The three sorCS genes are differentially ... - Wiley Online Library

Journal of Neurochemistry, 2004, 88, 1470–1476

doi:10.1046/j.1471-4159.2004.02286.x

The three sorCS genes are differentially expressed and regulated by synaptic activity Guido Hermey,*, Niels Plath,§ Christian A. Hu¨bner,à Dietmar Kuhl,§ H. Chica Schaller* and Irm Hermans-Borgmeyer* *Zentrum fu¨r Molekulare Neurobiologie, Universita¨t Hamburg, Hamburg, Germany Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark àInstitut fu¨r Humangenetik, Universita¨tsklinik Eppendorf, Hamburg, Germany §Molekulare Neurobiologie, Freie Universita¨t Berlin, Berlin, Germany

Abstract We have isolated the murine sorCS3 gene, a new member of the family of receptors containing a Vps10p-domain. Receptors of this family facilitate rapid endocytosis and are thought to be involved in intracellular sorting. SorCS3 and the highly homologous sorCS1 and sorCS2 genes were expressed in a combinatorial, mostly non-overlapping pattern in both the developing and mature central nervous system. During development, distribution and abundancy of their transcripts was regulated. Moreover, their expression was differentially influenced by neuronal activity in the hippocampus of adult mice.

Although kainic acid-induced seizures had no effect on sorCS2 mRNA levels, they dramatically increased the expression of sorCS1 and sorCS3. The activity-dependent induction of sorCS1 expression required de novo protein synthesis, whereas that of sorCS3 did not. Our results imply that the three sorCS genes have diverse, but partly overlapping functions in the developing and mature central nervous system. Keywords: brain development, cycloheximide, expression, kainic acid, sorting receptor, Vps10p-domain containing protein. J. Neurochem. (2004) 88, 1470–1476.

Endocytosis and sorting of surface membrane proteins are fundamental mechanisms by which neurones control their electrical and synaptic properties, signalling characteristics, morphology, and growth (Beattie et al. 2000; Kittler and Moss 2001; Blanpied et al. 2002). Identification and characterization of proteins involved in both, neuronal activity and the mediation of trafficking in neurones should improve our understanding of neuronal plasticity-related events. In recent years a novel family of sorting receptors containing a Vps10p-domain (Vps10p-D) was described in mammalia. It consists of four members that have been identified and characterized in humans and mice, sorLA (Jacobsen et al. 1996; Hermans-Borgmeyer et al. 1998), sortilin (Petersen et al. 1997; Navarro et al. 2001), sorCS1 (Hermey et al. 1999; Hermey et al. 2003), and sorCS2 (Nagase et al. 2000; Rezgaoui et al. 2001). A fifth family member, sorCS3, has been so far identified in humans only (Kikuno et al. 1999) and awaits further characterization. All five Vps10p-D receptors carry signals for rapid internalization and intracellular sorting in their cytoplasmic domains and consequently it was already demonstrated that sortilin, sorLA, and sorCS1 are capable of internalizing surface-

bound ligands (Jacobsen et al. 2001; Nielsen et al. 2001; Hermey et al. 2003). However, attention has focused on the Vps10p-D receptor family due to their involvement in intracellular trafficking processes. The two first described members of this family, sorLA and sortilin were shown to interact via their cytoplasmic parts with Golgi-localized, gamma-ear-containing, ARF binding proteins (GGAs) (Nielsen et al. 2001; Jacobsen et al. 2002), which are adaptor proteins believed to be involved in intracellular trafficking between the Golgi apparatus and late endosomes (Kirchhausen 2002), and the cytoplasmic domain of sortilin has been shown to mediate this type of transport (Nielsen et al. 2001; Tooze 2001). In addition, recent research demonstrated that the secretion of the hydrophobic conotoxin-TxVI is facilita-

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Received July 10, 2003; revised manuscript received November 10, 2003; accepted November 12, 2003. Address correspondence and reprint requests to Irm Hermans-Borgmeyer, ZMNH, Universita¨t Hamburg, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: [email protected] Abbreviations used: CHX, cycloheximide; KA, kainic acid; RAP, receptor-associated protein; Vps10p-D, Vps10p-domain.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 88, 1470–1476

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ted by binding of its propeptide to Vps10p-D receptors (Conticello et al. 2003). The five mammalian Vps10p-D receptors are typical type I transmembrane proteins characterized by the presence of an N-terminally located region with homology to the yeast vacuolar sorting protein vps10p in their extracellular/luminal portion. SorLA and sortilin are multiligand receptors which bind neuropeptides (Mazella et al. 1998; Jacobsen et al. 2001), apolipoprotein E (Jacobsen et al. 2001), and the endoplasmic reticulum resident receptor-associated protein (RAP) (Petersen et al. 1999; Jacobsen et al. 2001). In addition to the structural features that all family members share, sorLA, sortilin, sorCS1, and sorCS2 are highly expressed in the developing and mature murine central nervous system (Hermans-Borgmeyer et al. 1998, 1999; Hermey et al. 2001a; Rezgaoui et al. 2001). SorCS1 and sorCS2, whose expression during mouse embryonal nervous system development was analysed in detail (Hermey et al. 2001a; Rezgaoui et al. 2001), are expressed in a unique transient and dynamic pattern in regions where cells proliferate, as well as in areas where differentiated cells reside. In this study we isolated the cDNA encoding murine sorCS3 and analysed its expression pattern in the mature and developing brain in comparison to sorCS1 and sorCS2. Furthermore, we demonstrate that the expression of sorCS1 and sorCS3 in the hippocampus is regulated by neuronal activity.

Materials and methods Isolation of a cDNA encoding murine sorCS3 A cDNA encoding sorCS3 was isolated from an adult mouse brain library in kZAPII (Stratagene, La Jolla, CA, USA) with a probe corresponding to a SacI fragment (nucleotide 633–3115) derived from the sorCS1a cDNA. Screening and analysis were performed as described previously (Rezgaoui et al. 2001).

In situ hybridization In situ hybridization was essentially performed as described (Su¨sens et al. 1997). Antisense RNA probes labelled with [a-35S]UTP were generated according to the manufacturer’s instructions (Ambion, Austin, TX, USA). 10 lm cryosections of postnatal or 15 lm cryosections of adult brain were fixed, acetylated, dehydrated, and subjected to in situ hybridization at 55C for 18 h. RNaseA treatment was for 30 min at 37C and a high stringency wash was performed in 0.1 · saline sodium citrate buffer at 55C. Slides were exposed to X-ray films (Kodak Biomax MR, Amersham Biosciences, Freiburg, Germany) for 72 h before dipping into Kodak NTB-2 nuclear track emulsion, developed after 3–6 weeks and stained with Giemsa (Sigma, St Louis, MO, USA). Specificity of the signals was verified by comparing antisense with sense controls. The probe for sorCS1 comprised nucleotides 1023–1548 of the mouse cDNA cloned in pSK and linearized with XhoI for antisense transcription and BamHI for sense transcription. For sorCS2, a subclone (nucleotides 2280–3178) in pSK linearized with

NotI for the antisense and with EcoRI for the sense probe was used (Rezgaoui et al. 2001). The probes for sorCS3 comprised nucleotides 1850–2521 subcloned in pSK and linearized with BamHI and HindIII. Analysis of kainic acid (KA) experiments was performed using a phospho-imager (FujiBas 2000; Raytest, Sprockho¨vel, Germany) and the TINATM software (Raytest) for quantification. After 1 h exposure, changes in signal intensity were monitored. Analysed slides were then treated as described above. Kainic acid-evoked seizures Seven adult male C57B1/6J mice were used for KA injection according to institutional guidelines. Cycloheximide (CHX; 30 mg/kg, Sigma, Taufkirchen, Germany) and KA (27.5 mg/kg, Ocean Produce International, Darthmouth, Nova Scotia, Canada) were administered by i.p. injection. CHX was applied 0.5 h prior to KA. Three control animals were injected with similar amounts of isotonic saline. CHX and/or KA injected animals were killed 2–6 h after the onset of the first seizure, control animals 6 h after saline injection.

Results

Murine sorCS3, a novel member of the Vps10p-D receptor family An aim of our study was to isolate new members of the Vps10p-D receptor family expressed in the nervous system. Screening a cDNA library from adult mouse brain with a probe derived from murine sorCS1a (Hermey and Schaller 2000) resulted in the identification of one cDNA clone of 4003 bp. It contained the entire open reading frame of 3657 bp encoding 1219 amino acids (GeneBankTM Accession no. AF276314). Database analysis revealed a human orthologue with 88% overall identity, identified as KIA1059/ sorCS3 (Kikuno et al. 1999; Hampe et al. 2001). A comparison of the different murine Vps10p-D proteins showed that sorCS1 is the closest relative of sorCS3, and due to their common structure and homology, sorCS1–3 can be viewed as a subgroup of the Vps10p-D family. The sorCS3 Vps10p-D is 71% identical to sorCS1 and 49% to sorCS2, and in the leucine-rich regions 63% of the amino acids are identical to sorCS1, in contrast to only 35% of sorCS2. The genes for sorCS1 and sorCS3 map to the same locus in humans (10q23.3) and are close in mice (19D1 and 19D2). In summary, we isolated a murine cDNA encoding the third member of the sorCS subgroup of the Vps10p-D family, designated sorCS3, which exhibits highest homology to sorCS1. The three sorCS genes are expressed in an almost complementary pattern in the mature mouse brain We performed in situ hybridizations on neighbouring sections to determine the patterns of expression of the three sorCS genes in the mature murine brain. The three expression patterns were individual and unique, but overlapped partially in some regions (Figs 1a–c). Fibre tracts were


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Fig. 1 SorCS1–3 are expressed in distinct regions of the mature murine brain. Autoradiograms of neighbouring sagittal and coronal sections hybridized with sorCS1 (a), sorCS2 (b), or sorCS3 (c) specific probes, arranged in rostral to caudal direction, are shown. ac, arcuate nucleus; am, amygdala; cc, cerebral cortex; cb, cerebellum; cp, caudate putamen; gn, geniculate nucleus; hi, hippocampus; ic, inferior

colliculus; ig, induseum griseum; ip, interpeduncular nucleus; lc, locus coeruleus; ls, lateral septum; mh, median habenula; mo, medulla oblongata; ob, olfactory bulb; ot, olfactory tubercule; pi, piriform cortex; po, pons; s, septum; sc, superior colliculus; sh, septohippocampal nucleus; tt, tenia tecta.

devoid of hybridization signals, pointing to a mainly neuronal expression pattern of sorCS1–3. Transcripts for each of the three genes were widely distributed throughout the brain, whereas only some areas and nuclei exhibited prominent expression. Intense signals for sorCS1 were restricted to the cerebral and piriform cortex, the olfactory tubercule, the lateral septum, the superficial layer of the superior colliculus, the interpeduncular nucleus, and the locus coeruleus (Fig. 1a). SorCS2 was mainly detected in the neuronal constituents of the olfactory bulb, the piriform cortex, the amygdala, the tenia tecta, the median habenula, the induseum griseum, the septohippocampal nucleus, the hippocampus, the median geniculate nucleus, the interpeduncular nucleus, and some of the Purkinje cells of the cerebellum (Figs 1b and 2f). For sorCS3, prominent labelling was observed in the mitral cell layer of the olfactory bulb, the piriform and cerebral cortex, the hippocampus, the arcuate and interpeduncular nucleus, several motoric and sensoric hindbrain nuclei, and the molecular layer of the cerebellum (Figs 1c and 2f). Although hybridization signals for sorCS1–3 were often observed in the same areas of the brain, intense signals were usually not overlapping. This was especially obvious for sorCS2 and sorCS3 expression. In the hippocampus, sorCS1 was only weakly expressed, with a slightly higher abundance of transcripts in the subiculum and the CA1 region (Fig. 2a). SorCS2 hybridization signals highlighted the subiculum, the CA2 region, and the dentate gyrus, whereas predominant labelling with sorCS3 was restricted to the CA1 region (Fig. 2a). In the cerebral cortex, sorCS1 was abundant in layers 3 and 5 with the exception of the area of the visual cortex (Figs 1a and 2b). SorCS2 was present in the layer 5

with lower expression in the motor cortex and restricted to a subpopulation of cells (Figs 1b and 2b). SorCS3 was expressed in layer 5/6; a lower expression was found in layer 2/3 and the retrosplenial cortex (Figs 1c and 2b). In the superior colliculus, the zonal layer was highlighted by sorCS2 expression, the underlying superficial grey layer by sorCS1, whereas sorCS3 was expressed weakly in the intermediate layer (Fig. 2c). In the inferior colliculus, significant hybridization signals are only observed for sorCS2 and sorCS3, where sorCS2 was abundant in the dorsal cortex and sorCS3 in the external cortex (Figs 1 and 2c). The different subnuclei of the interpeduncular nucleus also exhibited differential expression, as did the raphe nucleus (Figs 2d and e). In the cerebellum, sorCS1 expression was low in the granular cell layer and the most caudally located Purkinje cell layer (Fig. 2f). SorCS2 expression was restricted to most of the Purkinje cell layer of lobule 7–9 (Figs 1b and 2f), and sorCS3 transcripts were present exclusively in the basket and stellate cells of the molecular layer (Fig. 2f). In summary, our data show that each of the three sorCS genes are predominantly expressed in distinct locations of the murine brain and that a complementary expression pattern is often observed. This is especially true for sorCS2 and sorCS3, whereas sorCS1 was sometimes coexpressed with one of the two other family members. The mature expression pattern of the sorCS genes is established during postnatal development We previously analysed the expression pattern of sorCS1 during embryonal and postnatal nervous system development of the mouse (Hermey et al. 2001a). We found that sorCS1 is



obvious in the thalamic and hypothalamic area, in the superior and inferior colliculus, and the cerebellum (Fig. 3 and data not shown). In the hippocampus, sorCS2 transcripts are present in all regions at P0, but the adult pattern is observed only at P9 (Fig. 3b). The typical expression pattern for sorCS3 in the hippocampus is detected at P3, but intensities of signals increase during further development (Fig. 3b). These results show that the expression of both genes during development is highly regulated.

Fig. 2 The three sorCS genes are expressed in a complementary pattern in the mature brain. Photoemulsion-dipped parallel sections through the hippocampus (a), the cerebral cortex (b), the superior and inferior colliculus (c), the interpeduncular (d) and the raphe nucleus (e), and the posterior lobules of the cerebellum (f) hybridized with the indicated probes are shown. In (a) false colours and overlays of sorCS1 (blue), sorCS2 (green), and sorCS3 (red) are shown. In the CA2 region only sorCS2 is present. CA1 and CA3, CA fields of the hippocampus; dg, dentate gyrus; gc, granular cell layer of the cerebellum; ic, inferior colliculus; lv, lateral ventricle; ml, molecular layer of the cerebellum; pc, Purkinje cell layer; sc, superior colliculus; su, subiculum.

predominantly expressed in periods of active morphogenesis and that the adult expression pattern is established during early postnatal development. Figure 3(a) gives an overview of the expression patterns of sorCS2 and sorCS3 at the day of birth (P0). The adult expression pattern of both genes arose during early postnatal development (compare Fig. 3a vs. Figs 1b and c) and in many areas of the brain signal intensity declined during maturation (Fig. 3). This was especially

SorCS1 and SorCS3 gene expression in the forebrain is regulated by synaptic activity As the expression pattern of the three sorCS genes during early postnatal development suggested that they might be regulated by neuronal activity, we analysed whether KA-induced limbic seizures alter their expression in the adult forebrain. Mice injected with KA were sacrificed 2, 4, and 6 h after the onset of KA-induced seizures, and cryostat sections were prepared and subjected to in situ hybridization with the three different sorCS probes. For sorCS1 and sorCS3 we observed an increase in labelling intensity in the dentate gyrus 4 h after the onset of seizures, which became even more prominent after 6 h (Fig. 4). At this time point most cells of the dentate gyrus exhibited strong signals for sorCS1 and sorCS3, whereas in the untreated controls only a subpopulation of neurones expressed these genes (compare Fig. 2a to Fig. 4b). In addition, a slightly stronger labelling in the CA1 region was observed for sorCS1. SorCS3 hybridization signals increased drastically in CA1 and slightly in CA3. SorCS2 expression in the hippocampus appeared not to be influenced by KA-induced limbic seizures (data not shown). To test whether the increase in expression of sorCS1 and sorCS3 in the hippocampus is dependent on protein synthesis, we pre-treated mice with cycloheximide before induction of seizures. Mice were sacrificed 6 h after onset of seizures, and expression was compared between sections derived from brains treated with cycloheximide only as a control and cycloheximide and KA treated animals. The induction of sorCS3 expression by KA treatment was present in the cycloheximide-treated preparations, whereas sorCS1 signals were not elevated when compared to the control (Fig. 4a). These data demonstrate that sorCS1 and sorCS3 gene expression in the hippocampus are regulated by neuronal activity and that stimulation of sorCS1 expression requires protein synthesis. Discussion

We identified murine sorCS3 as third member of the sorCS subfamily of Vps10p-D receptors, compared their expression patterns in postnatal and adult mouse brains, and examined changes in gene expression levels after KA-induced seizures in the hippocampus.



(a)

(b)

Fig. 3 The mature expression patterns of sorCS2 and sorCS3 are established during postnatal brain development. (a) Autoradiograms of parallel sagittal and coronal sections at P0 are shown. (b) Higher magnifications of the hippocampus (hi) at P0, P3, and P9, the colliculi (co) at P0, and the cerebellum (cb) at P0 and P3 showing the

expression of sorCS2 or sorCS3 are presented as photoemulsiondipped sections. cb, cerebellum; cc, cerebral cortex; cp, caudate putamen; dth, dorsal thalamus; hi, hippocampus; hyp, hypothalamus; ic, inferior colliculus; mo, medulla oblongata; ob, olfactory bulb; s, septum.

In the mature brain, the general expression patterns were complementary and non-overlapping, but in some areas sorCS1 distribution was similar to sorCS2 or sorCS3. The differential combinatorial pattern of gene expression levels of the sorCS genes defined well-recognized subgroups of neurones in distinct regions of the brain. An example is the pyramidal layer of the hippocampus, where sorCS2 transcripts were highly abundant in the CA2 region, whereas sorCS1 and sorCS3 transcripts were not detectable at significant levels. SorCS3 exhibits highest expression in the CA1 cells, and sorCS1 expression is low in CA1 and not present in CA3. All members of the Vps10p-D family characterized so far are more abundantly expressed in the developing than in the adult brain (Hermans-Borgmeyer et al. 1998, 1999; Hermey et al. 2001a; Rezgaoui et al. 2001). This points to a general role of these proteins in the development of neuronal structures. The postnatal expression of sorCS2 and sorCS3 demonstrated that the mature distribution of transcripts is established during the early postnatal period, as was described for sorCS1 (Hermey et al. 2001a). An example is the hippocampus, where the localization of sorCS2 and sorCS3 transcripts is dynamic until P9, when the mature expression pattern was established. In the same period, hippocampal neurones start to form synaptic connections and synaptic activity will give rise to adult patterns of synaptic connections (Ben-Ari 2001). These results hinted at a regulation of sorCS gene expression by neuronal activity and prompted us to investigate the effects of KA-induced limbic seizures on the expression of the three genes in the hippocampus.

Fig. 4 SorCS1 and sorCS3 expression in the hippocampus is regulated by electric activity. (a) Coronal sections of the mouse brain hybridized to sorCS1 and sorCS3 are shown as autoradiograms without previous KA injection (co), 4 (4 h) and 6 h (6 h) after the onset of KA-induced seizures, after cycloheximide (CHXco) and cycloheximide plus KA injection (CHX). Higher magnifications of the hippocampus without (co) and 6 h (6 h) after the onset of seizures are presented for sorCS1 and sorCS3 (b).



KA-induced epileptic seizures are considered to be an animal model of human temporal lobe epilepsy. In this model, continuous limbic seizures develop for several hours, leading to cell death of CA3 pyramidal cells and hilar neurones (Nadler et al. 1980), axonal sprouting, and synaptic reorganization. The developmental program of synaptogenesis may be re-activated in the mature epileptic brain (Barnes et al. 2003). KA-induced seizures resulted in an increase of sorCS1 and sorCS3 mRNA levels in the CA1 region of the hippocampus and in the dentate gyrus, whereas sorCS2 expression levels were unaffected. Furthermore, the induction of sorCS3 expression was not changed in the presence of a protein synthesis inhibitor, whereas parallel experiments evidenced that the induction of sorCS1 expression was dependent on new protein synthesis, indicating a differential induction by neuronal activity of the two genes. Neuronal cell death is associated with an enhanced release of adrenal steroid hormones (Dinkel et al. 2003), which are involved in the KA-induced elevation of expression of some genes (Barbany and Persson 1993; Riva et al. 1995). Therefore, we cannot exclude that the observed effects of KA-induced seizures on the expression of sorCS1 and sorCS3 are secondary, but the fact that we observe effects already 4 h after KA injection argues against this possibility. The KA-induced seizure model has been successfully used for the identification of plasticity-related genes (Nedivi et al. 1993). A difference in regional expression and response to neuronal activity, as observed for the sorCS genes, has been described as well for the members of the pim kinase gene family. All three pim kinase genes are expressed in the hippocampus, Pim1 and Pim3 are induced by synaptic activity, but only Pim1 plays a critical role in the formation of enduring forms of long-term potentiation (Konietzko et al. 1999). As the three sorCS proteins are similar in structure and highly homologous, the low redundant and combinatorial expression pattern and their differential induction by neuronal activity suggests that these receptors may serve related, slightly different functions and follow distinct regulatory pathways. It is established that the Vps10p-D receptors sortilin, sorLA, and sorCS1 are endocytic receptors (Jacobsen et al. 2001; Nielsen et al. 2001; Hermey et al. 2003). SorCS2 and sorCS3 present internalization motifs in their cytoplasmic domains and are therefore thought to be endocytic receptors, too. Sortilin conveys intracellular sorting (Nielsen et al. 2001; Tooze 2001) and this might be the second common function of Vps10p-D receptors. SorLA and sortilin are constitutively expressed in the hippocampus, but their expression is not up-regulated after KA-induced seizures (Hermans-Borgmeyer, unpublished results). Endocytic sorting of neurotransmitter and growth factor receptors and adhesion molecules regulates dendritic growth and synaptic strength (Beattie et al. 2000; Bozdagi et al. 2000; Ehlers 2000; Blanpied et al. 2002). The dendritic localization

of sorCS1 in the plasma membrane and vesicular structures (Hermey et al. 2001b) and its induction via neuronal activity is supporting the hypothesis that sorCS receptors are involved in postsynaptic endocytic processes, which are regulating signal transduction and give rise to signalling complexes, intracellular sorting, recycling, or degradation (Sorkin and von Zastrow 2002). Acknowledgements The authors thank Eva Vo¨cker for excellent technical assistance, Simon Hempel for help with the figures and Dr Michaela Schweizer for helpful suggestions and critical reading of the manuscript.

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