Sortilin-Related Receptor SORCS3 Is a Postsynaptic Modulator of Synaptic Depression and Fear Extinction

SORCS3 is an orphan receptor of the VPS10P domain receptor family, a group of sorting and signaling receptors central to many pathways in control of neuronal viability and function. SORCS3 is highly expressed in the CA1 region of the hippocampus, but the relevance of this receptor for hippocampal activity remained absolutely unclear. Here, we show that SORCS3 localizes to the postsynaptic density and that loss of receptor activity in gene-targeted mice abrogates NMDA receptor-dependent and -independent forms of long-term depression (LTD). Consistent with a loss of synaptic retraction, SORCS3-deficient mice suffer from deficits in behavioral activities associated with hippocampal LTD, particularly from an accelerated extinction of fear memory. A possible molecular mechanism for SORCS3 in synaptic depression was suggested by targeted proteomics approaches that identified the ability of SORCS3 to functionally interact with PICK1, an adaptor that sorts glutamate receptors at the postsynapse. Faulty localization of PICK1 in SORCS3-deficient neurons argues for altered glutamate receptor trafficking as the cause of altered synaptic plasticity in the SORCS3-deficient mouse model. In conclusion, our studies have identified a novel function for VPS10P domain receptors in control of synaptic depression and suggest SORCS3 as a novel factor modulating aversive memory extinction.


Introduction
VPS10P domain receptors are a unique class of sorting and signaling receptors expressed in the nervous system. Five receptors form this gene family in mammals designated sortilin [1], sorting-related receptor with A-type repeats (SORLA) [2,3] as well as SORCS1, SORCS2, and SORCS3 [4]. All family members are characterized by a 700 amino acid module in their extracellular domain, initially identified in the yeast sorting receptor VPS10P (vacuolar protein sorting 10 protein) [5]. In recent years, three VPS10P domain receptors have been studied in detail documenting the central role played by this gene family in control of neuronal viability and function (reviewed in 6). Thus, sortilin was shown to regulate neuronal cell death and survival through modulation of (pro)neurotrophin signaling [7,8,9,10]. SORLA and SORCS1 act as neuronal receptors for the amyloid precursor protein (APP) controlling proteolytic breakdown of this precursor into neurotoxic amyloid-β peptides, a pathological mechanism in Alzheimer's disease [11,12,13,14,15].
Contrary to other VPS10P domain receptors, the significance of SORCS3 for the nervous system is absolutely unclear. Together with SORCS1 and SORCS2, SORCS3 forms a closely related subgroup within the VPS10P domain receptor family characterized by the presence of one amino terminal VPS10P domain followed by an imperfect leucine-reach repeat in their extracellular regions [4]. SORCS3 is a 130 kDa neuronal orphan receptor distinctly expressed in the hippocampus and cortex, and to a lesser extend in the cerebellum [4,16]. Hippocampal expression in mice is markedly up-regulated by synaptic activity following induction of limbic seizures through kainic acid injection [16].
Here, we report the generation and functional characterization of a SORCS3-deficient mouse model to query the relevance of this receptor for hippocampal activity in vivo. SORCS3-deficient animals are viable and fertile, but exhibit profound alterations in synaptic plasticity as shown by loss of long-term depression (LTD). Altered synaptic plasticity coincides with defects in spatial learning and modulation of fear memory, indicating involvement of SORCS3 in anxiety-related hippocampal activities. The ability of the receptor to control the subcellular localization of PICK1, an adaptor that sorts glutamate receptors, suggests a molecular mechanism for SORCS3 in control of neurotransmitter receptor trafficking at the synapse.

Hippocampal synaptosomal preparations
Hippocampi of mice were dissected and homogenized in 320 mM sucrose, 4 mM HEPES (pH 7.4), with 10 strokes of a glass-teflon homogenizer (H, homogenate). The homogenate was centrifuged at 1000xg for 10 min and the postnuclear supernatant (S1) was further centrifuged at 10,000xg for 15 min to yield the crude synaptosomal pellet (P2) and a supernatant, of which the light membrane fraction (P3) was pelleted at 150,000xg for 30 min. After washing the pellet, P2 was lysed by hypo-osmotic shock and three strokes of a glassteflon homogenizer. The lysate was centrifuged at 25,000xg for 20 min to yield the lysed synaptosomal membrane fraction (LP1). The supernatant (LS1) was pelleted at 165,000xg for 2 hours to derive the crude synaptic vesicle fraction (LP2). LP1 was layered on top of a discontinuous gradient containing 0.8 to 1.0 to 1.2 M sucrose and centrifuged at 150,000xg for 2 hours. Synaptic plasma membranes (SPM) were recovered in the layer between 1.0 and 1.2 M sucrose, diluted to 0.32 M sucrose and centrifuged at 150,000xg for 30 min. After resuspension and addition of Triton X-100 (final concentration 0.5%), SPMs were incubated on a rotating wheel at 4°C for 15 min, followed by centrifugation at 32,000xg (20 min) to yielded the PSDI pellet. The pellet was resuspended in 0.5% Triton X100, incubated on a rotating wheel at 4°C for 15 min and centrifuged at 200,000xg (20 min) to obtain the PSDII pellet.

Affinity purification and characterization of SORCS3 interacting proteins
Proteins were isolated using synthetic peptides corresponding to the carboxyl terminal oligopeptides of SORCS3 (S 1207 ESTKEIPNCTSV; UniProt Q8VI51) or SORCS2 (S 1147 INSREMHSYLVG; Q9EPR5). Peptides were coupled to sepharose beads and incubated with total brain lysate (containing 1% Triton X-100) at 4°C over night. Beads were pelleted and bound proteins eluted by boiling in Laemmli buffer. After separation on SDS-PAGE, protein bands were stained with Coomassie, cut out from the gel, and identified by mass spectrometry.
For co-immunoprecipitation, constructs encoding SORCS3 and CS3 CTSV were transiently transfected into COS-7 cells. After 36 hours, the cells were sonicated in 150 mM NaCl, 50 mM Tris (pH8), 1% NP-40 substitute, and 1 mM 0.25% deoxycholate, and incubated with 1µg of IgG directed against PSD95 at 4°C over night. Immunoprecipitates were collected using Protein G agarose (Roche) according to manufacturer's protocols. Eluted Proteins were separated on SDS-PAGE and analyzed by Western blotting.
For pull-down studies, HEK293 cells stably expressing SORCS3 were lysed on ice in lysis buffer (150mM NaCl, 2 mM MgCl 2 , 0.1 mM EGTA, 2 mM CaCl 2 , 10 mM HEPES, 1% Triton X-100, pH 7.4) and centrifuged at 4°C. 200 µl of the supernatant were mixed with either 10 µg GST or 15 µg GST-PICK1, adjusted to a volume of 1 ml in lysis buffer (without Triton), and rotated overnight at 4°C. The following day, glutathione sepharose beads were added and rotated for 5 hours at 4°C. Beads were pelleted, washed four times in lysis buffer (modified to 0.4 M NaCl, without Triton X-100), eluted, and analyzed by SDS-PAGE.

Generation of SORCS3-deficient mice
For homologous recombination in embryonic stem (ES) cells, a targeting vector was constructed as detailed in the result section. In brief, regions homologous to the murine Sorcs3 locus were amplified by PCR from isogenic ES cell DNA. A neomycin cassette flanked by FRT and loxP sites was inserted 0.8 kb downstream stream of exon 1 and one loxP site was inserted 0.8 kb upstream of exon 3. After electroporation of the targeting construct into ES cells and selection with G418, clones with homologous recombination were identified by Southern blot analysis. Mice derived by standard blastocyst injection of targeted cell clones were bred to the Cre deleter strain [17] to remove the neomycin cassette and to derive animals heterozygous for the deleted Sorcs3 allele (Sorcs3 +/-). Sorcs3 +/-animals were backcrossed on C57Bl/6N for more than 10 generations and then bred to homozygosity for the deleted allele (Sorcs3 -/-). Experiments in this manuscript have been carried out in littermates including all behavioral and biochemical analyses and most of the electrophysiological studies. The only exception being the mGluR-dependent LTD study which was performed using separate C57Bl/6N inbred lines of wild type or Sorcs3 null background. Animal experimentation was performed after approval by local ethics committees (x9012/12, LAGESO, Berlin, Germany; 2011/561-119, University of Aarhus, Aarhus, Denmark).
For RT-PCR analysis of Sorcs3 transcription, hippocampi, cortices and cerebella from freshly sacrificed mice were homogenized in TRIzol Reagent (Life Technologies). RNA was isolated using RNeasy Mini Kit (Qiagen) and transcribed to cDNA using the High Capacity RNA-to-cDNA Kit (Life Technologies). Specific cDNAs were amplified using Taq Polymerase (New England Biolabs) and the following primer:
Coronal brain slices were placed in an interface recording chamber perfused with ACSF containing 0.5% albumin that was recycled (a total volume of 300 ml; flow rate 2 ml/min). Recording electrodes (resistance 10-20 MΩ) were pulled from borosilicate glass (outer diameter: 1.5 mm; inner diameter: 0.8 mm, King Precision Glass Inc.) and filled with ACSF. The stimulation electrode (Concentric Bipolar Electrode, CBARC75, FHC Inc.) was placed in the stratum radiatum in the CA1 of the hippocampus and Schaffer collaterals were stimulated using a Stimulus Isolator (A365, World Precision Instruments) and a Master8 (A.M.P.I.). The recording electrode was placed in the stratum radiatum and recordings were made using MultiClamp 700B (Axon instruments) and Digidata 1440A (Axon Instruments). Extracellular field recordings were performed at 33 ± 1°C. After obtaining a stable baseline, low-frequency or tetanic stimulations were applied. Stimulus intensity was adjusted to evoke fEPSP at 40-50% of maximum. NMDA receptor-dependent LTD was induced by a single pulse applied at a rate of 1 Hz for 20 min. mGluR-dependent LTD was induced by twin pulses separated by 50 ms applied at 1Hz for 18 min. We induced long-term potentiation (LTP) by two trains of high-frequency stimulation (100 Hz, 1 s, separated by 45 sec). Paired-pulse facilitation (PPF) was induced by twin pulses with interstimulus intervals of 25-300 ms. Where indicated, 10 µM of the GABA B receptor agonist baclofen (Tocris) was supplied to the recording chamber during the entire PPF experiment.

Behavioral studies
All behavioral experiments were carried out using 14-20 weeks old male mice between 9: 00 a.m. to 5: 00 p.m. The open field test was performed in chambers monitored by an automated video system (TSE). Mice were placed in the chambers for 90 min on three consecutive days. The distance traveled was analyzed in 5 min intervals.
The experimental setup for the Barnes maze paradigm of spatial learning consisted of a circular platform (92 cm in diameter) elevated 105 cm above the floor. The maze contains 20 equally spaced holes (5 cm in diameter; 7.5 cm distance between the individual holes). The centers of all holes were 4.5 cm away from the perimeter of the maze. A hidden escape box was placed under the target hole. To facilitate spatial orientation, visual cues were placed on the laboratory walls. On day 1, mice were allowed to freely explore the maze for 3 min and were subsequently placed in the escape box for 2 min. Mice were then placed in the center of the maze under a dark circular box for 10 sec to ensure random orientation at the beginning of the test. A fan was turned on as reinforcing stimulus during the test phase. Mice were tested for four consecutive days and their latency to enter the target hole was measured by video recording and analysis using the Any-Maze tracking software.
Extinction of fear memory was assessed using a Gemini Avoidance System which takes advantage of the natural preference of mice for the dark. The setup consists of a brightly lit room and a dark room separated by a guillotine door. On the training day, individual mice are placed in the bright room. When entering the dark room, the door closes and the mouse receives an electric foot shock (0.4 mA for 1 sec). Twenty-four hours later, the mice are returned to the bright room and the latency to enter the dark room is recorded as an indicator of memory of the shock. Extinction of fear was achieved by returning the animals once every 24 h into the setup and by monitoring their latency to cross into the dark room for a total of 5 consecutive days. To test remote fear memory, animals were shocked and than returned once into the setup after 16 days.
For statistical analysis of behavioral studies, each measure of learning (Barnes Maze) or extinction of fear memory was analyzed with a mixed model ANOVA using GraphPad prism with genotype as a between-subject factor and trial day as a within-subject factor. If the repeated measures analysis showed significant differences between genotypes, post hoc analyses were performed at individual time points using the two-tailed Student's t-test for independent samples. Differences were considered statistically significant if p<0.05.

SORCS3 is expressed in the postsynaptic density of hippocampal neurons
Previously, in situ hybridization studies localized Sorcs3 transcripts to the brain of mice. In the adult central nervous system, neuronal expression of Sorcs3 is most pronounced in the CA1 region of the hippocampus. Additional patterns of expression are seen in the mitral cell layer of the olfactory bulb, in the piriform and cerebral cortex, as well as in the molecular layer of the cerebellum [16]. No significant expression of the gene in peripheral tissues has been reported thus far. Here, we used subcellular fractionation to substantiate expression of the receptor protein in the murine hippocampus. As shown in Figure 1A, SORCS3 was detected in fractions encompassing the synaptic plasma membrane or the postsynaptic density (PSD), but not in synaptic vesicle-enriched fractions. The localization of SORCS3 at the PSD was further supported by unbiased proteomics approaches to identify proteins that interact with the cytoplasmic tail of the receptor in neurons. To do so, synthetic peptides encompassing the last 14 amino acids of the cytoplasmic domain of SORCS3 (CS3-CT) were coupled to sepharose resin and used to affinity purify interacting proteins from mouse brain homogenates. Two prominent proteins were purified on CS3-CT columns that were identified as PSD93 and PSD95 by mass spectrometry ( Figure  1B). These proteins were not recovered on columns containing a carboxyl terminal peptide of the related receptor SORCS2 (CS2-CT, Figure 2B). The ability of SORCS3 to interact with PSD95 was confirmed in transiently transfected COS7 cells showing co-immunoprecipitation of SORCS3 with an antiserum directed against PSD95 ( Figure 1C). The interaction site for PSD95 was mapped to the last four amino acids of the cytosolic part of SORCS3 containing a putative PDZ domain binding motif (CTSV) as a receptor mutant lacking this tetrapeptide failed to co-immunoprecipitate with anti-PSD95 IgG (C3 CTSV ; Figure 1C). Although obtained in vitro, the above data provide experimental support for a potential interaction of SORCS3 with PSD95 in vivo.

Generation of a SORCS3-deficient mouse model
To explore the relevance of SORCS3 for hippocampal activity, we next generated a mouse model carrying a targeted disruption of Sorcs3. To do so, loxP recombination sites flanking exon 1 of the Sorcs3 locus on mouse chromosome 19D1 were introduced into the genome of mice using standard embryonic stem cell technology (Figure 2A). Exon 1 encodes 206 amino acid residues encompassing the Start-ATG, the   Figure 2B). Trace amounts of aberrant transcripts encompassing exons 13-15 were still produced from the targeted gene locus as shown by RT-PCR ( Figure 2B). However, no wild type protein or any truncated receptor product were detected in hippocampus, cortex, or cerebellum of mice homozygous for the disrupted Sorcs3 locus confirming successful inactivation of SORCS3 expression in our mouse model ( Figure 2C).
Mice homozygous for the Sorcs3 null allele (Sorcs3 -/-) were viable and fertile, and showed no discernable abnormalities upon external inspection. Also, histological analysis did not reveal any obvious histoanatomical alterations of the SORCS3deficient hippocampus as compared to control tissue ( Figure  2D). Finally, the level of expression of proteins in the PSD was not affected by lack of SORCS3 as shown in subcellular fractionation experiments for the related receptor SORCS2, for PSD95, or for subunits of AMPA receptors (GluR1, GluR2), NMDA receptors (NR1 and NR2B), or the metabotropic glutamate receptor mGluR5 ( Figure 2E). Similarly, the levels of co-receptors to other VPS10P domain receptors such as the neurotrophin receptors P75 NTR and TrkB were unaltered ( Figure  2E).

SORCS3 deficiency results in loss of long-term depression
Given the prominent expression of SORCS3 in the CA1 region of the hippocampus, we performed electrophysiological recordings to explore the consequences of receptor deficiency for synaptic plasticity in this brain area. Long-term potentiation (LTP) is a major form of long-lasting synaptic plasticity [18]. During LTP, AMPA receptors are inserted into the PSD, resulting in an increase in synaptic strength [19,20]. However, field recordings of the stratum radiatum in the CA1 region of the hippocampus following stimulation of Schaffer collaterals failed to reveal any difference in early LTP in slices from SORCS3-deficient compared with wild type brains (Figure 3).
Long-term depression (LTD) is the second major form of long-lasting synaptic plasticity and considered important for synaptic retraction. During LTD, AMPA receptors undergo internalization from the PSD causing a decrease in synaptic strength. The most widely studied form of LTD is NMDA receptor-dependent. Here, the synaptic activation of NMDA receptors triggers the induction of LTD [19,21]. As shown in Figure 4A and B, NMDA receptor-dependent-LTD was absent in hippocampal slices from Sorcs3 -/-animals. Another form of LTD is mGluR-dependent whereby synaptic activation of metabotropic glutamate receptors triggers LTD [19,22]. Remarkably, mGluR-dependent LTD was also lost in SORCS3deficient mice ( Figure 4C and D).
To exclude possible presynaptic effects of SORCS3 deficiency, we tested paired-pulse facilitation (PPF), a form of short-term synaptic plasticity [23]. In PPF, postsynaptic field potentials evoked by extracellular stimulation are increased at the second pulse due to enhanced release of synaptic vesicles from the presynaptic terminal [24]. As shown in Figure 5, there was no difference in PPF in mutant as compared to wild type mice. The GABA B receptor agonist baclofen reduces the release probability primarily by inhibiting calcium channels, leading to a larger pool of release-ready vesicles for the second pulse [25]. Therefore, PPF is increased. Application of baclofen did not differently affect PPF in SORCS3-deficient mice compared with control animals, indicating intact presynaptic GABA B receptor signaling cascades ( Figure 5).

Impaired learning and fear memory in mice lacking SORCS3
To explore the consequences of altered synaptic plasticity for learning and memory, we subjected SORCS3 null mice to a number of behavioral tests. In the open-field test, Sorcs3 -/-mice failed to show any difference in locomotion compared to controls ( Figure 6). However, when tested for spatial learning and memory using the Barnes maze (see methods for details), SORCS3-deficient animals performed significantly poorer than control animals with an obvious inability to learn the location of the escape target hole over a 4-day test period (Figure 7A and B) and a significantly increased number of nose poke errors on the fourth test day ( Figure 7C).
Fear memory is another form of long-lasting memory involving the hippocampus. Accordingly, we applied an inhibitory avoidance test to investigate fear memory in our mouse model. This setup takes advantage of the natural preference of mice for the dark and consists of a brightly lit room and a dark room separated by a guillotine door. On the training day, mice are placed in the bright room. When entering the dark room, the animals receive an electric foot shock. Twenty-four hours later, the mice are returned to the bright room and their latency to enter the dark area is recorded as an indicator of memory of the adverse event. Both wild type and  Figure 8A, day 1). Subsequently, extinction of fear was tested by returning the animals once every 24 h into the setup and by monitoring their latency to cross into the dark room for a total of 4 consecutive days. As seen in Figure 8A, Sorcs3 -/-mice displayed a significantly accelerated extinction of short-term fear memory as indicated by their willingness to enter the dark room faster than control animals on days 2 through 4 of the trial. This behavioral abnormality was not due to increased forgetting over time as the remote fear memory retrieval of SORCS3-deficient mice was identical to that of wild type controls when shocked and returned to the setup once after 16 days ( Figure 8B).

SORCS3 affects localization of PICK1 at the postsynapse
Based on the alterations seen in SORCS3-deficient mice, SORCS3 seems to play a role in weakening of synaptic strength. Possibly, up-regulation of Sorcs3 by synaptic activity serves as a mechanism to balance synaptic hyperactivity [16].
One hypothesis concerning the molecular mechanism of SORCS3 action is involvement of this receptor in intracellular protein trafficking. As NMDA receptor-dependent and mGlu receptor-dependent LTD are lost in mutant mice, SORCS3 ought to act in a fundamental (trafficking) mechanism common to both forms of LTD (Figure 4). A cellular mechanism underlying both NMDA receptordependent and -independent forms of LTD is the endocytosis of AMPA receptors from the postsynaptic membranes, processes controlled by interaction of the glutamate receptors with anchoring proteins (reviewed in 26). PSD95 is mainly responsible for tethering of AMPA receptors to the synaptic membrane, with evidence showing that synaptic depression requires dephosphorylation of serine-295 of PSD95 [27]. On the other hand, PICK1 (protein interacting with C-kinase 1) emerges as a sorting adaptor implicated in removal of AMPA receptors from the synaptic cell surface during LTD [28,29,30,31,32]. Especially, PICK1 has been shown to be essential for the clustering and intracellular retention of the AMPA receptors, a mechanism that underlies the expression phase of the LTD [32,33]. Accordingly, we tested whether PICK1 may represent an adaptor protein also interacting with the cytoplasmic domain of SORCS3. Interaction of PICK1 with SORCS3 was confirmed by pull-down of the receptor from transiently transfected HEK293 cells using a recombinant GST-PICK1 fusion protein ( Figure 9A). Total levels of PICK1 were unchanged in hippocampus, cortex or cerebellum of SORCS3deficient mice ( Figure 9B). However, subcellular fractionation studies documenting reduced levels of PICK1 in the PSD of SORCS3-deficient compared to wild type hippocampi. This observation supported the functional significance of PICK1 and SORCS3 interaction for adaptor sorting in vivo ( Figure 9C and D).

Discussion
VPS10P domain receptors emerge as central regulators of neuronal viability and function. Their mode of action commonly involves the ability to interact with cytosolic adaptors to sort target proteins, such as Trk receptors [9] or APP [11,15,34,35]. Studies presented herein support this notion by elucidating a hitherto unknown function for the orphan receptor SORCS3 in Figure 8. Lack of SORCS3 results in increased fear extinction in mice. (A) Mice were exposed to an inhibitory avoidance experiment as described in the method section. SORCS3-deficient mice display a more rapid extinction of fear over four consecutive days albeit at an intact acquisition of fear memory (shock). In contrast, wild type mice show no reduction in fear memory during the course of the experiment. Statistical analysis was performed by two-way ANOVA documenting a significant effect of genotype on learning performance: F=5.57, p<0.5. Subsequently, post hoc analyses were performed at individual time points using the two-tailed Student's t-test for independent samples (*p<0.05, **p<0.01). (B) SORCS3-deficient and wild type mice display similar remote fear memory 16 days after the foot shock. doi: 10.1371/journal.pone.0075006.g008 control of LTD, an activity potentially involving PICK1dependent sorting processes as the postsynapse.
SORCS3 is a member of the VPS10P domain receptor gene family that shows the most restricted pattern of expression, with the main site of expression being the hippocampus. The receptor is also unique in as much as it is the only VPS10P domain receptor to harbor a PDZ domain binding motif (X-S/T-X-V), arguing for a specific role of this protein at the postsynapse of hippocampal neurons not shared by the other receptors. A role for SORCS3 in synaptic activity was now confirmed by documenting distinct defects in synaptic plasticity related to LTD in mice genetically deficient for this receptor. Postsynaptic dysfunctions coincide with distinct behavioral anomalies in affected animals including defect in spatial learning and memory, and in enhanced extinction of contextual fear.
Spatial learning as tested in the Barnes maze requires hippocampal function. Also, modulation of fear through repeated exposure to the context of an adverse event is controlled by hippocampal activities [36], providing a behavioral correlate for the alteration in synaptic plasticity seen in the hippocampus of Sorcs3 -/-mice. Thus, applying antagonists of NMDA receptor subunits to specifically erase LTP versus LTD, blockade of LTD (but not of LTP) in the CA1 region of the hippocampus of rats resulted in impaired consolidation of spatial memory in the Morris water maze [37]. These data are in line with studies in knockout mouse models wherein selective loss of LTD [38,39] but not of LTP [40,41] affected spatial learning. Our data in SORCS3-deficient mice support the concept that LTD in the CA1 region is of particular importance for establishing long-term spatial memory, a function that apparently involves SORCS3 activity.
LTD is also implicated in other forms of learning such in the extinction of aversive memories. For example, mice overexpressing Rap2, a Ras-related GTPase at the synapse, display normal LTP but enhanced LTD, which, in turns, results in impaired extinction of contextual fear [42]. In line with such an inverse correlation, loss of LTD in Sorcs3 -/-animals coincides with accelerated inhibition of contextual fear memory ( Figure 8). Several psychiatric disorders such as major depression or posttraumatic stress disorder syndrome are caused in part by a failure to erase aversive memory. Given the functional implication of Sorcs3 in modulation of fear memory in mice, this gene may represent a novel risk gene for anxietyrelated disorders in humans. Although highly speculative at present, one genetic study reported a de novo duplication of the chromosomal region 10q23 encoding SORCS1 and SORCS3 in an individual with bipolar disorder [43]. Intriguingly, bipolar disorder is known to share high comorbidity with posttraumatic stress disorder syndrome [44].
What may be the molecular mechanism of SORCS3 action in hippocampal neurons? Based on analogy to other VPS10P domain receptors, sorting of target proteins at the postsynapse seems a plausible hypothesis. This model is supported by the ability of the receptor to interact in vitro with PSD95 and PICK1, adaptors implicated in glutamate receptor localization. In a simple scenario, our data suggest a model whereby SORCS3 interacts with PICK1 to modulate AMPA receptor sorting at the PSD. Inhibitory peptides or antibodies blocking the interaction of PICK1 with GluR2/3 attenuate LTD induction in cerebellar Purkinje cells in culture [45]. Furthermore, inactivation of PICK1 by gene ablation [28,30,46] or by pharmacological intervention [29] impairs LTD in the cerebellum as well as LTP and LTD in the CA1 region of the hippocampus. Because LTD but not LTP are affected by loss of SORCS3, the interaction of PICK1 with this receptor seems relevant for removal of AMPA receptors from but not for targeting to the postsynaptic membrane compartment. Total levels of PICK1 are not changed in the hippocampus of Sorcs3 -/-mice ( Figure 9B). However, we observed a distinct loss of the adaptor protein from the PSD fraction, arguing for a redistribution of the protein between subcellular compartments in the absence of SORCS3 ( Figure  9C and D). So far we failed to detect any major changes in levels of AMPA receptor subunits in the PSD of SORCS3deficient hippocampi using subcellular fractionation studies ( Figure 2E). However, more drastic interventions, such as induction of synaptic activity by kainic acid injection, may be required to better appreciate altered localization of AMPA receptors in SORCS3-deficient hippocampal neurons.
In conclusion, we have identified a hitherto unknown function for SORCS3 as regulator of LTD, further underscoring the central roles played by VPS10P domain receptors in control of neuronal activities. Further elucidation of this pathway will help in understanding the molecular mechanisms underlying repression of synaptic activity and how alterations in components of this pathway may contribute to anxiety-related disorders in humans.