt8A, a Ligand for LRP6, Promotes Excitatory Synapse Development

There are 19 Wnt ligands, few of which have been studied in the context of excitatory neurotransmission in the CNS. Wnt7a is the only well characterized canonical Wnt ligand for its role in activity dependent spine development and synapse formation in the mammalian CNS in vivo. However, expression analysis of different Wnts in the Allen brain atlas revealed that Wnt8A is expressed at relatively higher levels in comparison to other Wnt ligands in the forebrain area (Figure 4A). Although a functional interaction between Wnt8A and LRP6 has been described(Itasaki et al., 2003), but the role of Wnt8a in synapse development in unknown.

Figure 4. Wnt8A induces excitatory synaptogenesis.

Figure 4

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A) Wnt8A is significantly enriched in forebrain (Image is from Allen brain atlas; http://mouse.brain-map.org/gene/show/20652). B) Wnt8A conditioned media was prepared by overexpression of Wnt8A-myc in HEK cells. Neurons were treated with Wnt8A conditioned media or mock conditioned media for 24 hours. C) Neurons treated with Wnt8A show a significant increase in PSD95 and vGluT1 puncta but no effect on Gephyrin puncta. D) Quantification of PSD95, vGluT1 and Gephyrin puncta revealed a selective and significant increase in excitatory synapses. (n= 15 each, P < .005, t test). E) shRNA mediated knock down of LRP6 diminishes the effect of Wnt8A. F) Quantification of vGLUT1 positive PSD95 puncta. Merged frames in panel C include PSD95 (Red), vGluT1 (Green) and Gephyrin (Blue), and GFP (Green), PSD95 (Red) and vGluT1 (Blue) in panel E. See also Figure S4 and S5.

First, to confirm the interaction between Wnt8A and LRP6 we tested if Wnt8A can associate with endogenous LRP6. HEK cells that have endogenous LRP6 were transfected with Wnt8A-myc cDNA. HEK cell lysate was subjected to immunoprecipitation with anti-myc antibodies, and probed for endogenous LRP6. In agreement with previous findings we observed pull down of endogenous LRP6 in Wnt8A-myc transfected cells but not in mock transfected cells (data not shown).

Next, to determine whether Wnt8A has synaptogenic activity neurons (DIV11) were treated with conditioned media for 24 hours, and analyzed for changes in synapse number. We observed that cultured neurons treated with Wnt8A condition media had an increased density of PSD95 and vGluT1 puncta but unchanged levels of Gephyrin puncta (Figure 4C,D), suggesting that Wnt8A specifically promotes formation and maturation of excitatory synapses. Next, we asked if LRP6 is required for the synaptogenic action of Wnt8A. To test this we transfected neurons with shRNA against LRP6 and treated neurons with mock conditioned media or Wnt8A conditioned media. Confirming the role of LRP6 in synaptogenic action of Wnt8A, transfected neurons showed no increase in PSD95 and vGluT1 puncta (Figure 4E–F). Wnt8A potently increased the number of excitatory synapses in neurons transfected with scrambled shRNA (Suppl. Figure 4). To investigate if Wnt8a is necessary for formation of excitatory synapses we took a loss of function approach where we generated lentiviral particles carrying GFP and shRNA against Wnt8a. Neurons were infected with lentivirus on DIV 4 and tested for Wnt8A expression at DIV 11. As shown in supplementary figure 5A, we saw a significant reduction in Wnt8A levels in neuronal cultures. Next, we infected 3 sets of neuronal cultures at DIV4 - one set was infected with control lentiviral particles, and two sets were infected with Wnt8A targeting lentiviral particles. One of these two sets infected with Wnt8A targeting lentiviral particles received Wnt8A conditioned media every 24 hours from DIV10 to 14 to compensate for the loss of endogenous Wnt8A. Neurons were fixed and immunostained at DIV14. We observed a significant decrease in number of excitatory synapses in response to Wnt8A knockdown that could be rescued by exogenous application of Wnt8a (Supplementary Figure 5B–C). Together these data demonstrate that Wnt8a is necessary for formation of excitatory synapses, and it regulates excitatory synapse formation through LRP6. Since LRP6 phosphorylation is one of the key events in relay of Wnt signaling, we asked if Wnt8a could lead to changes in phosphorylation status of LRP6. To test this we prepared Wnt8A conditioned media from Wnt8A transfected HEK cells, and control conditioned media from mock-transfected HEK cells. Neurons were treated with Wnt8a conditioned media for 3 hours, lysed and probed with Phospho-LRP6 antibodies that recognized LRP6 phosphorylated at Serine-- residue (Tamai et al., 2004). We observed an increase in phosphorylation of LRP6 in response to Wnt8a treatment suggesting a direct regulation of LRP6 phosphorylation by Wnt8a in neurons (Figure 5A,B). Next, we asked if LRP6 phosphorylation is one of the key steps required for synaptogenesis. To test this we generated an LRP6 phosphomutant, LRP6S1490A, where we replaced Serine-1490 with Alanine. As shown in Figure 5C, LRP6S1490A was targeted to dendritic spines in a fashion similar to that of wild type LRP6. To determine if LRP6 phosphorylation is critical for synapse formation we transfected neurons with an shRNA against LRP6 in conjunction with wild type LRP6 or LRP6S1490A. Wild type LRP6 efficiently rescued the loss of endogenous LRP6 but LRP6S1490A failed to rescue the effect of shRNA on synapse formation as well as on the formation of dendritic spines (Figure 5D,E). These results suggest that phosphorylation of LRP6 at Serine-1490 is crucial for the assembly of synaptic apparatus.

Figure 5. LRP6 undergoes Wnt dependent phosphorylation to promote synaptogenesis.

Figure 5

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A–B) Wnt8A treatment leads to enhanced phosphorylation of LRP6. C) Transient transfection of VSVG tagged LRP6 cDNA shows that LRP6S1490A is targeted to dendritic spine in a fashion similar to that of wild type LRP6. Merged frame in panel C include GFP (Green), LRP6 (Red) and vGluT1 (Blue). D–E) Wild type but not LRP6S1490A can rescue the effect of shRNA. Data in panel E was normalized to the average of wild type LRP6 rescue data set. (n= 14 each, P < .05, t test). Merged frames in panel C include GFP (green), PSD95 (Red) and vGluT1 (Blue).

LRP6 is required for Functional Synapse Development in Vivo

Studies in dissociated hippocampal neurons allow a wide range of manipulations and analysis at a higher resolution but have the limitation of belonging to a two-dimensional neuronal network system that may not truly recapitulate the complex development of three-dimensional brain architecture. To test if the role of LRP6 in synapse development observed in vitro holds true for the developing intact brain, and also if loss of LRP6 in fact leads to reduction in the number of functional synapses in vivo, we knocked down LRP6 in layer II/III cortical neurons by in utero electroporation of shRNA-LRP6 and dsRed at E15.5. Acute slices from animals at the age of P21-P28 were prepared, and immunolabeled with antibodies against dsRed to analyze spine growth. Spines are the primary site for excitatory synapses and an accurate morphometric parameter to assess synapse density in vivo (Knott et al., 2006). Confirming in vitro results we observed a significant reduction in the number of spines on dendrites of layer II/III neurons in somatosensory cortex in animals expressing shRNA-LRP6 in comparison to the animals expressing scrambled shRNA (Figure 6). In addition, the effect on spine numbers caused by the shRNA-LRP6 could be rescued by shRNA-resistant human LRP6.

Figure 6. LRP6 is required for spine formation in vivo.

Figure 6

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A–C) In utero electroporation of shRNA and dsRed at E15.5 stage results is efficient labeling of pyramidal neurons in layer II/III neurons in somatosensory cortex of P28 animals. D–E) Neurons transfected with shRNA-LRP6 and dsRed have a significantly decreased number of spines in comparison to neurons transfected with scrambled shRNA and dsRed. This reduction in spine number is rescued by hLRP6. (n=12, P < .05, t test).

To test further if the reduction in spine number truly reflects a decrease in the number of functional synapses, we prepared slices from animals expressing either shRNA-LRP6 or scrambled shRNA and carried out analysis of mini excitatory postsynaptic currents (mEPSCs). Further validating our earlier studies we found that neurons expressing shRNA-LRP6 had a significant reduction in mEPSC frequency with relatively unchanged amplitude in comparison to neighboring untransfected neurons (Figure 7). In contrast, neurons expressing scrambled shRNA had no difference in frequency in comparison to neighboring untransfected neurons. Moreover, the effect caused by the shRNA-LRP6 could be rescued by shRNA-resistant human LRP6. Together these results establish LRP6 as a bona fide hit of our screen that is critical for development of functional excitatory synapses in vitro as well as in vivo.

Figure 7. LRP6 is required for formation of functional AMPAR containing synapses in vivo.

Figure 7

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A) Representative traces from neurons transfected with scrambled, shRNA-LRP6 or shRNA-LRP6+hLRP6, and neighboring untransfected neurons in corresponding group, in layer II/III neurons in somatosensory cortex. Scale 200 msec, 10pA. B) Neurons electroporated in utero with scrambled shRNA did not show any difference in miniature excitatory postsynaptic currents (mEPSC) frequency in comparison to neighboring untransfected neurons (n=8 each). C) LRP6 knockdown led to a significant reduction in mEPSC frequency (n = 9 each, P < .05, t test). A small and non-significant increase was observed in mEPSC amplitude. D) Effect of shRNA against rat LRP6 can be rescued by hLRP6 (n=15 control, n=19 rescue).

DISCUSSION

We have optimized a high throughput assay using primary neuronal cultures that can be employed for a diverse range of screening methods. With the exception of automated image analysis for complex synaptic patterns, we have carried out a loss of function screen to discover synaptogenic proteins in a fully automated manner. Traditionally larger scale screening has required the use of simpler culture systems. Hence, most screens are limited to non-neuronal cells or neuroblastoma cell lines (Jain and Heutink, 2010). The fragile nature of neurons and lack of versatile methods for gene delivery have posed significant challenges to the use of primary neuronal cultures in a high throughput assay. Optimization of high content culturing, genetic manipulations, immunostaining and image acquisition of primary neuronal networks is the first major contribution of this work, and overcomes the conventional challenges of a larger screen requiring simpler cellular systems, to a significant extent. Notably, assays that have a simple read out parameter such as ratio of total CREB to phosphorylated CREB fluorescent signal intensities (Figure 1), or expression of GFP or lack thereof, can be analyzed in high throughput fashion enabling truly large-scale high throughput screens in neurons. Reporter assays such as Wnt regulated TCF/LEF reporter assay can also be successfully employed in this platform to discover neuron specific genetic components of the cascade or drug like small molecule modulators of these pathways in neurons. This platform can be further optimized to discover regulators of activity dependent protein trafficking, e.g. chemical LTP (Long Term Potentiation) paradigms can be employed in conjunction with surface labeling of AMPA receptor subunits. Recent advances in high throughput electroporation of neurons have made it feasible to overexpress any given set of genes in 96 wells format, making it possible to carry out gain of function genetic screens using this platform.

Lack of reliable high throughput image analysis tools to process images of mature neurons that have complex patterns of synaptic markers still remains a challenge and requires development. Typically statistical parameters such as Z-score and MAD scores provide indispensible tools to identify a hit, and to calculate false discovery rate (Chung et al., 2008; Konig et al., 2007; Zhang et al., 1999). The limitation of currently available software to quantify synapse number with high stringency has forced us to manually inspect the wells, and select ROIs for analysis. Computational tools available at present allow quantitative analysis of manually selected regions of interest along the dendritic regions but fail to generate a quantitatively consistent read out when applied globally to images across the 96-well plates. The advancement of a computational tool to quantify synaptic labeling will significantly enhance the throughput of similar screens in the future. In summary our system can be readily used for a wide variety of neuronal assays, and refinement of computational tools to analyze complex synaptic patterns will further enhance its capabilities.

One of the hits discovered in our loss of function shRNA screen is LRP6, a Wnt co-receptor. We present compelling data for a role for LRP6 in the development of functional excitatory synapses, and its direct association with the postsynaptic density in vivo. Further, we show that a ligand for LRP6, Wnt8A has synaptogenic activity. Given the complexity of Wnt signaling in mammals it is important to understand the localization and role of each component of this signaling cascade. In contrast to invertebrates such as Drosophila or C. elegans that have 5 Wnt ligands and 4 Fzd receptors, Wnt signaling in mammals is comprised of 19 Wnt ligands and 10 Fzd receptors (Ciani and Salinas, 2005). In recent years elegant studies on the neuromuscular junction have paved the way to an understanding of Wnt signaling in synapse development (Korkut and Budnik, 2009). In mammals different Wnt ligands have been shown to be important in axonal remodeling, dendritic growth (Wayman et al., 2006), synaptic development (Ciani et al., 2011; Gogolla et al., 2009; Varela-Nallar et al., 2010) and long-term potentiation (Chen et al., 2006). However, to date no receptor member has been described to play a functional role in synapse development in the mammalian CNS in vivo. Despite a well-established canonical model in which LRP6 acts as an obligatory co-receptor for Fzd receptors, the role of LRP6 in synapse development and its localization has remained elusive. Here we show that the key Wnt co-receptor LRP6 is predominantly present at excitatory synapses where it selectively regulates the development of functional excitatory synapses. These findings indicate either that canonical Wnt signaling is exclusively dedicated to excitatory synapses or that canonical Wnt signaling at excitatory and inhibitory synapses can employ a different suite of signaling receptors. Since non-canonical Wnt5A has been reported to play regulatory roles at excitatory and inhibitory synapses (Varela-Nallar et al., 2010), it is possible that a combination of canonical and non-canonical pathways regulates distinct synapse subtypes in forebrain. However, more experiments are required to draw a distinction between the roles of different Wnts in the development of different synapse subtypes.

We also discovered Wnt8A as a novel synaptogenic factor that acts via its interaction with LRP6. A close analysis of different Wnt ligands for their localization in forebrain, and established interactions with LRP6 led us to investigate Wnt8A for its potential role in excitatory synaptogenesis. We found a role for Wnt8A that is specific to excitatory synapses. Wnt8A has been described as a canonical Wnt ligand that binds multiple Fzd receptors, and the Wnt8-Fzd complex further associates with LRP6 to transduce the signal. Interestingly many of the Fzd receptors have PDZ binding motifs, and can bind to PSD95 (Hering and Sheng, 2002). This suggests a mechanism where Wnt8A triggers Wnt-Fzd-LRP6 complex formation and recruiting intracellular signaling complex at the phosphorylated carboxy terminus of LRP6 that can act further as a postsynaptic organizer. We have, in fact, discovered that Wnt8a triggers phosphorylation of LRP6 at Serine-1490. Our results further show that indeed phosphorylation of LRP6 is critical for synapse formation.

Our characterization of LRP6 suggests a specific role for canonical Wnt signaling in excitatory synapse development. It also opens many questions for future investigation. Most importantly it will be necessary to identify which Fzd receptors associate with LRP6 to promote excitatory synapse development. Fzd receptors are the primary interaction partners for Wnt ligands, and are shared between canonical and non-canonical Wnt ligands. It is possible Fzd receptors have a wide spread localization across excitatory and inhibitory synapses, and that LRP6 confers specificity to canonical Wnt signaling pathways promoting excitatory synapse development and function. Based on this hypothesis, LRP6 may play a key role in balancing excitatory and inhibitory synaptic weights in neuronal networks. Besides regulating the development of different synapse subtypes, it is important to note that cyclin Y-dependent L63/PFTK kinase phosphorylates LRP6 in a ligand independent manner during the cell cycle (Davidson et al., 2009). Interestingly cyclin Y has been shown to dynamically regulate synapse elimination during network remodeling (Park et al., 2011). This suggests that LRP6 may not be only required for synaptogenesis but may play a broader role in context of activity-dependent remodeling at the network level independent of Wnt ligands. Our data provides a step forward in understanding the localization and function of a key Wnt receptor that acts as a hub for canonical Wnt signaling, as well as uncovers a novel role for another Wnt ligand. However, extensive and systematic studies are required to further decipher the combinatorial codes defined by Wnt-Fzd-LRP6 complexes that act in a context-dependent manner. In fact our optimized platform for high throughput assay to study synapse development can play an important role in delineating signaling cascades under a given set of parameters in the future.

EXPERIMENTAL PROCEDURES

Animal care

All animals were treated in accordance with the Johns Hopkins University Animal Care and Use Committee guidelines.

cDNA and Antibodies

LRP6 cDNA was obtained from Addgene (Addgene plasmid 27242)(Tamai et al., 2000). ORF was amplified and sub cloned into pCAG plasmid. Phosphomutant LRP6S1490A was generated by using site directed mutagenesis kit from Stratagene. Wnt8a cDNA was obtained from Addgene (Addgene plasmid 35916)(Najdi et al., 2012), and amplified to sub clone into pCDNA3.1-Myc plasmid.

The following antibodies (and resources) were used in this study. PSD95 K28/43 (NeuroMabs, 1:2500), Gephyrin IgG1 (Synaptic System, 0.5 μg/ml), vGluT1 guiney pig (Millipore, 1:5000), vGAT rabbit polyclonal (Synaptic System, 1:1000), MAP-2 chicken (Novus Biologicals, 1:20,000), LRP6 C5C7 (Cell Signaling, 1:2500 for Western Blot only), LRP6 rabbit monoclonal (Epitomics, 1:2000 for ICC, 1:500 for Western Blot), LRP6-pS1490 (Cell Signaling 1:1000 for Western Blot). All the primary antibodies were incubated at 4° C overnight. Secondary antibodies were conjugated with Alexa Fluor dyes and used at 1:500 dilutions.

Neuronal Cultures and Immunocytochemistry

Primary hippocampal neurons were prepared from mouse or rat as described elsewhere (Brewer and Cotman, 1989). In brief, for high throughput assays neurons were dissociated from E16.5 mouse or E18 rat hippocampi and plated into 96 well optical plates using the multidrop apparatus (Thermo Scientific). For high content assays mouse neurons were plated at the density of 10,000 neurons per well and rat neurons were plated at 6000 neurons per well. Neurons were maintained at 37°C in a 5% CO2 incubator. Half of the media was changed every 4th day. A similar protocol was followed for small scale culturing in 12-well plate on Poly-L-Lysine coated coverslips. For automated immunolabeling a multidrop, plate washer and a robotic arm were employed using optimized programs. After the last step of washing neurons were preserved in fixation solution.

CREB Phosphorylation Assay

Rat neurons from E18 embryos were used for a pilot chemical screen to identify small molecules that can modulate CREB pathway. On DIV14 neurons were treated with Sigma LOPAC chemical library compounds for one hour at final concentration of 10 μM. Neurons were depolarized with 50 mM KCl in ACSF for 20 min. Neurons were fixed and immunostained with antibodies against CREB and Phospho-CREB. Images were acquired as described above.

Image acquisition and Analysis

High content image acquisition was carried out at BD Pathway Imaging Station. Images were acquired using a 40X objective in 4X4 montage format that covered a network of 25–35 neurons per well. Wells that showed obvious reduction in puncta count were analyzed by using Image J where regions of interest (ROIs) were selected on the basis on MAP2 staining and overlapped with PSD95 and Gephyrin labeled images. After background subtraction and thresholding puncta were counted using Analyze Particle tool. For the validation step ROIs were selected on the basis of GFP expression and analyzed as described above. The length of dendrite was measured using NeuronJ plugin. The data were normalized to the average of control group and plotted as percent of average of control.

ShRNA Loss of Function Genetic Screen

Mouse neurons prepared from E16.5 embryos were used for a loss of function genetic screen. Lentiviral particles suspension from MISSION shRNA library was used at 10,000 TU/ml to infect neurons at DIV 4. Neurons were fixed and immunostained at DIV13 for somatodendritic marker MAP2, and synaptic markers PSD95 and Gephyrin. Images were acquired by BD Pathway microscope in laser autofocus mode in 4X4 montages per well. For follow up studies LRP6 was knocked down in dissociated neurons with shRNA (5′-CGCACTACATTAGTTCCAAA-3′) that has common target in rat and mouse but not in human LRP6. Effect of shRNA was rescued by human LRP6.

In utero Electroporation and Immunohistochemistry

In utero electroporation was carried out as described (Tabata and Nakajima 2001). In brief, uterine horns of E15.5 mice were surgically exposed under anesthesia, and ~3 μg DNA mixed with fast green was injected into lateral ventricles. DNA was electroporated with 5 pulses at 950 ms interval at 40V for 50 ms with a tweezers electrode. Embryos were then placed back and abdominal wall was sutured. At postnatal day 28 mice were perfused with PBS followed by 4% paraformaldehyde in PBS. Brains were fixed for 24 hours followed by cryoprotection by 30% sucrose in PBS. 150μm thick slices were cut in coronal plane with a vibratome, and immunostained with anti-dsRed antibodies. Optical section in Z planes were acquired using Zeis 510 laser scanning confocal microscope.

Electrophysiology

3 – 5 week old in utero-electroporated mice were anaesthetized by isoflurane inhalation, and decapitated. Brains were quickly dissected in ice-cold buffer (212.7 mM sucrose, 10 mM glucose, 2.6 mM KCl, 1.23 mM NaH2PO4, 26 mM NaHCO3, 0.5 mM CaCl2 and 5 mM MgCl2). Brains were sliced into 300 μm thin slices with a vibratome in the same solution, and transferred to normal artificial cerebrospinal fluid (ACSF - 124 mM NaCl, 5 mM KCl, 1.23 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 2 mM CaCl2 and 1 mM MgCl2). Slices were allowed to recover for 1 hr. at 30°C, and maintained at room temperature (22–25 °C). Neurons were targeted for whole-cell patch-clamp recording with borosilicate glass electrodes of 3–6 MΩ resistance. The electrode internal solution consisted of 130 mM cesium methanesulphonate, 10 mM HEPES, 0.5 mM EGTA, 8 mM CsCl, 5 mM TEA-Cl, 1 mM QX-314, 10 mM Na phosphocreatine, 0.5 mM Na-GTP and 4 mM Na-ATP. Cortical pyramidal neurons with or without fluorescence were selected from layer II-V of primary somatosensory cortex through entorhinal cortex. For AMPA receptor-mediated miniature EPSCs, external solution was supplemented with 1 μM tetrodotoxin, 50 μM d,l-APV (2-amino-5-phosphonovalerate) and 100 μM picrotoxin. Data were acquired with a Multiclamp 700A, and Clampex 8 program (Molecular Devices) at 10 kHz., Current traces were low-pass filtered at 1 kHz prior to mEPSC detection and analysis. mEPSCs were detected and analyzed using Mini Analysis (Synaptosoft) or Clampfit 10 program (Molecular Devices).

Supplementary Material

01

HIGHLIGHTS.

  • Semi-Automated high content approach for neuron-based functional screen.

  • LRP6 required for excitatory synapse development in vitro and in vivo.

  • LRP6 is selectively localized to excitatory synapse.

  • Wnt8A is synaptogenic factor that acts via its interaction with LRP6

Acknowledgments

This work was supported by grants from the National Institutes of Health and the Howard Hughes Medical Institute to R.L.H. and National Institute of Health Grant 5U54MH084691 to M.L.

Footnotes

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Contributor Information

Kamal Sharma, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Se-Young Choi, Department of Physiology, Seoul National University School of Dentistry, Seoul 110-749, South Korea.

Yong Zhang, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Thomas J.F. Nieland, Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA

Shunyou Long, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Min Li, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Richard Huganir, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

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