Expression and adhesion profiles of SynCAM molecules indicate distinct neuronal functions - PubMed (original) (raw)

Expression and adhesion profiles of SynCAM molecules indicate distinct neuronal functions

Lisa A Thomas et al. J Comp Neurol. 2008.

Abstract

Cell-cell interactions through adhesion molecules play key roles in the development of the nervous system. Synaptic cell adhesion molecules (SynCAMs) comprise a group of four immunoglobulin (Ig) superfamily members that mediate adhesion and are prominently expressed in the brain. Although SynCAMs have been implicated in the differentiation of neurons, there has been no comprehensive analysis of their expression patterns. Here we examine the spatiotemporal expression patterns of SynCAMs by using reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistological techniques. SynCAMs 1-4 are widely expressed throughout the developing and adult central nervous system. They are prominently expressed in neurons throughout the brain and are present in both excitatory and inhibitory neurons. Investigation of different brain regions in the developing and mature mouse brain indicates that each SynCAM exhibits a distinct spatiotemporal expression pattern. This is observed in all regions analyzed and is particularly notable in the cerebellum, where SynCAMs display highly distinct expression in cerebellar granule and Purkinje cells. These unique expression profiles are complemented by specific heterophilic adhesion patterns of SynCAM family members, as shown by cell overlay experiments. Three prominent interactions are observed, mediated by the extracellular domains of SynCAMs 1/2, 2/4, and 3/4. These expression and adhesion profiles of SynCAMs together with their previously reported functions in synapse organization indicate that SynCAM proteins contribute importantly to the synaptic circuitry of the central nervous system.

(c) 2008 Wiley-Liss, Inc.

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Figures

Fig. 1

Fig. 1. Detection of specific heterophilic interactions between SynCAMs in a cell overlay assay

A. Detection of soluble SynCAM extracellular domain interactions with full-length SynCAM proteins by cell overlay. HEK 293 cells expressing the indicated full-length SynCAM-CFP fusion proteins on their surface (red) serve as substrate for the indicated soluble fusion proteins of SynCAM extracellular domains with IgG (green). Identical protein amounts of each purified SynCAM extracellular domain were overlaid on HEK 293 cells, and bound protein was detected with Protein A-Alexa 594. Fluorescence microscopy images are shown, with the merged image on the left of each panel and the signal from the overlaid ligand in grayscale on the right. Images are representative of the quantification results below. Scale bar = 30 µm. B. Quantification of SynCAM cell overlay. Fluorescence images of SynCAM-CFP expressing HEK 293 cells overlaid with individual, soluble SynCAM extracellular domains were analyzed for the signal intensities of expressed SynCAM-CFP and of retained extracellular domains (n = 6 images per condition, with 3 images each obtained in two independent experiments; each analyzed image contained up to several hundred cells). Expression of each full-length SynCAM in the overlaid HEK293 cells was comparable (data not shown). Strong and reciprocal heterophilic interactions of SynCAM 1 and 2, of SynCAM 2 and 4, and of SynCAM 3 and 4 were detected. C. Diagram depicting the three SynCAM heterophilic complexes identified in this study.

Fig. 2

Fig. 2. Quantitative real-time RT-PCR of SynCAMs 1–4 in the central nervous system

The four SynCAMs transcripts were detected in all brain regions examined, though each had a distinct expression profile during development. Results were normalized to actin and are presented as n-fold increase in mRNA expression of each SynCAM over four developmental stages (P4, P8, P15, and adult) in five different brain regions (cortex, hippocampus, olfactory bulb, cerebellum, and hindbrain). Results are expressed relative to SynCAM 1 levels in olfactory bulb at P4. The legend is shown on the top right, with solid lines indicating expression in forebrain regions. A. mRNA encoding SynCAM 1 was the most prominent of the four SynCAM transcripts in hippocampus at P4. B. mRNA encoding SynCAM 2 was increasingly expressed during postnatal development in all brain regions. C. mRNA encoding SynCAM 3 was increasingly expressed during postnatal development in all brain regions and was most prominent in cerebellum after P8. D. mRNA encoding SynCAM 4 was most prominent in cerebellum and hindbrain after P8 but displayed a low relative expression in hippocampus.

Fig. 3

Fig. 3. SynCAM 1–4 in situ hybridization in sagittal sections at P15 and adult stages

All SynCAMs are widely expressed throughout the central nervous system at both developmental time points. A, C, E, G. Expression overview at P15. SynCAM 1 and 2 are expressed in more caudal regions of the central nervous system, with relatively weaker staining observed for SynCAM 2 in all brain regions. High expression of SynCAM 3 was observed in cerebellum. B, D, F, H. Expression overview in adult. Expression patterns for all SynCAM proteins are similar to those at P15. Scale bar = 5 mm.

Fig. 4

Fig. 4. SynCAM 1–4 in situ hybridization in hippocampus at P15 (A, C, E, G) and adult (B, D, F, H)

A. At P15, SynCAM 1 is expressed throughout the hippocampus, with strongest expression seen in the pyramidal cells of the CA fields and the granule cells of the dentate gyrus (DG). Expression was also seen in some cells of the corpus callosum. B. In the adult, SynCAM 1 expression was identical to that seen at P15. C. At P15, SynCAM 2 is most strongly expressed in the pyramidal cells of the CA fields. Reduced expression is seen in the granule cells of the dentate gyrus. Expression is also seen in many interneurons in the hippocampal formation. D. In adult, expression of SynCAM 2 is still high in the pyramidal cells of the CA fields and relatively low in the granule cells of the dentate gyrus. E. At P15, SynCAM 3 is strongly expressed in the pyramidal cells of the CA fields, with slightly elevated expression observed in the cells of CA3. Granule cells of the dentate gyrus show somewhat weaker expression. Interneurons of the hippocampal formation also express SynCAM 3. F. In the adult, expression of SynCAM 3 appears identical to P15. G. SynCAM 4 appears uniformly expressed in the pyramidal cells of the CA fields and the granule cells of the dentate gyrus at P15. Expression of SynCAM 4 is also seen in the interneurons throughout the hippocampal formation. Additionally, SynCAM 4 is expressed in some cells of the corpus callosum. H. In the adult, SynCAM 4 expression is identical to that seen at P15. Sections were obtained in a sagittal plane that was relatively medial. DG, dentate gyrus. Scale bar = 100 µm.

Fig. 5

Fig. 5. SynCAM expression is predominantly neuronal and observed in the vast majority of neurons

A–C. Coexpression (B) of SynCAM 1 observed by in situ hybridization (A) and NeuN by immunohistochemistry (C) in the hippocampus at P15. D–F. Coexpression (E) of SynCAM 1 (D) and NeuN (F) in P15 cortex. G–I. Coexpression (H) of SynCAM 3 (G) and NeuN (I) in main and accessory olfactory bulbs at P15. NeuN does not label the mitral cell bodies of the olfactory bulb. J–L. Coexpression (K) of SynCAM 2 (J) and NeuN (L) in P15 cerebellum. NeuN does not label the Purkinje cell bodies of the cerebellum. M–O. Coexpression (N) of SynCAM 1 (M) and NeuN (O) in P2 spinal cord. Scale bars = 100 µm.

Fig. 6

Fig. 6. SynCAM 1–4 in situ hybridization in mouse cortex at P15 (A, C, E, G) and adult (B, D, F, H)

A. At P15, SynCAM 1 is expressed throughout cortex, but is expressed at a slightly elevated level in layer V. B. Expression of SynCAM 1 in the adult cortex shows a similar enrichment in layer V. C. At P15, SynCAM 2 expression was slightly elevated in layers II/III and V as compared to the other layers. D. In adult cortex, SynCAM 2 remained highly expressed in layer V with uniformly lower expression in other cortical layers. E. SynCAM 3 was uniformly expressed across all cell layers at P15. F. In the adult, SynCAM 3 was also expressed evenly in all layers of cortex. G. SynCAM 4 was broadly expressed across all layers at P15. H. SynCAM 4 expression was also uniformly expressed in all layers in the adult. Sections were obtained approximately in the middle of the rostral-caudal axis. Scale bar = 100 µm.

Fig. 7

Fig. 7. SynCAM 1–4 in situ hybridization in the main and accessory olfactory bulbs at P15 (A, B, E, F, I, J, M, N) and adult (C, D, G, H, K, L, O, P)

A. At P15, SynCAM 1 is expressed in the mitral and tufted cells of both the main (OB) and accessory (AOB) olfactory bulbs. Expression is also seen in the granule and deep juxtaglomerular cells. B. Enlarged panel (as indicated by the box in A) showing expression of SynCAM 1 in the different layers of the OB at P15. C. In the adult, SynCAM 1 expression was similar to that at P15, with expression also seen in periglomerular cells. D. Enlarged image of SynCAM 1 expression in adult OB. E. At P15, SynCAM 2 is predominantly expressed in mitral cells of the main olfactory bulb, with significantly weaker expression observed in the accessory olfactory bulb. F. Magnified view of SynCAM 2 expression at P15. G. In the adult olfactory bulb, nearly all cell populations appear to express SynCAM 2 at relatively uniform levels. H. Enlarged image of SynCAM 2 expression in adult OB. I. At P15, SynCAM 3 expression is strongest in the mitral cells of both the main and accessory olfactory bulbs. Faint expression is seen in other cell types. J. Magnified view of SynCAM 3 expression in the OB at P15. K. In the adult, SynCAM 3 expression remains strong and relatively uniform in the mitral and tufted cells of the main olfactory bulb. Granule cells demonstrate a relative increase in signal as compared to P15. L. Magnified panel of adult SynCAM 3 expression in the OB. M. At P15, strongest expression of SynCAM 4 was seen superficially in the periglomerular cells and olfactory ensheathing cells, with other cell types also showing expression. N. Enlarged image of SynCAM 4 expression in the OB at P15. O. In the adult, the pattern of SynCAM 4 expression was similar to that at P15. P. Magnified view of adult SynCAM 4 OB expression. Sections were obtained in a sagittal plane that was relatively medial. OB, main olfactory bulb; AOB, accessory olfactory bulb; ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell body layer; IPL, internal plexiform layer; GCL, granule cell layer. Scale bars = 100 µm (in M, O), 30 µm (in N, P) apply to the respective columns of panels.

Fig. 8

Fig. 8. SynCAM 1–4 in situ hybridization in cerebellum at P15 (A, C, E, G) and adult (B, D, F, H)

A. At P15, SynCAM 1 is most strongly expressed in Purkinje cells. Weak expression is seen in the granule cells. SynCAM 1 expression is also observed in the molecular layer (ml), indicating expression in interneurons or glia. B. In the adult, SynCAM 1 expression is similar to that seen at P15. C. At P15, SynCAM 2 is weakly expressed in Purkinje cells. D. In the adult, SynCAM 2 expression is uniformly weak throughout the cerebellum. E. At P15, SynCAM 3 is strongly expressed in the granule cells of the cerebellum and not detected in Purkinje cells. F. In the adult, SynCAM 3 expression remains high in the granule cells and is at this developmental time point also observed in Purkinje cells. SynCAM 3 labeling is additionally detectable in cells in the molecular layer, potentially interneurons or glia. G. At P15, SynCAM 4 expression is strong in the Purkinje cells, with no apparent expression in the granule cell layer H. In the adult, expression of SynCAM 4 remains strongest in the Purkinje cells and relatively weak in the granule cells. SynCAM 4 expression the molecular layer is increased in comparison to that at P15, to a level nearly as high as in the granule cells. Sections were obtained in a sagittal plane that was relatively medial. GCL, granule cell layer; PCL, Purkinje cell layer; ML, molecular layer. Scale bar = 100 µm.

Fig. 9

Fig. 9. SynCAM 1–4 in situ hybridization in spinal cord at P2 (A, C, E, G) and adult (B, D, F, H)

A. At P2, SynCAM 1 is expressed in neurons as well as presumptive oligodendrocytes of the white matter (WM) in both dorsal (D) and ventral (V) spinal cord. B. In the adult, expression of SynCAM 1 expression is restricted to neurons in the dorsal and ventral areas of the spinal cord. Expression is evident in the motor nuclei of the ventral horn. C. At P2, SynCAM 2 is expressed in neurons throughout the dorsal and ventral areas. D. In the adult, expression of SynCAM 2 is seen uniformly throughout the dorsal and ventral areas. Expression is now also observed in presumptive oligodendrocytes of the white matter. E. At P2, SynCAM 3 is expressed in neurons throughout the spinal cord. F. In the adult, SynCAM 3 is uniformly expressed in both dorsal and ventral spinal cord. Expression is also seen in presumptive oligodendrocytes. G. At P2, SynCAM 4 is expressed in neurons with higher levels of expression in dorsal spinal cord. H. In adult, SynCAM 4 expression is limited to neurons, with uniform staining in dorsal and ventral areas. Expression is apparent in the motor nuclei of the ventral horn. All sections are from the lumbar region of the spinal cord. D, dorsal; V, ventral; WM, white matter. Scale bars = 100 µm (in G, H) apply to the respective columns of panels.

Fig. 10

Fig. 10. Co-expression analysis of SynCAM 1–4 and GAD67 by double in situ hybridization in P15 hippocampus

A–C. Expression of SynCAM 1 and GAD67 in P15 hippocampus. SynCAM 1-expressing cells (A) and a small number of GAD67-positive inhibitory interneurons (C) colocalize in the hippocampal formation. The center panel shows merged images (B). D–F. SynCAM 2 expression (D) is observed in a small number of GAD67-expressing cells (F). G–I. Expression of SynCAM 3 (G) labels some GAD67-positive inhibitory interneurons (I). J–L. SynCAM 4 (J) shows partial coexpression with GAD67 (L), with several GAD67-positive inhibitory interneurons also expressing SynCAM 4. Scale bar = 100 µm.

Fig. 11

Fig. 11. Co-expression analysis of SynCAM 1–4 in P15 cerebellum

A–C. Expression analysis of SynCAM 1 and GAD67 by double in situ hybridization in P15 cerebellum. SynCAM 1 expression (A) colocalizes with GAD67 (C) in the Purkinje cells of the cerebellum. GAD67-positive cells throughout the rest of the cerebellum do not express SynCAM 1. The center panel shows merged images (B). D–F. SynCAM 2 (D) is detected by double in situ hybridization in GAD67-positive Purkinje cells (F). Similarly to SynCAM 1, SynCAM 2 does not label GAD67-positive inhibitory neurons in the cerebellum, indicating that its expression in the granule cell layer is restricted to excitatory neurons. G–I. SynCAM 3 (G) is expressed exclusively in the granule cells of the cerebellum at P15 as shown by double in situ hybridization, with no coexpression with inhibitory Purkinje cells or interneurons that express GAD67 (I). J–L. SynCAM 4 (J) appears coexpressed by double in situ hybridization with GAD67-positive Purkinje cells (L). Other inhibitory interneurons positive for GAD67 do not coexpress SynCAM 4 throughout the cerebellum. M–O. SynCAM 4 is expressed in Purkinje cells of P15 cerebellum. SynCAM 4 in situ hybridization signal (M) colocalizes with Purkinje cells immunopositive for the Ins(1,4,5)P3 receptor (O). The center panel shows merged images (N). P–R. SynCAM 4 is not prominently expressed in Bergmann glia cells of the Purkinje cell layer in P15 cerebellum. SynCAM 4 in situ hybridization signal (P) does not colocalize with Bergmann glia cells immunopositive for the marker BLBP (R). IP3R, Ins(1,4,5)P3 receptor. Scale bar = 100 µm.

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References

    1. Arase N, Takeuchi A, Unno M, Hirano S, Yokosuka T, Arase H, Saito T. Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8+ T cells. Int Immunol. 2005;17:1227–1237. - PubMed
    1. Benson DL, Colman DR, Huntley GW. Molecules, maps and synapse specificity. Nat Rev Neurosci. 2001;2:899–909. - PubMed
    1. Biederer T. Bioinformatic characterization of the SynCAM family of immunoglobulin-like domain-containing adhesion molecules. Genomics. 2006;87:139–150. - PubMed
    1. Biederer T, Sara Y, Mozhayeva M, Atasoy D, Liu X, Kavalali ET, Südhof TC. SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science. 2002;297:1525–1531. - PubMed
    1. Blue ME, Parnavelas JG. The formation and maturation of synapses in the visual cortex of the rat. II. Quantitative analysis. J Neurocytol. 1983;12:697–712. - PubMed

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