Regulated expression and subcellular localization of syndecan heparan sulfate proteoglycans and the syndecan-binding protein CASK/LIN-2 during rat brain development

Abstract

The syndecan family of cell surface heparan sulfate proteoglycans interacts via their cytoplasmic C-terminal tail with the PDZ domain of CASK/LIN-2, a membrane-associated guanylate kinase homolog. The syndecan-CASK interaction may be involved in intercellular signaling and/or cell adhesion. Here we show that syndecan-1 to syndecan-4 have distinctive mRNA distributions in adult rat brain by in situ hybridization, with syndecan-2 and -3 being the major syndecans expressed in neurons of the forebrain. At the protein level, syndecan-2 and -3 are differentially localized within neurons; syndecan-3 is concentrated in axons, whereas syndecan-2 is localized in synapses. The synaptic accumulation of syndecan-2 occurs late in synapse development. CASK is a cytoplasmic-binding partner for syndecans, and its subcellular distribution changes strikingly during development, shifting from a primarily axonal distribution in the first 2 postnatal weeks to a somatodendritic distribution in adult brain. This change in CASK distribution correlates temporally and spatially with the expression patterns of syndecan-3 and -2, consistent with the association of both of these syndecans with CASK in vivo. In support of this, we were able to coimmunoprecipitate a complex of CASK and syndecan-3 from brain extracts. Our results indicate that specific syndecans are differentially expressed in various cell types of the brain and are targeted to distinct subcellular compartments in neurons, where they may serve specialized functions. Moreover, CASK is appropriately expressed and localized to interact with both syndecan-2 and -3 in different compartments of the neuron throughout postnatal development.

Fig. 1.

In situ hybridization analysis of syndecan mRNAs in adult rat brain. A–D, Syndecan-1 (A); syndecan-3 (B); syndecan-2 (C); syndecan-4 (D). E, F, Sense strand negative controls for syndecan-2 (E) and syndecan-4 (F). Syndecan-1 and -3 sense stand negative controls also gave no significant ISH signal (data not shown).G, H, Higher magnification (emulsion autoradiography) views of syndecan-2 (G) and syndecan-4 (H) distribution in the hippocampus, showing glial expression of syndecan-4. cc, Corpus callosum; Ch, choroid plexus; DG, dentate gyrus; GC, granule cell layer of the cerebellum;St, striatum; syn, syndecan;Th, thalamus. Scale bars, 400 μm.

Fig. 2.

Characterization of Syn-2C and Syn-3C antibodies.A, Amino acid alignment of the cytoplasmic C-terminal tails of syndecan-1 to -4 is shown. Syn-2C and Syn-3C antibodies were raised against the underlined peptide sequences in the cytoplasmic domains of syndecan-2 and -3, respectively. Dashes represent gaps in the sequence introduced to maximize alignment of conserved sequences.B, Specificity of Syn-2C and Syn-3C antibodies was tested by immunoblotting using COS-7 cells transfected with syndecan-1, -2, -3, or -4 or vector alone, as indicated. Syn-2C recognizes heterologously expressed syndecan-2 and cross-reacts with syndecan-4. Syn-3C-1 specifically recognizes two protein bands (∼83 kDa,arrow; ∼130 kDa, arrowhead) in COS-7 cells transfected with syndecan-3 but not other syndecans. Similar results were obtained with Syn-3C-2 antibodies (data not shown). Syn-3ecto antibodies also recognize the ∼83 kDa band, as well as a broad high–molecular weight smear that likely represents more fully modified, cleaved forms of syndecan-3. C, Specificity of Syn-2C and Syn-3C antibodies on immunofluorescence staining of COS cells transfected with syndecan-1, -2, -3, or -4, as indicated, is shown. D, Syn-3C antibodies immunoprecipitate syndecan-3 from rat brain extracts. Immunoprecipitation with affinity-purified Syn-3C antibodies (Syn-3C-1 and Syn-3C-2are from different rabbits) was performed using P8 rat brain extracts. Immunoprecipitates were immunoblotted with Syn-3ecto or Syn-3C antibodies, as indicated. Control immunoprecipitations were performed with nonimmune purified rabbit IgGs (IgG). The ∼150 kDa band is indicated by an arrow, and the 150–250 kDa smear is indicated by the bracket. Aninput lane contains 10% of the detergent extract used for immunoprecipitation (IP).

Fig. 3.

Subcellular fractionation and biochemical association of syndecan-3 and CASK in rat brain. A,Immunoblotting of subcellular fractions of rat brain with Syn-3C, Syn-3ecto, and CASK antibodies. B, Coimmunoprecipitation of CASK and syndecan-3 from P7 rat brain extracts by affinity-purified Syn-3C-1 antibodies. Negative control precipitations were performed with purified nonimmune rabbit IgG (lane 2) or after preincubation of Syn-3C-1 antibody with antigenic peptide (pep) (lane 4).Input lanes contain 10% (for Syn-3C immunoblot) or 5% (for CASK and PSD-95 immunoblots) of the detergent extract used for immunoprecipitation. Immunoprecipitates were probed for syndecan-3, CASK, and PSD-95. The arrow andbracket are described in Figure2D. H, Total homogenate; other fractions are described in Materials and Methods.

Fig. 4.

Localization of syndecan-3 in P7 rat brain by DAB immunohistochemistry. A, Syn-3C immunoreactivity is highly concentrated in axonal pathways [e.g., the fimbria (fi) and alveus (alv) of the hippocampal formation], the cc, and axon tracts running through the thalamus (arrowheads). B–E, Staining patterns of antibodies Syn-3C (B, D) and Syn-3ecto (C, E) are compared in the hippocampal formation (B, C) and cerebral cortex (D, E). F, Preincubation with Syn-3C antigenic peptide blocks the Syn-3C immunostaining of the corpus callosum and alveus; weak background staining of cell bodies of CA1 remains. G, Syn-3C antibodies strongly label the white matter (w) of the cerebellum. dg, Dentate gyrus; P, Purkinje cell layer. Scale bar: A, 400 μm; B--E,G, 350 μm; F, 150 μm.

Fig. 5.

Axonal localization of Syn-3C immunoreactivity in adult rat brain, revealed by immunofluorescence microscopy. Localization of syndecan-3 in axons is shown in the dentate gyrus of the hippocampus (A), the corpus callosum (B), the stria terminalis (C), the white matter of the cerebellum (an axon bundle is cut in cross section) (D), and basket cells of the cerebellum (E). In E1and E2, the same field is double labeled by Syn-3C (E1) and synaptophysin (E2) antibodies.M, Molecular layer of the cerebellum; mf, mossy fiber tract; p, Purkinje cell bodies. Scale bar, 40 μm.

Fig. 6.

Expression and subcellular distribution of CASK during rat brain development. A, Developmental expression of CASK in rat brain. Ten micrograms of brain membrane fractions from rats at the indicated ages were immunoblotted with CASK and PSD-95 antibodies. B, C,Immunohistochemistry for CASK in forebrain (B) and cerebellum (C) at the indicated postnatal ages. Scale bars: A, 1 mm; B, 0.5 mm.

Fig. 7.

Syndecan-3 is localized in axons but excluded from synapses. A, Axonal localization of syndecan-3 in cultured cortical neurons. Double immunofluorescence labeling of cultured cortical neurons with Syn-3C and MAP2 (a), Syn-3C and Tau (b,c), and Syn-3C and CASK monoclonal K56A/50 (d) is shown. In c, Syn-3C antibodies were preincubated with syndecan-3 antigenic peptide. Eachset of images (a1–a3,b1–b3, etc.) represents the same field visualized for syndecan-3 (Cy3, red; left) or for MAP2, Tau, or CASK (FITC, green; middle). The right image is an overlay of the first two (showing colocalization in yellow). B, Subcellular segregation of syndecan-2 and -3 in adult rat brain, revealed by double immunostaining with synaptophysin. Sections were double labeled with Syn-2C and synaptophysin antibodies (synap.) (a, c) or Syn-3C and synaptophysin antibodies (b, d). Confocalimages were collected from the granule cell layer (GCL) of the cerebellum (a,b) and region CA4 of the hippocampus (c,d). Syndecan-2 and -3 were visualized by Cy3 (red), and synaptophysin was visualized by FITC (green) secondary antibodies. Scale bars, 20 μm.

Fig. 8.

Coordinate layer-specific expression of syndecan-2 and synaptophysin in developing rat cerebellum. Double-label immunofluorescence of the cerebellum at P3, P7, P15, and P22 with Syn-2C and synaptophysin antibodies (A) or with Syn-2C and calbindin antibodies (B). Synaptophysin and calbindin were used as markers for synapses and Purkinje cells, respectively. Each pair ofimages represents confocal images of the same field visualized for one or the other antibody, as indicated. Scale bars, 100 μm.

Fig. 9.

Synaptic localization of syndecan-2 occurs late in synapse development. Relationship of Syn-2C and synaptophysin staining in the IGL of the cerebellum and in theCA3 region of the hippocampus during postnatal development examined by double-label immunofluorescence confocal microscopy at P3, P7, P15, and P22. Syn-2C is visualized by Cy3 (red), and synaptophysin is visualized by FITC (green). The composite images show colocalization in yellow.P, Pyramidal cell layer; sl, stratum lucidum. Scale bars: IGL, 10 μm; CA3,20 μm.