Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75 NTR . CNS neurons, but not glia, express SorCS2 as a single-chain protein that is essential for proBDNF-induced growth cone collapse in developing dopaminergic processes. SorCS2- or p75 NTR -deficient in mice caused reduced dopamine levels and metabolism and dopaminergic hyperinnervation of the frontal cortex. Accordingly, both knockout models displayed a paradoxical behavioral response to amphetamine reminiscent of ADHD. Contrary, in PNS glia, but not in neurons, proteolytic processing produced a two-chain SorCS2 isoform that mediated proNT-dependent Schwann cell apoptosis. Sciatic nerve injury triggered generation of two-chain SorCS2 in p75 NTR -positive dying Schwann cells, with apoptosis being profoundly attenuated in Sorcs2 −/− mice. In conclusion, we have demonstrated that two-chain processing of SorCS2 enables neurons and glia to respond differently to proneurotrophins.

Surprisingly, Schwann cell (SC) apoptosis induced by sciatic nerve injury requires p75expression (), yet sortilin is absent from these cells (). This observation suggested that SC death is either independent of proNTs or requires a yet unknown p75coreceptor. Now, we establish the sortilin-related receptor SorCS2 as a proNT binding partner and coreceptor to p75that displays a remarkably complementary cell type-specific and subcellular expression compared to sortilin. We find that SorCS2 is unique among the members of the Vps10p-D receptor family because it exists in single- and two-chain forms that engage in axonal retraction and transmission of apoptotic signals, respectively. This example shows that proteolytic processing of a receptor can regulate two disparate functions critical for balancing trophic and apoptotic signals in the nervous system.

The Vps10p-domain (Vps10p-D) family of neuronal receptors comprises sortilin, SorLA, and SorCS1, SorCS2, and SorCS3. We previously reported that sortilin, the archetype Vps10-D receptor (), plays a central role in regulating cell fate (). It is required for proNTs to induce apoptosis by forming a ternary death-inducing receptor-ligand complex with p75and either proNGF, proBDNF, or proNT3 (). Accordingly, sortilin knockout mice exhibit reduced proNGF-dependent apoptosis under conditions where neuron death normally prevails, including pruning of retinal ganglion cells, senescence of sympathetic neurons, and injury to corticospinal neurons ().

The neurotrophin (NT) family comprises nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4;). Among other functions, these factors stimulate neuronal survival, differentiation, axon guidance, and synaptic strengthening by engaging members of the receptor tyrosine kinases Trks in conjunction with p75. NTs are commonly secreted as precursors denoted proneurotrophins (proNT). In contrast to their mature counterparts, proNTs induce apoptosis, growth cone collapse, and facilitate synaptic retraction by a mechanism that requires p75but is independent of Trk receptors (). The capacity of proNTs to induce apoptosis is considered particularly important in conditions of acute and insidious neuronal and glial cell degeneration such as spinal cord and peripheral nerve injury, seizure, and aging ().

During embryonic and early postnatal development, axons navigate to their targets through a process controlled by attractive and repulsive cues and signals regulating growth cone morphology. Once innervation is completed, a period of pruning follows that serves to nourish axons and neurons that made proper synaptic contacts while eliminating those that failed to do so. Many trophic cues persist during adulthood and secure neuronal integrity but apoptotic signals become dormant (). However, following injury to the nervous system, apoptosis signaling may be reactivated, causing death of the lesioned neurons and their neighboring glia cells (). The mechanisms that switch between trophic and apoptotic responses are incompletely understood, but signaling by neurotrophins and their precursors is likely to be involved ().

Neuronal loss is also a key feature of nerve injury. We therefore stained DRGs for Casp3 activation 17 days after lesioning. As opposed to SorCS2, which was confined to satellite cells (cf. Figure 1 E), sortilin was present exclusively in neurons ( Figure 7 J). In marked contrast to the protective effect of SorCS2-deficiency on SC apoptosis, it did not safeguard the corresponding neurons from caspase-3 activation ( Figure 7 K). Conversely, sortilin-deficiency did not affect SC apoptosis, although it reduced activated Casp3neurons by ∼80% ( Figures 7 I and 7K). The data suggest that two-chain SorCS2 and sortilin subserve similar apoptosis-inducing activities but do so in distinct cell types. Whereas SorCS2 operates in Schwann cells, sortilin induces apoptosis in the corresponding neurons.

Because all components of the extracellular apoptotic machinery (two-chain SorCS2, proNTs, and p75) were induced after sciatic nerve injury, we compared caspase-3 activation (Casp3) and DNA fragmentation (TUNEL) in adult WT and Sorcs2animals 17 days after surgery. The neuronal composition of the DRG, the morphology of the sciatic nerve, number of SCs, and peripheral innervation as determined by nociception, tactile sensation, and motor function were identical in naive WT and SorCS2 KO animals prior to the injury ( Figure S7 ). Notably, SC apoptosis was substantially reduced distal to the lesion in the SorCS2-deficient mice as shown by a reduction in Casp3and TUNEL-positive cells of ∼46% and ∼51%, respectively ( Figures 7 H and 7I and S8 C and S8D). For comparison, mice devoid in sortilin expression (Sort1) showed no protection against caspase-3 activation ( Figures 7 H and 7I). In P2 animals, the number of TUNEL-positive cells was diminished by 34% in the SorCS2 KOs 24 hr after injury, demonstrating the importance of the receptor for SC apoptosis in the neonatal period ( Figure S6 E).

Taking this further, we investigated whether injury may stimulate SorCS2 and p75heterodimerization by use of in situ proximity ligation assay. This assay is based on hybridization of circular DNA probes coupled to antibodies. When probes are located less than 30 nm apart they will hybridize, and interacting proteins can be visualized with PCR using fluorescent-labeled nucleotides. Notably, we observed a red punctate staining on the injury side, reflecting juxtaposition of the receptors in the plasma membrane. No such labeling was seen in the contralateral uninjured nerve ( Figure 7 G).

SC apoptosis followed by demyelination is a prominent feature of peripheral nerve injury. Given the role of proNTs and p75in this process (), we explored whether SorCS2 might also be involved. With WB analysis, we first studied expression of single- and two-chain SorCS2 in ipsi- and contralateral nerve fibers after a sciatic nerve ligation of adult mice. On the injured side, there was a pronounced and selective increase in two-chain SorCS2 that was particularly prominent distal to the lesion site ( Figures 7 D and S8 A). Whereas on the unlesioned side SorCS2 was confined to the epineurium, injury triggered a remarkable expression of the receptor in S100SCs in the distal stump. In contrast, sortilin was completely absent from these cells ( Figure 7 E). Injury also produced a striking coexpression of proBDNF, proNGF, and p75in the SorCS2-positive SC ( Figure 7 F), confirming previous reports that these proteins and their mRNAs are upregulated following a sciatic nerve lesion (). We also observed a similar induction of SorCS2, p75, and proNTs in P2 mice analyzed 24 hr after the injury ( Figure S6 B and data not shown).

The pro-protein convertase PC1 is induced in the transected sciatic nerve and is present in cultured Schwann cells: comparison with PC5, furin and PC7, implication in pro-BDNF processing.

Because sortilin is proapoptotic when coexpressed with p75), we explored whether SorCS2 might also engage in apoptosis induction. To do so, RN22 Schwannoma cells that express endogenous p75were transfected with WT SorCS2 and cell death was scored after the addition of proNGF. As in the primary PNS glia cell cultures (cf. Figure 1 G), SorCS2 was efficiently processed into its two-chain form in Schwannoma cells ( Figure 7 A). Treatment of the transfectants with proNGF for 72 hr increased cell death by more than 4-fold relative to mock transfected cells ( Figure 7 B). Intriguingly, whereas transfection with WT SorCS2 substantially increased Schwannoma cell death over that observed in control cells, SorCS2-one failed to do so. Similarly, in a rescue experiment using primary SorCS2 KO Schwann cells, transfection with two-chain, but not one-chain SorCS2, rendered the cells sensitive to proBDNF-induced apoptosis ( Figure 7 C). These observations indicate that two-chain processing is obligatory for SorCS2 to mediate cell death.

(H) Immunohistochemistry for activated caspase-3 in injured sciatic nerves distal from the site of injury in WT, Sorcs2 −/− , and Sort1 −/− mice (17 days after injury). Data indicate reduced apoptosis in SorCS2-deficient animals.

(D) Two-chain SorCS2 and p75 NTR are upregulated following sciatic nerve lesion (24 hr after surgery). Two-chain SorCS2 levels are increased both proximal (P) and distal (D) to the lesion (ipsilateral side) as determined with WB analysis (755% ± 243% and 2,018% ± 401%, respectively, compared to unlesioned nerve, n = 3, 50 μg tissue homogenate per lane; 12- to 16-week-old mice were used).

(C) Treatment with 10 ng/ml proBDNF for 24 hr induced apoptosis in primary SCs from perinatal SorCS2 KO mice transfected with SorCS2-two but not SorCS2-one as determined by counting pycnotic and disintegrated nuclei (n = 4).

(B) Treatment with 100 ng/ml proNGF for 72 hr induces cell death in RN22 cells when transfected with SorCS2-wt (n = 4) but not with SorCS2-one as measured using quenched fluorescence-based assay for the number of live cells per well (n = 4).

The combined effect of altered dopamine levels and metabolism and hyperinnervation of the frontal cortex would predict an abnormal response to psychostimulants. Thus, we subjected 12- to 16-week-old Sorcs2mice to the open field test in the absence or presence of amphetamine treatment and measured their mobility during a 40 min trial period. Strikingly, vehicle-treated SorCS2 KOs were significantly more active than their WT controls (p = 0.04; Figures 6 F and 6G). Furthermore, whereas amphetamine increased the distance traveled in WT animals from 56.2 ± 4.47 m to 158 ± 24.8 m (p = 0.005), we observed a paradoxical calming effect on Sorcs2mice (from 76.7 ± 7.78 m to 49.7 ± 8.97 m, p = 0.03), a key symptom of attention deficit hyperactivity disorder (ADHD) in humans (). In agreement with the cooperativity of SorCS2 and p75in proBDNF binding, p75-deficient animals (Ngfr) were also hyperactive (p = 0.02) and showed a blunted response to amphetamine ( Figures 6 F and 6G).

We next studied dopaminergic activity by measuring dopamine and its metabolites in the frontal cortex and striatum with high-performance liquid chromatography ( Figures 6 D and 6E). We found a marked reduction in dopamine levels of frontal cortex in SorCS2 and p75deficient animals (p = 0.02 and p = 0.03, respectively). In the striatum, the absolute dopamine concentrations were unaltered (data not shown) but dopamine metabolism was decreased in both KO models compared to WT mice. These changes possibly reflect compensatory adaptations to abnormal DA connectivity.

Defects in neuronal guidance during development would predict abnormal connectivity in the adult. Hence, we inspected the anatomy of the DA system in 12-week-old WT and Sorcs2mice. TH-positive neurons were readily seen in all discrete dopaminergic cell groups of both genotypes ( Figure S5 and data not shown). The number of neurons in the substantia nigra and VTA determined by stereologic quantification was also unaltered in the adult knockout mice, indicating that SorCS2 expression does not affect neuronal viability or migration of DA precursors ( Figure 6 A). In addition, the volume of the striatum as estimated using the principle of Cavalieri was similar for both genotypes (1.00 × 10± 1.37 × 10μmfor WT and 0.95 × 10± 1.42 × 10μmfor KO, n = 8). Even so, we found that the frontal cortex, which receives DA input from the VTA, was strikingly hyperinnervated as shown with optical density image analysis ( Figures 6 B and 6C). TH-positive fibers of WT animals occupied 17% of the infralimbic area, a value that had increased to 23% in the SorCS2 KOs. Notably, mice lacking p75expression (Ngfr) exhibited a hyperinnervation that was identical to that observed in Sorcs2mice (22% versus 23%; Figure 6 C).

(F) Track blots showing increased spontaneous motor activity in Sorcs2 −/− and Ngfr −/− mice. While WT mice respond to amphetamine by hyperactivity, knockout animals are calmed by this treatment. Each image displays the trace (40 min) of one representative mouse for each condition.

(D and E) Reduced total dopamine levels and metabolism in the frontal cortex and striatum, respectively, of SorCS2 and p75 NTR KOs compared to WT as determined with high-performance liquid chromatography of 12-week-old WT and KOs, n = 8 per group. Dopamine metabolism was estimated as the dopamine levels divided by the sum of its metabolites.

(C) The area covered by TH + DAB staining was quantified in the infralimbic cortex from WT mice, SorCS2 KO, and p75 NTR KO (14-week-old mice, four animals in each group). The results were evaluated using the Mann-Whitney U test.

(B) Qualitative anatomical analysis of the dopaminergic system that revealed a marked increased density of TH fibers in the infralimbic cortex of SorCS2 KO animals (14 weeks old, six animals of each genotype) as shown with DAB immunohistochemistry on horizontal sections.

Given the heterodimerization of p75and SorCS2, we examined p75expression at day E15.5. Like SorCS2, p75was present in THneurons of the midbrain ( Figure 5 D), suggesting that they may cooperate in maturation of dopaminergic neurons. ProNGF was undetectable in this structure, but we observed abundant proBDNF expression in cells demarcating the DA neurons compatible with a function in axon guidance ( Figure 5 E). Hence, we investigated whether proBDNF can provoke DA growth cone collapse in E14.5 midbrain explants. Intriguingly, proBDNF induced a substantial collapse of THneurons in explants from wild-type animals whereas explants from SorCS2 knockouts were completely unresponsive ( Figures 5 F and 5G).

To investigate whether SorCS2 may engage in establishing neuronal connectivity during development, we analyzed the embryonic expression pattern by in situ hybridization and immunohistochemistry at embryonic day 14.5 (E14.5) and E15.5, respectively ( Figure S4 ). As opposed to the adult brain, SorCS2 was absent from the embryonic cerebellum, cortex, and dorsal hippocampus at E15.5 ( Figure S4 J). In contrast, mRNA transcripts were abundant not only in the mesencephalic flexure of the midbrain area ( Figure 5 A, asterisk), but also in the ventral hippocampus, the spinal cord, and in nonneuronal tissues such as heart and lung ( Figure S4 ). Immunofluorescence microscopy revealed enrichment of SorCS2 but not sortilin in tyrosine-hydroxylase positive (TH) neurons of the developing midbrain, which matures into the ventral tegmental area (VTA) and substantia nigra of the mature dopaminergic (DA) system ( Figures 5 D and S4 K). To identify the isoform expressed in these neurons, we dissected out the embryonic midbrain and generated primary neuronal cultures. In accordance with the immunohistology, SorCS2 was prominent in TH-positive neurons with single-chain SorCS2 as the main isoform expressed ( Figures 5 B and 5C).

(F and G) SorCS2 mediates proBDNF-induced growth cone collapse. Midbrain E14.5 explants were cultured 2DIV prior to the addition of 10 ng/ml proBDNF for 20 min (F). The percentage of collapsed growth cones was subsequently scored by morphology (G). A minimum of 200 growth cones from at least 12 individual explants were evaluated for each condition (n = 4). Error bars represent SEM. ∗ p < 0.05.

(B and C) Expression of single-chain SorCS2 (green) in cultured embryonic TH + (red) neurons (E14.5DN, 7DIV) as analyzed by immunofluorescence (B) and WB using αECD (C).

A role for SorCS2 in axonal guidance was recently proposed because antireceptor antibodies attenuated growth cone retraction in cultured hippocampal neurons (). To extend these studies, we exploited growth cone morphology in neonatal cerebellar granule cells (CGN) because these cells express high levels of single-chain SorCS2 but not the 104 kDa two-chain variant ( Figure 4 A). As opposed to sortilin, which was mainly found in vesicular structures concentrated in the soma, SorCS2 was also abundant in axons and growth cones ( Figure 4 B). In the neurites, SorCS2 intensively colocalized with p75in extending filopodia-rich projections and in collapsed growth cones, supporting a role in regulating growth cone decision ( Figure 4 C). To demonstrate a function for SorCS2 in axon retraction in vivo, we generated a SorCS2-deficient mouse and demonstrated with WB and immunofluorescence that expression of both receptor isoforms had been disrupted in CNS neurons (brain extracts, CGN cultures) and in PNS glia (sympathetic and dorsal root ganglia, respectively; Figures S2 1 K, and 4 A). Knockout mice were viable, fertile, had a normal life span, and showed no histological abnormalities of the CNS (data not shown). First, we treated cultured CGNs from wild-type and knockout mice with proBDNF for 20 min to induce growth cone collapse in a p75-dependent manner () and quantified retracted axons by labeling with the microtubule marker phalloidin ( Figure 4 D). Whereas control CGNs responded to proNT stimulation by a marked increase in growth cone collapse of ∼57% (26.3% ± 1.76% versus 41.3% ± 2.36%, p < 0.01), Sorcs2neurons failed to do so (28.7% ± 1.86% versus 33.7% ± 2.91%, p = 0.22). Likely, because of impaired sensitivity to endogenous proBDNF, knockout neurites were greatly extended compared to wild-types after 7 days in culture ( Figure 4 E). Of note, wild-type (WT) and knockout (KO) neurons showed identical localization of p75in soma and neurites, suggesting that this effect was not accounted for by changes in the subcellular distribution of p75 Figure S3 ). Remarkably, in SorCS2 knockout neurons proBDNF-induced growth cone collapse was rescued by expression of SorCS2-one, the isoform normally expressed in CNS neurons, but not by SorCS2-two, the predominant form in PNS ( Figure 4 F).

(F) SorCS2-one, but not SorCS2-two, rescues proBDNF-induced growth cone collapse in transfected SorCS2 KO hippocampal neurons (3DIV). At least 50 transfected neurons were evaluated per coverslip on four independent coverslips per condition. The inset shows WB analysis of SorCS2-one and SorCS2-two in transfected KO neurons.

(D) ProBDNF (10 ng/ml, 20 min) induces growth cone collapse in cultured CGN (3DIV) from WT mice but from not KO neurons. Growth cones were depicted with phaloidin staining (red). The experiment was performed four times with similar results. Arrowheads indicate collapsed growth cones.

(A) WB showing that SorCS2 is expressed as a single-chain isoform in cultured cerebellar granule neurons (CGN; 6 DIV) from WT mice (+/+). SorCS2 KO neurons (cf. Figure S3 ) are used as negative and transfected CHO cells as positive control. CHO cells produce mainly the two-chain form.

Finally, we investigated whether SorCS2 and p75cooperate in proNT binding. To this end, cells expressing SorCS2 and p75separately or together were incubated with low concentrations (3 nM) of proNGF at 4°C followed by anti-proNGF immunostaining ( Figure 3 I). Binding to SorCS2 or p75individually was barely detectable but surface labeling was greatly enhanced when the receptors were coexpressed. We inferred that coexpression of SorCS2 and p75is required for efficient proNT binding.

Sortilin forms a complex with p75and we speculated SorCS2 might do the same (). Cells expressing wild-type SorCS2 and p75were subjected to coimmunoprecipitation using αECD or αCT antibodies followed by anti-p75immunoblotting ( Figure 3 D). SorCS2 robustly pulled down the highly glycosylated mature form of p75, which indicates complex formation at the cell surface. Importantly, p75and SorCS2 also coprecipitated in cerebellum lysates of 8-week-old mice, signifying that complex formation can also occur when receptors are expressed at endogenous levels ( Figure 3 E). In the reverse situation, p75coprecipitated both single- and two-chain SorCS2 from HEK293 cells transfected with p75and wild-type SorCS2 ( Figure 3 F). Hence, two-chain processing does not affect the ability of p75and SorCS2 to form heterodimers. In marked contrast to the interaction of p75with sortilin (), proNTs did not strengthen the association with SorCS2 (data not shown). The interaction was direct and mediated by the extracellular receptor domain, because the p75ectodomain fused to IgG-Fc bound immobilized SorCS2-sol with a Kof ∼10 nM. No binding was observed for IgG ( Figures 3 G and 3H).

Given that sortilin can bind proNGF, proBDNF, and proNT3 with high affinity, we exploited whether this also applies to SorCS2. The ectodomain of the receptor was immobilized on a biosensor chip and tested for proNT binding by surface plasmon resonance (SPR) analysis. ProNGF and proBDNF showed a robust interaction with SorCS2-sol corresponding to a Kof ∼5 nM, but mature NGF and BDNF bound only poorly ( Figure 3 A and data not shown). The proNT prodomains accounted for this interaction because the propeptides of proNGF, proBDNF, and proNT3, but not that of the glial-cell derived neurotrophic factor precursor (GDNFpro), bound SorCS2 avidly ( Figure 3 B). We next asked whether the SorCS2 isoforms can bind proNTs when exposed on the plasma membrane by incubating cells with 20 nM proNGF at 4°C for 120 min followed by anti-proNGF immunostaining. Because all three lines stained to a similar extent, we concluded that binding of proNTs is not restricted to one of the SorCS2 isoforms ( Figure 3 C).

(I) Binding of proBDNF (3 nM) to the surface of transfected HEK293 cells is greatly enhanced by the presence of both SorCS2 and p75 NTR . The level of p75 NTR expression was not altered by the presence of SorCS2 (data not shown).

(D) Coimmunoprecipitation (co-IP) of p75 NTR with SorCS2 in transfected HEK293 cells is shown. After crosslinking with a reducible crosslinker, SorCS2 was immunoprecipitated with αCT or αECD. Coprecipitated p75 NTR was depicted with WB analysis. Asterisk indicates two unspecific bands recognized by the p75 NTR antibody upon co-IP using αCT.

To study the dynamics of receptor processing in HEK293 cells, we metabolically labeled wild-type SorCS2 or the mutants in the presence of Brefeldin A to inhibit protein export from the endoplasmic reticulum. After labeling, the cells were washed and nascent receptor molecules chased for up to 22 hr ( Figure 2 F). As predicted, the propeptide of SorCS2-pro was liberated much slower and to a lesser extent than that of the wild-type receptor. In contrast, disrupting (SorCS2-one) or optimizing (SorCS2-two) the cleavage site atRKRdid not affect propeptide cleavage. Rather, it greatly shifted the balance between the one- and two-chain receptor isoforms and did so in a sequential manner. We concluded that propeptide cleavage is a prerequisite for subsequent two-chain processing of SorCS2.

Taken together, we inferred that in CNS neurons SorCS2 exists as a single-chain receptor of 122 kDa whereas in PNS glia it is processed into a two-chain variant comprising a 104 kDa N-terminal domain that is noncovalently tethered to a smaller C-terminal fragment of 18 kDa ( Figure 2 E).

We next asked whether the 104 kDa extracellular and 18 kDa C-terminal fragments of SorCS2 remain associated after processing. Indeed, both receptor fragments coimmunoprecipitated (samples not crosslinked) when using αCT or αECD antibodies; i.e., when the 18 kDa fragment was pulled down the 104 kDa band came along and vice versa ( Figures 2 C and 2D). This interaction was noncovalent because the 104 kDa band was also present when SorCS2 was analyzed with αECD western blotting of cell lysates in the absence of reducing agents ( Figure S1 D). Moreover, in cells expressing two-chain SorCS2 double-immunostaining with αCT and a monoclonal anti-ECD antibody, the extracellular domain and the cytoplasmic tail colocalized at the plasma membrane and in intracellular vesicles, indicating that the 104 kDa fragment remain associated with the plasma membrane ( Figure S1 E). Finally, surface biotinylation experiments further revealed that both isoforms were equally represented on the cell surface ( Figure S1 F), and when chased with surface-bound αECD, internalization and retrograde transport of the antibody to the trans-Golgi network was similar between the two receptor isoforms. This suggests that cellular trafficking of the 122 kDa single-chain receptor and 104 kDa extracellular domains is governed by the same intracellular sorting motifs ( Figure S1 G).

The combined data suggested that SorCS2 can undergo two processing events. The 130 kDa SorCS2, denoted the proform, can be cleaved immediately after its propeptide to generate a 122 kDa single-chain receptor and, second, in the extracellular domain close to the plasma membrane to produce two fragments; one of 104 kDa comprising most of the extracellular domain and one of 18 kDa encompassing the transmembrane domain and cytoplasmic tail.

SorCS2 contains four putative cleavage sites for furin-like proprotein convertases: three sites conform to the consensus sequence RXXR and localize to the propeptide at aa 66–69 (site 1), 84–87 (site 2), and 117–119 (site 3), respectively, and one highly conserved R/KKR motif positioned at residues 1,028–1,030 (site 4) in the juxtamembrane leucine-rich repeat domain ( Figures S1 A–S1C). To identify the positions subject to proteolytic processing, we expressed a soluble and truncated receptor variant (SorCS2-sol) that comprises the entire extracellular domain. N-terminal sequencing revealed three distinct N termini starting at Ser, Ala, and Ser, respectively, demonstrating that SorCS2 can be cleaved in both the first, third, and fourth consensus motifs ( Figures 2 A and S1 A–S1C). In support of this, disruption of sites 1 and 3 at Serand Alaby substitution of alanines for the RXXR motif (SorCS2-pro) considerably diminished propeptide processing of the full-length receptor as determined with western blotting with αECD and αCT ( Figure 2 B, left and middle). Disrupting the fourth consensus cleavage site at RKR (SorCS2-one) preceding Serand positioned 48 residues proximal to the membrane-spanning region, completely eliminated expression of the 104 kDa fragment ( Figure 2 B, left and middle). In the converse situation, we produced a mutant in which the Sercleavage site was changed from RKR to RRKR by substituting Thrto arginine to introduce an ideal proprotein convertase cleavage site (SorCS2-two). The 104 kDa cleaved receptor was now the sole isoform recognized by αECD and no high-molecular-weight bands were detectable with αCT, suggesting immediate proteolytic processing of the 122 kDa single-chain receptor ( Figure 2 B, left and middle). In agreement, the 18 kDa peptide, which was absent from cells expressing SorCS2-one, was clearly visible in SorCS2-two ( Figure 2 B, right).

(F) HEK293 cells stably transfected with SorCS2-wt, SorCS2-pro, SorCS2-one, or SorCS2-two were pulsed for 4 hr in the presence of Brefeldin A and chased at 37°C for the indicated times. Subsequently, proteins were immunoprecipitated using αECD, and visualized using fluorography. The proform (Pro), single-chain (Single), and two-chain (Two) forms of SorCS2 are indicated.

(C and D) Coimmunoprecipitation of the C-terminal fragment with the N-terminal fragment followed by WB using αCT or αECD are given. Nonimmune rabbit IgG was used as negative control for immunoprecipitation.

(A) N-terminal sequencing of SorCS2-sol isoforms following purification from conditioned medium of transfected CHO cells. The first six amino acids of each isoform are indicated.

The nucleotide sequence of SorCS2 predicts a protein of 1,159 amino acids (aa) comprising a short propeptide of 69 residues (aa 51–119; based on homology with sortilin) followed by a Vps10p-D (aa 120–785), a polycystic kidney disease domain (aa 786–876), a leucine-rich domain (aa 877–1,078), a transmembrane spanning region (aa 1,079–1,099), and a short cytoplasmic tail of 60 aa ( Figures S1 A and S1B available online). When expressed in HEK293 cells, we observed three bands in WB analysis (αECD), a faint band of approximately 130 kDa in addition to the 122 kDa and 104 kDa bands seen in the DRG glia, demonstrating that the two isoforms are not produced by alternative splicing ( Figure 1 H). SorCS2 has seven potential N-linked glycosylation sites, but heterogeneous glycosylation did not account for the triple band as it still persisted, albeit at lower molecular weight, following PNGase-mediated deglycosylation ( Figure 1 I). Neither treatment with neuraminidase nor O-glycosidase changed their relative migration in the gel (data not shown). When probing with an antibody against the propeptide sequence of SorCS2 (αPRO), only the 130 kDa protein was recognized, indicating that the propeptide had been liberated from the 122 and 104 kDa isoforms that were detected by αECD ( Figure 1 H). An antibody against the cytoplasmic tail (αCT) detected the 130 and 122 kDa bands in addition to a band of 18 kDa, indicating the existence of a C-terminal receptor fragment ( Figure 1 J). The 18 kDa band was also present in DRGs but not in brain homogenates ( Figure 1 K).

We first characterized SorCS2 expression in the adult murine CNS with immunohistochemistry using a polyclonal antibody against the extracellular part of SorCS2 (αECD). In the cerebellum, cortex, and hippocampus, αECD intensely stained Purkinje cells and NeuN-positive pyramidal neurons but not GFAP-positive glia cells ( Figures 1 A–1D). SorCS2 localization was clearly evident in neuronal processes and to a much a larger extent than the related receptor sortilin, which was mainly concentrated around the soma ( Figure 1 A). The opposite expression pattern for SorCS2 was observed in the peripheral nervous system (PNS). Here, SorCS2 levels were high in satellite cells of dorsal root ganglia (DRG) and in cultured glia cells of superior cervical ganglia and absent from the corresponding neurons ( Figures 1 E and 1F). We confirmed this divergent expression pattern with western blot (WB) analysis of cultured neurons and glia cells derived from the CNS and PNS, respectively. Robust immunoreactive bands were present in the hippocampal neurons and DRG glia, but not in cortical glia and neurons from the PNS ( Figure 1 G). Remarkably, while SorCS2 in hippocampal neurons mainly migrated as one distinct band, a double band of 122 and 104 kDa was present in DRG glia, with the 104 kDa band being most intense. The data suggested that SorCS2 exists in two variants that are differentially expressed in the CNS and PNS.

(K) WB using αCT showing the presence 122 kDa form of SorCS2 in E14.5 dopaminergic neurons (7DIV) and brain homogenate from WT mice (left). The 122 kDa and 18 kDa bands are also present in DRG homogenates (right). No specific signals are observed in homogenate from SorCS2 KOs (−/−).

(J) WB of lysates from SorCS2 transfected cells using αECD (left) or an antibody against the cytoplasmic tail (αCT) showing the presence of an additional band of 18 kDa (right).

(H) WB of SorCS2 in HEK293 using polyclonal antibodies against the extracellular domain (αECD) or the propeptide (αPRO) of the receptor are shown.

(G) Anti-SorCS2 (αECD) western blot analyses (WB) of cultured DRG (PNS) glia, DRG (PNS) neurons, hippocampal (CNS) neurons, and cortical (CNS) glia cells are shown. Immunoreactive bands representing SorCS2 isoforms are indicated by their molecular weight. Detection of actin served as loading control.

(F) Dissociated culture of superior cervical ganglia neurons and glia from P0 mice shows that SorCS2 (green) is present in glia and not neurons. Right: is a merger of immunofluorescense and differential interference contrast images. Neurons were identified based on morphology (white arrowheads).

(A–C) Immunofluorescence microscopy for SorCS2 and sortilin in adult cerebellum and for SorCS2 in CA1 of the hippocampus and cortex (10-week-old mice). Coronal sections are shown.

Discussion

Nykjaer and Willnow, 2012 Nykjaer A.

Willnow T.E. Sortilin: a receptor to regulate neuronal viability and function. Song et al., 2010 Song W.

Volosin M.

Cragnolini A.B.

Hempstead B.L.

Friedman W.J. ProNGF induces PTEN via p75NTR to suppress Trk-mediated survival signaling in brain neurons. Sculpturing the neuronal network and fine-tuning of synaptic contacts rely on an intricate balance between guidance cues, growth cone morphogens, and trophic and apoptotic signals regulating neuron and glia cell fate. The mechanisms that govern these activities are incompletely understood but neurotrophins have long been considered critical. According to the neurotrophic factor hypothesis, depriving neurons of their trophic support leads to synaptic weakening and/or neuronal degeneration. However, signals elicited by proNTs may augment these activities or may even override signals that are simultaneously provided by NTs (). These observations imply that proteolytic processing of proNTs to their mature forms, by switching receptor specificity, can elicit opposing signaling events to regulate cell fate. Our findings add another level of complexity by which trophic and apoptotic signaling can be regulated. First, we identify SorCS2 as a proNT receptor capable of inducing both growth cone collapse and apoptosis in vivo. Second, we show that cell fate decision is not limited to proteolytic processing of the ligand but also depends on receptor processing. While single-chain SorCS2 regulates axon guidance of developing midbrain dopaminergic processes, two-chain processing in PNS glia transforms the receptor into a transducer of apoptotic signals. To our knowledge, this is the first example to demonstrate that cell type-specific receptor processing may regulate two distinct biological functions.

+ neurons in E14.5 midbrain explants from WT but not Sorcs2−/− explants. Second, transfection of KO neurons with single-, but not two-chain, rescued the proBDNF sensitivity. In adult Sorcs2−/− mice, DA functionality of the mesolimbic system was severely afflicted despite an unaltered number of midbrain dopaminergic neurons. Conceivable, this is the consequence of the faulty prefrontal innervation rather than acute effects on synaptic transmission because studies have demonstrated that SorCS2 expression in the VTA is limited to the period during which dopaminergic neurons differentiate and axonal processes reach their targets ( Hermey et al., 2001 Hermey G.

Schaller H.C.

Hermans-Borgmeyer I. Transient expression of SorCS in developing telencephalic and mesencephalic structures of the mouse. Rezgaoui et al., 2001 Rezgaoui M.

Hermey G.

Riedel I.B.

Hampe W.

Schaller H.C.

Hermans-Borgmeyer I. Identification of SorCS2, a novel member of the VPS10 domain containing receptor family, prominently expressed in the developing mouse brain. During development, single-chain SorCS2 is abundant in dopaminergic precursors of the developing midbrain. Several lines of evidence underpin the biological relevance of SorCS2 for growth cone collapse during development. First, proBDNF potently induced axonal retraction of THneurons in E14.5 midbrain explants from WT but not Sorcs2explants. Second, transfection of KO neurons with single-, but not two-chain, rescued the proBDNF sensitivity. In adult Sorcs2mice, DA functionality of the mesolimbic system was severely afflicted despite an unaltered number of midbrain dopaminergic neurons. Conceivable, this is the consequence of the faulty prefrontal innervation rather than acute effects on synaptic transmission because studies have demonstrated that SorCS2 expression in the VTA is limited to the period during which dopaminergic neurons differentiate and axonal processes reach their targets ().

Liston et al., 2011 Liston C.

Malter Cohen M.

Teslovich T.

Levenson D.

Casey B.J. Atypical prefrontal connectivity in attention-deficit/hyperactivity disorder: pathway to disease or pathological end point?. Poelmans et al., 2011 Poelmans G.

Pauls D.L.

Buitelaar J.K.

Franke B. Integrated genome-wide association study findings: identification of a neurodevelopmental network for attention deficit hyperactivity disorder. Conner et al., 2008 Conner A.C.

Kissling C.

Hodges E.

Hünnerkopf R.

Clement R.M.

Dudley E.

Freitag C.M.

Rösler M.

Retz W.

Thome J. Neurotrophic factor-related gene polymorphisms and adult attention deficit hyperactivity disorder (ADHD) score in a high-risk male population. Ribasés et al., 2008 Ribasés M.

Hervás A.

Ramos-Quiroga J.A.

Bosch R.

Bielsa A.

Gastaminza X.

Fernández-Anguiano M.

Nogueira M.

Gómez-Barros N.

Valero S.

et al. Association study of 10 genes encoding neurotrophic factors and their receptors in adult and child attention-deficit/hyperactivity disorder. Syed et al., 2007 Syed Z.

Dudbridge F.

Kent L. An investigation of the neurotrophic factor genes GDNF, NGF, and NT3 in susceptibility to ADHD. Lesch et al., 2008 Lesch K.P.

Timmesfeld N.

Renner T.J.

Halperin R.

Röser C.

Nguyen T.T.

Craig D.W.

Romanos J.

Heine M.

Meyer J.

et al. Molecular genetics of adult ADHD: converging evidence from genome-wide association and extended pedigree linkage studies. A number of studies have reported that patients with ADHD commonly exhibit miswiring of the prefrontal cortex and striatum accompanied by altered dopaminergic function (). It is noteworthy that the majority of ADHD risk genes identified in genome-wide associations studies (GWAS) are associated with regulation of neurite outgrowth (). Also, results of recent studies suggested that SNPs in the coding regions of proNT3, proNGF, and proBDNF are genetically linked to ADHD. In particular, SNPs in their prodomains that harbor the SorCS2 binding motif were uncovered in several independent cohorts (). Perhaps most strikingly, a recent genome-wide association study examining 500.000 SNPs identified a polymorphism in the SORCS2 locus that was strongly linked to risk of ADHD ().

Chen et al., 2007 Chen Z.L.

Yu W.M.

Strickland S. Peripheral regeneration. −/− mice clearly emphasizes the relevance of two-chain processing in vivo. As opposed to CNS neurons that express single-chain SorCS2, PNS glia produce the two-chain variant of the receptor. Studies in Schwannoma cells and primary SCs established that the apoptotic response to proNTs was specific to two-chain SorCS2. Injury to the PNS often causes permanent neurological deficits due to disruption of the myelin-axonal unit, resulting in a poor capacity of the neurons to regenerate (). We found that nerve injury was accompanied by a selective upregulation of two-chain SorCS2 in the Schwann cells. The substantial reduction in SC apoptosis in lesioned Sorcs2mice clearly emphasizes the relevance of two-chain processing in vivo.

NTR and proNTs. Thus, single-chain and two-chain SorCS2 can physically interact with p75NTR and cooperate in proNT binding. Single-chain SorCS2 and p75NTR receptors colocalize in axonal filopodia and are expressed in DA neurons of the ventral midbrain flexure. ProBDNF is in the vicinity because it is abundant in the demarcating tissue in which axons project. Furthermore, SorCS2 and p75NTR KO mice had reduced DA levels and metabolism in the frontal cortex and striatum, respectively, were hyperinnervated with TH-positive fibers, hyperactive, and exhibited a paradoxical calming response to amphetamine. Importantly, these data are supported by recent studies reporting that p75NTR is obligate for cultured hippocampal neurons to respond to proNT by growth cone retraction ( Deinhardt et al., 2011 Deinhardt K.

Kim T.

Spellman D.S.

Mains R.E.

Eipper B.A.

Neubert T.A.

Chao M.V.

Hempstead B.L. Neuronal growth cone retraction relies on proneurotrophin receptor signaling through Rac. Sun et al., 2012 Sun Y.

Lim Y.

Li F.

Liu S.

Lu J.J.

Haberberger R.

Zhong J.H.

Zhou X.F. ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA. NTR, proBDNF, and proNGF were strongly induced and proximity ligation assay showed clustering of SorCS2 and p75NTR in SCs. The 46% reduction in lesion-induced SC apoptosis observed for Sorcs2−/− mice matches values previously reported for p75NTR-deficient mice or mice treated with polyclonal antibodies targeting proNGF/NGF ( Petratos et al., 2003 Petratos S.

Butzkueven H.

Shipham K.

Cooper H.

Bucci T.

Reid K.

Lopes E.

Emery B.

Cheema S.S.

Kilpatrick T.J. Schwann cell apoptosis in the postnatal axotomized sciatic nerve is mediated via NGF through the low-affinity neurotrophin receptor. Syroid et al., 2000 Syroid D.E.

Maycox P.J.

Soilu-Hänninen M.

Petratos S.

Bucci T.

Burrola P.

Murray S.

Cheema S.

Lee K.F.

Lemke G.

Kilpatrick T.J. Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy. NTR jointly regulate SC apoptosis in lesioned nerves of the PNS. Surprisingly, despite the disparate functions of single- and two-chain SorCS2 in CNS neurons and PNS glia, both activities require complex formation with p75and proNTs. Thus, single-chain and two-chain SorCS2 can physically interact with p75and cooperate in proNT binding. Single-chain SorCS2 and p75receptors colocalize in axonal filopodia and are expressed in DA neurons of the ventral midbrain flexure. ProBDNF is in the vicinity because it is abundant in the demarcating tissue in which axons project. Furthermore, SorCS2 and p75KO mice had reduced DA levels and metabolism in the frontal cortex and striatum, respectively, were hyperinnervated with TH-positive fibers, hyperactive, and exhibited a paradoxical calming response to amphetamine. Importantly, these data are supported by recent studies reporting that p75is obligate for cultured hippocampal neurons to respond to proNT by growth cone retraction (). Finally, following peripheral nerve injury, expression of two-chain SorCS2, but not the single-chain form, was upregulated. In addition, p75, proBDNF, and proNGF were strongly induced and proximity ligation assay showed clustering of SorCS2 and p75in SCs. The 46% reduction in lesion-induced SC apoptosis observed for Sorcs2mice matches values previously reported for p75-deficient mice or mice treated with polyclonal antibodies targeting proNGF/NGF (). The combined observations support that SorCS2 and p75jointly regulate SC apoptosis in lesioned nerves of the PNS.

Munck Petersen et al., 1999 Munck Petersen C.

Nielsen M.S.

Jacobsen C.

Tauris J.

Jacobsen L.

Gliemann J.

Moestrup S.K.

Madsen P. Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding. Like sortilin, SorCS2 is produced as a proreceptor containing a sequence of 69 amino acids after the signal peptide. In sortilin, the propetide prevents premature ligand binding in the biosynthetic pathway. Once exiting the late trans-Golgi compartment, the propetide is liberated by furin-mediated cleavage, conditioning the receptor for full functional activity (). The role of propeptide in SorCS2 is so far unknown but it is plausible that it functions in a similar manner to shield the ligand-binding site. In support of a regulatory function, two-chain processing did not take place until the propeptide had been released. In another scenario, the propetide might facilitate receptor folding and secure expedited transport of SorCS2 through the biosynthetic pathway. Studies are currently ongoing to address these possibilities.

de Wit et al., 2011 de Wit J.

Hong W.

Luo L.

Ghosh A. Role of leucine-rich repeat proteins in the development and function of neural circuits. Cole et al., 2008 Cole C.

Barber J.D.

Barton G.J. The Jpred 3 secondary structure prediction server. The enzyme(s) and cellular compartment(s) involved in the two-chain cleavage of SorCS2 are currently unknown. However, once processed, the two chains remain associated by a noncovalent interaction. SorCS2 belongs to the group of leucine-rich repeat transmembrane proteins, members of which many are involved in axon guidance, including Slit, Slitrk, the Nogo receptors, and TrkA, -B, and -C. In these proteins, the leucine-rich repeat domains create a versatile structural framework for protein-protein interactions. The repeats are formed by two β strands connected by a loop region of variable sequence and structure and are arranged in a curved horseshoe-like structure. Globular ligands fit into this structure but their access is permitted or prohibited by the relative position of the β strands (). Two-chain conversion occurs in the leucine-rich repeat domain of SorCS2 and secondary structure analysis using the Jpred3 algorithm () predicts that the cleavage site is located in a loop between the two β strands (data not shown). Cleavage at this site would likely add additional degrees of freedom to the tertiary structure of the domain allowing or preventing access of new ligands or yet unknown coreceptors. This could provide the molecular basis for the different biological functions of single- to two-chain conversion of SorCS2.

In conclusion, we have found that SorCS2 exists in two isoforms with distinct activities. The single-chain from is expressed in CNS neurons and is required for growth cone collapse of dopaminergic neurons, but in PNS glia, proteolytic processing converts the receptor into a transducer of apoptosis signals. To the best of our knowledge, this is the first example to demonstrate that a receptor, taking advantage of the same ligand and same coreceptor, can exhibit disparate functions depending on its proteolytic processing. Our findings add a new dimension to our understanding of the mechanisms that govern receptor multifunctionality.