Binding of Y-P30 to Syndecan 2/3 Regulates the Nuclear Localization of CASK

The survival promoting peptide Y-P30 has documented neuroprotective effects as well as cell survival and neurite outgrowth promoting activity in vitro and in vivo. Previous work has shown that multimerization of the peptide with pleiotrophin (PTN) and subsequent binding to syndecan (SDC) -2 and -3 is involved in its neuritogenic effects. In this study we show that Y-P30 application regulates the nuclear localization of the SDC binding partner Calcium/calmodulin-dependent serine kinase (CASK) in neuronal primary cultures during development. In early development at day in vitro (DIV) 8 when mainly SDC-3 is expressed supplementation of the culture medium with Y-P30 reduces nuclear CASK levels whereas it has the opposite effect at DIV 18 when SDC-2 is the dominant isoform. In the nucleus CASK regulates gene expression via its association with the T-box transcription factor T-brain-1 (Tbr-1) and we indeed found that gene expression of downstream targets of this complex, like the GluN2B NMDA-receptor, exhibits a corresponding down- or up-regulation at the mRNA level. The differential effect of Y-P30 on the nuclear localization of CASK correlates with its ability to induce shedding of the ectodomain of SDC-2 but not -3. shRNA knockdown of SDC-2 at DIV 18 and SDC-3 at DIV 8 completely abolished the effect of Y-P30 supplementation on nuclear CASK levels. During early development a protein knockdown of SDC-3 also attenuated the effect of Y-P30 on axon outgrowth. Taken together these data suggest that Y-P30 can control the nuclear localization of CASK in a SDC-dependent manner.


Introduction
In recent years a number of bioactive peptides have been identified that affect neurite outgrowth and provide neuroprotection. The survival promoting peptide Y-P30 is one of these factors [1] and was identified later on also as a peptide that promotes the survival of organotypic thalamic cultures [2]. Y-P30 is a 30 aminoacid peptide that derives from a larger precursor that also includes the anti-bacterial peptide dermcidin [3]. The dermcidin gene was not identified in the genome of rodents [4], albeit peptides of the precursor have been identified in various proteomic screens in rodent tissues [2,[5][6][7][8][9][10]. Despite the unclear status of the expression of the peptide in non-primate species it is well documented that Y-P30 has profound neuroprotective, cell migration and neurite outgrowth promoting effects [1,2,[11][12][13][14]. The neurite outgrowth promoting activity of Y-P30 appears to depend on binding to the trophic factor pleiotrophin (PTN) and the cell adhesion molecule syndecan (SDC), which results in a trimeric signaling complex that following neuronal polarization selectively stimulates the growth of axons [11]. Multimerization of Y-P30 and PTN may result in larger SDC clusters, which in turn might be involved in neuronal signaling [11].
SDC-2 and -3 can associate via their C-terminal binding motif with the MAGUK family member Calcium/calmodulin-depen-dent serine kinase (CASK) [15,16]. The interaction occurs with the post-synaptic density 95/discs large/zonula occludens-1 (PDZ) domain of CASK and requires the C-terminal PDZ-binding motif of both SDC (Fig. 1a+b). It has been previously reported that CASK might be imported in the nucleus in early development [17]. The underlying mechanisms are unknown. But it was shown that SDC-2 and -3 play a role during neuronal development for the localization of CASK either in the nucleus or at the cell membrane [15][16][17]. Nuclear CASK regulates gene expression via association with the transcription factor T-box transcription factor T-brain-1 (TBR-1), which binds to a T-box motif in many developmentally regulated genes like reelin, a secreted glycoprotein important for cell migration, cortical wiring and stabilizing cortical cytoarchitecture [18,19], or the GluN2B NMDA-receptor subunit that determines synaptic maturation [20]. We therefore asked in this study whether the neurite outgrowth promoting activity during development might be related to the regulation of nucleocytoplasmic shuttling of CASK.

Ethics Statement
In the present experiments, animal care and procedures were approved and conducted under established standards of the German federal state of Sachsen-Anhalt, Germany in accordance with the European Communities Council Directive (86/609/ EEC).

Preparation of dissociated primary cell cultures from hippocampus and cortex
Neuronal primary cultures were prepared as described previously [21]. In brief, rat cortices and hippocampi were prepared from embryos (Long Evans rats) at stage E19 and subsequently transferred into ice cold Hanks Balanced Salt Solution without Mg 2+ /Ca 2+ (HBSS, Gibco, Karlsruhe, Germany). After triple washing with 5 ml HBSS, 2.0 ml HBSS containing 0.5% trypsin (Sigma) was added, followed by incubation for 20 minutes at 37uC. The tissue was then washed 5 times with 5 ml HBSS and finally transferred into 2 ml tubes with HBSS, containing 0.01% DNAse-I (Invitrogen). For dissociation the respective tissue was pressed three times slowly through a 0.9 mm-gauged needle followed by 3 passages through a 0.45 mm-gauged needle. The remaining cell suspension was poured through a 70 mm cell strainer (BD Biosciences, San Jose, USA) into a 50 ml tube and filled up with 18 ml Dulbecco's modified Eagle Medium (DMEM; Gibco), containing 10% FCS, 2 mM glutamine and 1% Penicillin/Streptavidin (DMEM+). After estimating cell quantity, the suspension was diluted with DMEM+ according to the experimental required density. For axonal growth assays and morphological analyses the cells were plated in a density of 10.000/cm 2 , for biochemical assays in a density of 80.000/cm 2 in culture flasks. Y-P30 was applied in a final concentration of 6 mg/ml to the bath medium for the indicated periods of time.

Microscopic evaluation and morphological analysis
Images were taken using a Zeiss-Axioplan II imaging fluorescent microscope (Zeiss, Jena, Gemany), Spot RT camera and Meta View software (Visitron Systems, Puchheim, Germany). Morphological analysis of neurons was carried out with ImageJ, a Java-based image-processing program developed at the National Institutes of Health (USA; http://rsb.info.nih.gov/ij/index.html), as reported previously [22].
Expression of GFP-tagged Syndecan-2 and 3 in COS-7 cells COS-7 cells (Cell Lines Service GmbH, Eppelheim, Germany) were transfected with Lipofect 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturers instructions. Transfection efficiencies and expression rates were monitored by fluorescent microscopy. At the appropriate time points the cells were scraped with 10 ml ice-cold TBS and subsequently centrifuged for 5 minutes at 10006g and 4uC. Afterwards the cell pellets were washed in TBS and centrifuged again. The remaining cell pellets were processed directly or shock frozen in liquid nitrogen.

SDS-PAGE and Western-Blot experiments
For SDS-PAGE or immunoblot experiments protein fractions were solubilized with 46SDS sample buffer (250 mM Tris-HCl, pH 6.8, 1% SDS, 40% glycerol, 20% ß-mercaptoethanol, 0.004% brome phenol blue) and cooked for five minutes. Afterwards they were separated on 5-20% SDS-Polyacrylamide gradient gels (except for SDC-3 where 4-12% gradient gels were used) and subsequently transferred to nitrocellulose membranes (90 min, 200 mA). The transfer buffer contained 25 mM Tris, 192 mM glycine, 0.02% SDS and 20% methanol. After blotting the membranes were blocked with 5% dry milk and 0.1% Tween 20 in 16TBS for 2 hours. Subsequently the membranes were incubated at 4uC over night with primary antibodies in the respective concentration in 16TBS containing 0.1% Tween 20. Thereafter the blots were incubated for 90 minutes at room temperature with HRP-conjugated secondary antibodies (1:4000) and finally developed using ECL-Films. Phosphoprotein purification was done according to manufacturers instructions (Qiagen, Hilden, Germany). Antibodies against SDC-3 (Abnova (PAB9044); dilution 1:1500) and GluN2B (NeuroMab, Cat.No. 75-101; dilution 1:1000), mouse anti-CASK (Acris, Germany; dilution 1:1000) were used for immunoblotting. While its GUK-domain interacts with the transcription factor Tbr1, the PDZ domain is responsible for binding to the C-terminus of SDC. (b) Essential for this binding are the final four amino acids that are conserved within all SDC. The binding of Y-P30 is mediated via heparan sulfate side chains (HSSC). (c) Supplementation of primary cortical neurons with Y-P30 at DIV 18 leads to the accumulation of CASK in the nucleus as visualized in confocal images following CASK antibody staining. Scale bar is 20 mm. (d) For quantitative analysis fractions of nuclei (P1) and remaining cellular components (P2) were prepared and analysed with quantitative immunoblotting. In order to normalize the relative amounts of CASK, the P1 fraction was analysed in relation to the NeuN signal, the P2 fraction to the Actin signal. (e) In mature primary cortical cultures (DIV 18) supplementation with Y-P30 leads to a significant increase of the CASK concentration in P1 fractions after 3 and 6 h whereas the CASK concentration in P2 declines albeit not significantly at the same time points. (g) Interestingly, a decrease of the CASK concentration in P1 fractions of young neurons (DIV 8) 3 and 6 h after supplementation with Y-P30 was observed. (h, i) The effect of Y-P30 on the nuclear localization of CASK was abolished in mature neurons (DIV18) treated with Heparitinase I and Chondroitinase A, B, C, cleaving the HSSCs. Note that all fractionation assays were done in the presence of 7.5 mM anisomycin. For Western-blots 20 mg of protein were loaded per lane. Relative concentrations of CASK were analysed by measuring the optical density of the respective signal and normalized as described above. Black boxes indicate treatment with Y-P30. N = 4-11 in each group. ** p,0.001, * p,0.05. doi:10.1371/journal.pone.0085924.g001 Fractionation of nuclei from primary cortical cultures Nuclear fractions were prepared essentially according to a modified and shortened protocol of Reyes et al. [23]. In brief, after washing with 5 ml 16TBS, pH 7.8, primary cortical cultures of one flask (1.8 Mio cells/flask) were scraped with 1 ml ice cold HNB (0.5 M sucrose, 15 mM Tris/HCl pH 7.5, 60 mM KCL, 0.25 mM EDTA (pH 8), 0.125 mM EGTA (pH 8), 0.5 mM Spermidine, 0.15 mM Spermin, EDTA-free Complete Protease-Inhibitor-Cocktail (Roche)) and centrifuged for 5 min at 5006g at 4uC. The remaining pellet was re-suspended in 750 ml HBN and homogenized with 12 strokes at 900 rpm in a Potter S homogenizer. The homogenate was transferred into 1.5 ml Eppendorf tubes and supplemented with 375 ml HBN containing 1% Nonidet P40 (Sigma). After incubation for 5 min on ice the homogenate was spun for 4 min at 10006g. The resulting pellet (P1) contained the cell nuclei and was directly solubilized and boiled for 5 min at 95uC with 100 ml SDS-sample buffer. Remaining cell components, in particular the membrane fractions were precipitated via a second centrifugation step at 208006g for 20 min (P2) and solubilized in 60 ml SDS-sample buffer as described above.

Precipitation of extracellular proteins from COS-7 cells and subcellular membrane fractionation
The culture medium (DMEM+) of COS-7 cells, expressing Syndecan-2int.myc-GFP, was completely removed and after washing with pre-warmed HBSS replaced with 5 ml HBSS+ containing 20 mg/ml Brefeldin A (Sigma, Taufkirchen, Germany). After 30 min, the cells were supplemented with 20 mM Y-P30 (final concentration) or with the appropriate volume of 5 mM Tris-HCl, pH 7.4 as a control. In order to inhibit matrix metalloproteinase activity either 50 nM GM6001 (Calbiochem, Darmstadt, Germany) or 20 nM of the specific MMP9/13-Inhibitor I (Calbiochem) were added. Three hours later the HBSS-medium was harvested and centrifuged at 30006g. 5 ml of the remaining supernatant were mixed with 20 ml freezing ethanol and incubated at 220uC over night. On the next day, the precipitated proteins were centrifuged at 4uC and 10.000 rpm for 10 min and the resulting pellet was washed twice with 15 ml 80% ethanol (220uC). Residual ethanol was removed by lyophilizing the pellets for 5 min. Afterwards the protein pellet was dissolved for 3 h at 4uC in 100 ml ultrapure water containing 26EDTA-free complete proteinase inhibitor (PI, Roche) and finally solubilized by adding 100 ml 26SDS sample buffer and boiling for 5 min at 95uC.
For subcellular membrane fractionation corresponding pellets from COS-7 cells were re-suspended in 800 ml HOM-buffer (5 mM Hepes, pH 7.4, 0.32 M sucrose, PI) and subsequently homogenized with 15 strokes at 900 rpm in a Potter S homogenizer. The resulting homogenates were centrifuged in 1.5 ml Eppendorf tubes for 10 min at 10006g at 4uC. Afterwards, the supernatants (S1) were transferred in fresh tubes and kept on ice. The pellets were re-suspended again and the procedure repeated. In the next both supernatants of the respective sample (S1 and S19) were combined and spun for 30 min at 20.0006g. The resulting pellets were again re-suspended in 800 ml HOMbuffer, homogenized with 12 strokes at 900 rpm and centrifuged for 30 min at 20.0006g. The obtained pellets represent a crude membrane fraction and were re-suspended in 800 ml 5 mM Tris, pH 8.1, containing 0.32 M sucrose and PI and were subsequently loaded on top of a 1.1 M/1.4 M sucrose step gradient and finally centrifuged for 2 h at 85.0006g and 4uC in a ultra centrifuge. Subsequently, the membrane-containing fraction was transferred into a fresh tube and the sucrose concentration adjusted to 0.32 M with 5 mM Tris buffer+PI, pH 8.0. In a final centrifugation step the membrane fractions were spun for 1 h at 150.0006g at 4uC. The remaining pellets, containing highly enriched cellular membranes, were directly solubilized in 16SDS sample buffer and boiled for 5 min at 95uC.

mRNA purification and reverse transcription
For Real-Time PCR experiments two million cells/cultureflasks of primary cortical cultures were harvested at the appropriate time-points and treatments. After washing twice with 5 ml ice cold 16PBS, pH 7.4, cells were scraped in 1 ml of the same buffer and afterwards centrifuged for 5 min at 10006g and 4uC. The resulting pellets were re-suspended in 0.6 ml OL1buffer, containing 0.43 M ß-ME, from the Oligotex mRNA purification kit (Qiagen, Hilden, Germany) that was used for mRNA purification. For efficient cell disruption, the suspension was centrifuged for two minutes at maximum speed through QIAshredder homogenizers (Qiagen). The obtained mRNAs were immediately quantified and reverse transcribed into cDNA, using the Sensiscript RT kit (Qiagen) according to the manufacturers protocol. Reverse primer OdT18 (Promega) and random nanomers (Sigma, Taufkirchen, Germany) were used in separate reaction mixes. The success of the reverse transcription was verified via PCR reaction using specific primers for GAP-DH (not shown). Finally the corresponding cDNAs of one sample were combined and used as one template.

Quantitative Real-Time PCR (qRT-PCR)
Quantitative Real-Time PCR was performed using the Light-Cycler 1.5 Instrument from Roche (Roche Applied Science, Mannheim, Germany). For the reaction mix the LightCycler TaqMan Master kit (Roche) was used according to the manufacturers instructions. Primer and probe reagents were ready-made reagents using FAM-dye (Pre-designed TaqMan Assay Reagents; Applied Biosystems) with the following assay-and corresponding GenBank accession numbers: Hprt1 Rn01527840_m1/ NM_012583.2, Reelin Rn00589609_m1/NM_080394.2. For NR2B analysis the reagent was designed from the Custom TaqMan Gene Expression Assay Service (Applied Biosystems) with following primer-and probe sequences: GluN2B-237s fw 59-CAAGCCTGGCATGGTCTTCT-39, rev 59-GGATTGGCGCTCCTCTATGG-39, probe 59-FAM-CCAT-CAGCAGAGGTATCT-NFQ-39 (M91562.1). qRT-PCR reactions were started with an initial denaturation step for 10 min at 95uC, followed by 45 cycles of 95uC for 10 sec, 60uC for 60 sec and 72uC for 1 sec. Relative amounts of Reelin and GluN2B had been normalized with Hprt for mRNA amount variations. The mRNA expression is presented as the change of relative quantities and was analyzed using the 2-DDCt method [24].

Lentivirus production
The generation of lentiviruses was done by co-transfecting HEK293T-cells, grown in DMEM+, with the respective shuttle plasmid (pFUGW or pSDC2/3shRNA-FUGW) and two packing plasmids (pSPAX2 and pHCMV-VSVg) using Lipofectamine 2000 (Invitrogen) according to the manufacturers protocol. After 12 hours 60% of the DMEM+ were exchanged against DMEM2 in order to reach a final FCS concentration of 4%. 24 hours later the virus-containing medium was collected, briefly centrifuged at 2000 rpm for 5 min and the clear supernatant filtered using a 0.45 mm-filter. Finally, the viruses were spun down in an ultracentrifuge and the remaining virus-containing pellet was gently dissolved in 100 ml NB+, divided in aliquots and stored at 280uC. The viral titer was estimated as infective units via the determination of the GFP-fluorescence in infected HEK293T cells.

Statistical Analysis
Statistical analyses were performed using Student's t-test.

Results and Discussion
To test the idea that Y-P30 might regulate the nuclear localization of CASK we applied the peptide for three and six hours to cortical primary neurons at the concentrations of 6 mg/ml that promotes neurite outgrowth [11] and then performed immunocytochemical stainings and quantitative immunoblotting experiments. We found that bath application of the peptide at DIV18 resulted in an increased nuclear accumulation of CASK in comparison to controls. This increase was evident in immunostainings (Fig. 1c) as well as on immunoblots aimed at quantifying CASK protein content in a P1 fraction that contains purified neuronal nuclei (Fig. 1d-f). Surprisingly, however, the opposite effect was observed at DIV8. At this developmental stage supplementation of the culture medium for three or six hours with Y-P30 resulted in significantly reduced nuclear CASK levels (Fig. 1g). The nuclear accumulation of CASK at DIV18 was blocked when we incubated the cultures with Heparitinase I and Chondroitinase A,B,C to remove sugar side chains prior to Y-P30 administration (Fig. 1h+i), a treatment that abolishes binding of Y-P30 and PTN to SDC-2 and -3 [11].
Nuclear CASK is supposed to regulate the expression of TBR1 target genes like reelin or the GluN2B subunit of the NMDAreceptor. GluN2B containing NMDA receptors predominate during early development and synaptogenesis, whereas following synaptic maturation the number of GluN2A-containing receptors increases. In qPCR experiments we found that bath application of Y-P30 indeed regulated the mRNA expression levels of reelin and the GluN2B-subunit. In early development at DIV 6 reelin and GluN2B transcript levels were significantly lower in cultures treated with Y-P30 as compared to control conditions (Fig. 2a). This effect was less apparent at DIV 8 for GluN2B and reversed for reelin at DIV 12 (Fig. 2b+c). Interestingly, at DIV 18 administration of the peptide to the medium induced elevated mRNA levels for both reelin and GluN2B (Fig. 2d). Thus, in young neurons, where Y-P30 reduces the nuclear levels of CASK, also CASK/TBR1 regulated gene expression is reduced while this is no longer found in older cultures. To corroborate these findings we performed quantitative immunoblotting experiments and probed these blots with a GluN2B antibody. We found that at DIV6 GluN2B protein levels were indeed lower in Y-P30 treated cultures (Fig. 3a), whereas at DIV18 bath application of Y-P30 had no significant effect (Fig. 3b).
Taken together these results lead to the question about the underlying mechanism of the differential effect of Y-P30 on the nuclear localization of CASK during neuronal development. We reasoned that this differential effect might be related to the fact that the two family members that are abundant in neurons, SDC-2 and -3, are expressed at different levels during development [16,26]. SDC-3 is prominently expressed during early development and mainly localized in axons whereas SDC-2 protein levels increase later during synaptogenesis when SDC-2 is mainly found in dendrites and synapses [16,26]. To address the question whether binding to SDC-2 and -3 mediates the effect of Y-P30 on the nuclear localization of CASK we employed an shRNA approach to knock down both SDC (Fig. 4a+c). Since available SDC antibodies were not found to be suitable for immunostainings we transfected HEK-293T cells with SDC-myc constructs and the corresponding shRNA plasmid and checked for knock down efficiency (Fig. 4b). Thereafter we transfected hippocampal primary neurons with the most efficient shRNA construct for SDC-2 or -3 and compared the effects of Y-P30 application at DIV 8 and 18 to control cultures. We found that knock down of SDC-2 at DIV18 (Fig. 4d) and SDC-3 at DIV8 completely abolished the effect of Y-P30 on the nuclear localization of CASK (Fig. 4e).
It has been reported that SDC-3 is phosphorylated at several sites and that the expression of the phosphorylated form of SDC-3 decreases during neuronal development [27], which might alter its function. We therefore checked for Y-P30 induced phosphorylation of SDC-3 but could not identify apparent differences at DIV8 and DIV18 (Fig. 5).
In previous work we observed that Y-P30 promotes neurite outgrowth of thalamic neurons in primary cultures [11]. We followed up on these results and examined in more detail whether the application of the peptide might selectively stimulate axonal outgrowth of hippocampal and cortical primary neurons in early development. To this end we applied Y-P30 for 24 h or 36 h in cortical cultures 24 h after plating. We then determined the length and branching of the longest neurite (Fig. 6a). Bath application of Y-P30 in cortical cultures resulted in faster extension of axons  (Fig. 6b). In addition, the number of branches was increased 12 h after application of the peptide (Fig. 6c).
We next employed a SDC-3 knock down and analyzed axon length and branching in cultures that were treated with Y-P30 at DIV 3 or DIV 6 (Fig. 6d). A SDC-3 knock down had only minor effects on axon outgrowth and branching as compared to nontransfected or scrambled-transfected control cells (Fig. 6e+f). Interestingly, however, the Y-P30 induced axon outgrowth was significantly reduced in cells transfected with the SDC-3 knock down construct (Fig. 6e).
These data suggest that the effects of Y-P30 on axon outgrowth as well as on the nuclear localization of CASK depend upon the expression of SDC-2 and -3. However, it is puzzling that binding to SDC-3 during early development and to SDC-2 during synaptogenesis has fundamentally different effects on the nuclear localization of CASK and CASK/TBR-1 mediated gene expression. We therefore asked next what might be the underlying mechanism and reasoned that it might a differential shedding of extracellular domain of SDC-2 as compared to SDC-3. Previous work has shown that SDC-3 is susceptible to intramembranous cleavage by c-secretase [28] and that Y-P30 has proteolytical activity on its own [29]. We therefore overexpressed SDC-2 and -3 that harbor a myc-tag in their extracellular domain in HEK-293 cells and examined cleavage of the extracellular domain after application of Y-P30. It turned out that Y-P30 induced cleavage of the extracellular domain of SDC-2 ( Fig. 7a) but not of SDC-3 (data not shown). An inhibitor of Matrix-metalloproteinase 9 (MMP9) blocked the effects of Y-P30 on shedding of the extracellular domain of SDC-2, suggesting that proteolytical activity of Y-P30 alone is not sufficient to induce cleavage (Fig. 7).
In previous work we found that the neuritogenic effects of Y-P30 are based on binding of the peptide to PTN and SDC-2 and -3 [11]. In the present study we have analyzed potential mechanisms by which the interaction of Y-P30/PTN with SDC can regulate neurite outgrowth. We found that during early development Y-P30 application in primary neurons reduces the nuclear localization of CASK whereas the opposite was found in older neurons. This effect of Y-P30 needs the association with SDC-3 in young and SDC-2 in older neurons. The underlying signaling mechanism probably involves the nuclear localization of inhibitor I were used. After 3 h the culture media were collected, the containing proteins precipitated with ethanol and subsequently analysed with quantitative immunoblotting. In order to analyse the successful over expression and membrane-incorporation of the tagged fusion proteins, the respective cells were harvested, fractionated and the membrane proteins evaluated on western blots. A representative quantitative immunoblot analysis of the SDC-2 ecto-domain from the culture medium is shown in (a). Note that supplementation with Y-P30 increases the amount of the myctagged SDC-2 ecto-domain, whereas GM6001 as well as MMP9/13I abolished the Y-P30 dependent cleavage. The total expression and incorporation of the SDC-2 construct was analysed in membranes after subcellular fractionation using western-blot analysis (b). The relative amounts of the detected SDC-2 ecto-domains from the culture media are depicted in (c) as % to the control. N: 3-6; *** p,0.0001. (d) Illustrates an immunofluorescence image of the SDC-2intmyc-GFP expression in COS7 cells, showing a clear merge of the GFP-fluorescence from the C-terminus of the fusion protein and the myc-tag, incorporated into the ecto-domain of SDC-2. Scale bar is 20 mm. doi:10.1371/journal.pone.0085924.g007 CASK and a differential effect of Y-P30 on ectodomain cleavage of SDC-2 and -3. In young cultures Y-P30 binding appears to reduce ectodomain shedding of SDC-3 and thereby probably stabilizes a SDC-3/CASK complex that shifts the distribution of CASK from the nucleus to the plasma membrane. In older neurons Y-P30 binding to SDC-2 has the opposite effect and intramembranous cleavage of SDC-2 might release CASK for nuclear import.
One interesting question regarding these actions of Y-P30 concerns the mechanism of SDC cleavage. Ecodomain shedding of SDC has been described for all family members in different tissues and is considered to be important for SDC signalling [30]. In many cases matrix metalloproteases are involved in ectodomain shedding [31,32] but only few co-factors like insulin [33] have been shown to be involved in this cleavage event. It is tempting to speculate that following shedding intramembranous cleavage of SDC-3 by c-secretase [28] might release the intracellular domain. In turn this would allow for nuclear transport of CASK [17,26]. Unfortunately very little is known about the mechanisms of transport to the nucleus and whether transport is regulated by ligand binding to SDC. Nuclear CASK associates with TBR-1 and this transcription factor has been shown to regulate the expression of genes important during neuronal development [26,34]. In line with this role we found increased expression of reelin and GluN2B mRNA at cortical neurons at DIV 18. In summary, we propose that in development the neuritogenic effects of the peptide as well as its effect on gene expression might be related to SDC-binding and the control of nuclear import of CASK.
Apart from Y-P30 the only other known soluble ligand of SDC is the ubiquitously expressed trophic factor PTN. It was shown that the SDC interaction with CASK depends on SDC homodimerization [35]. This is interesting since Y-P30/PTN profoundly oligomerize and subsequent SDC binding might ease clustering and homo-dimerization, which in turn might affect signaling. The signaling role of PTN is not well investigated. We speculate that the presence of PTN might be necessary for the effects of Y-P30 on ectodomain shedding or prevention of SDC cleavage. The association of both peptides with SDC might occur also in other tissues and with other family members like SDC-1 and -4. Along these lines it was indeed shown that Y-P30 is expressed in breast cancer and that ectodomain shedding of SDC might be causally related to a poor prognosis in this and potentially other types of cancer [36,37].
At present it is unclear whether the Y-P30 peptide might play a role during neuronal development in human brain. The expression of Y-P30 mRNA has been reported in human brain regions [36]. However, recent reports indicate that the human Y-P30/ dermcidin peptide as well as the mRNA are extremely stable for several weeks and can be used as abundant markers for human sweat in forensic medicine [38]. Thus, the contamination of cell cultures and tissue sections during processing by human skin in analogy to keratin is a serious concern in all studies looking at Y-P30 expression. In light of the difficulties to identify a rodent Y-P30/dermcidin gene it is therefore conceivable that reports by us [2] and potentially also of others on the presence of the peptide in rodents are confounded by contaminations with the human peptide.

Conclusion
The survival promoting peptide Y-P30 promotes neurite outgrowth in a manner that involves SDC/Cask signaling. Interference with the nuclear localization of CASK using the peptide might be of interest for pharmacological applications to promote neurite outgrowth and cell survival.