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. 2010 Aug 10;5(8):e12003.
doi: 10.1371/journal.pone.0012003.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Nathalia Vitureira  1 , Rosa Andrés, Esther Pérez-Martínez, Albert Martínez, Ana Bribián, Juan Blasi, Shierley Chelliah, Guillermo López-Doménech, Fernando De Castro, Ferran Burgaya, Kelly McNagny, Eduardo Soriano
Affiliations

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Nathalia Vitureira et al. PLoS One. .

Abstract

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PC is expressed during mouse brain development.
(A–C) Distribution of PC mRNA (A) and protein (B–C) at P5. Low-power micrographs of the forebrain (A,B) and cerebellum (C) showing wide expression of PC mRNA and protein in laminar structures and brain nuclei at P5. (D–F) Confocal micrographs illustrating patterns of PC expression in laminated regions, including the hippocampus (D), cerebellum (E) and olfactory bulb (F). Note the pericellular pattern of staining, also evident in the inset in D (D'). (G–L) Distribution of PC protein in developing neurons in culture. Dissociated (G–I) and hippocampal explant cultures (J–L) were incubated with antibodies against PC and the neuronal-specific β-tubulin III (TUJ-1) marker. The merged images are also shown. Neurites (G–I) and axonal processes (J–L) are PC-immunoreactive. Note the punctuate distribution of PC along the hippocampal axons and the localization in axonal growth cones (J'–L'). (J'–L') are magnifications of the boxed areas. CA1, CA3, pyramidal cell regions of the hippocampus; CC, corpus callosum; CPu, caudate-putamen of the striatum; Cx, cerebral cortex; DG, dentate gyrus; EGL, external granular layer; FN, facial nuclei IGL; internal granular layer; PCx, piriform cortex; Pyr, pyramidal cell layer of the hippocampus; P, Purkinje cell layer; WM, white matter; ON, olfactory nerve; GL, glomerular layer; EPL, external plexiform layer. Scale bars: 500 µm (A,B); 125 µm (C); 150 µm (D); 40 µm (E,F); 25 µm (J–L); 20 µm (G–I).
Figure 2
Figure 2. Expression, sialylation and protein interaction of neural PC.
(A) Western Blot showing PC immunoreactivity in wt brain (E18), kidney (P4) and podxl −/− brain (E18); actin is shown as loading control. (B) Developmental profile of PC-immunoreactivity at several developmental stages; actin immunoreactivity is shown as a loading control. (C) Neuraminidase treatment of hippocampal dissociated cultures (7DIV), E16 brain lysates and adult kidney extracts. Note that the 140-kDa band is sensitive to Neuraminidase digestion in all cases since the mobility of PC is markedly decreased (arrow). (D,E) Ezrin and PC immunoprecipitations of E16 forebrain homogenates. Ezrin immunoprecipitation probed with an α-PC antibody yields co-immunoprecipitation of PC. PC immunoprecipitation yields co-association with Ezrin (D). Co-immunoprecipitation experiments with an antibody recognizing the ERM protein family (Ezrin/Radixin/Moesin) yields identical results (E). (F,G) PC and NHERF1/2 immunoprecipitation assays in forebrain lysates result in co-association of both proteins. (H,I) PC immunoprecipitation results in co-association with the small Rho GTPases RhoA and RhoG; the reverse immunoprecipitation with anti-RhoA/G antibodies also reveals PC protein in the immunoblots. Immunoprecipitation with control, irrelevant antibodies (GFP) did not give positive signals in the immunoblots. Anti-βIII-Tubulin antibodies were used as loading controls.
Figure 3
Figure 3. Neuronal migration is not altered in podxl (−/−) embryonic brains.
(A,B) Nissl-stained sections showing normal cytoarchitecture in the E18 podxl (−/−) forebrain. Medium power magnifications showing normal cytoarchitecture in the neocortex (C,D), hippocampus (E,F), olfactory bulb (G,H) and cerebellum (I,J) of podxl (−/−) embryos. (K,N) BrdU-immunoreacted sections showing normal distribution of E12- and E15-labeled neurons in the neocortex of podxl (−/−) embryos at E18. E12-labeled BrdU-positive nuclei (arrows) are correctly positioned in the deep cortical layers (K,L). E15-labeled BrdU-positive neurons are positioned in the upper cortical layers (M,N). Cortical layers are indicated to the right. (O,P) Sections immunoreacted with the Tbr1 antibody show normal distribution of upper layer neurons in the neocortex of podxl (−/−) E18 embryos. (Q,R) Explants from the lower rhombic lip co-cultured with aggregates of Netrin-1 expressing cells demonstrate a similar pattern of neurophilic chain migration and chemoattraction in wt and podxl (−/−) explants. CA1, CA3, pyramidal cell regions of the hippocampus; CP, cortical plate; Cx, cortex; DCN, deep cerebelar nuclei; DG, dentate gyrus; EGL, external germinative layer; F, fimbria fornix; GR, granular layer of the olfactory bulb; Hip; hippocampus; IPL, internal plexiform layer; MCL, mitral cell layer; MZ, marginal zone; N, netrin-expressing cells; P, Purkinje cell layer; PCx, pyriform cortex SP, subplate; St, striatum; VZ, ventricular zone. Scale bars = 500 µm (A,B); 100 µm (C–F, I–R); 50 µm (G–H).
Figure 4
Figure 4. PC regulates neurite outgrowth in vitro.
(A–C) Examples of wt and podxl (−/−) hippocampal explants growing on laminin, and of wt explants growing in the presence of PC-Ectodomain. Note the formation of a dense meshwork in PC-deficient conditions. (A'–C') are high-power magnifications of (A–C). Examples of hippocampal wt (D) and podxl (−/−) (E) neurons growing on poly-D-lysine, and wt neurons growing in the presence of PC-Ectodomain (3DIV) (F). Neurons were labeled with the TUJ-1 antibody. Histograms showing that podxl (−/−) neurons (G, H) and wt neurons growing on PC-Ectodomain (J, K) have increased neurite lengths (G, J) and branching points per neuron (H, K), compared to controls. (I, L) Distribution of neurons (per number of branching points) in the different conditions. UT, untreated control; Fc, control substrate with rabbit Fc; PC-Ecto, PC-ectodomain substrate. Mann-Whitney test, statistical significance, * P<0.05, ** P<0.01, *** P<0.001. Data are expressed as mean ± s.e.m. Scale bar = 25 µm (A–C); 15 µm (D–F).
Figure 5
Figure 5. Expression of PC in presynaptic terminals.
(A,C) Hippocampal cultures (7DIV) were incubated with antibodies against PC (A) and against Synapsin (B). The merged images are shown in (C). (D) Western Blot showing enriched expression of PC in adult forebrain synaptosomes (SS), in comparison to total brain homogenates (H). Western blots for synaptic vesicles (Synaptophysin and VAMP-2) and axonal membranes (SNAP-25) are shown. (E) Purified synaptosomes were subfractionated in a sucrose gradient. Fractions were collected and analyzed by immunoblot. Note that PC is detected in the fractions enriched in synaptic vesicle markers (F10–F13), whereas it is absent from cytosolic fractions (F3–F5) and expressed at low levels in membrane fractions (F6–F9). Immunoblots for several synaptic markers and for Tubulin are shown. Immunoblot for Myelin Basic Protein is also shown as Myelin is present in the fractions enriched in membranes. (F-,G) Electron micrographs showing localization of PC in presynaptic terminals in the adult hippocampus using post-embedding immunogold labeling. at, axonal terminal; ds, dendrite spine. Scale bars = 15 µm (A–C); 0.25 µm (F,G).
Figure 6
Figure 6. PC is required for correct synaptogenesis in the hippocampus.
(A–C) PC expression in presynaptic terminals. Hippocampal cultures (7DIV) were incubated with antibodies against PC (A) and against synaptophysin (B). The merged image is shown in (C). (D–I) Hippocampal neuronal cultures from wt and podxl (−/−) embryos immunolabelled for Synapsin I and II and the specific dendritic marker MAP2, showing synaptic appositions on MAP2-positive dendrites. (J,K) Histograms showing that cultures of podxl (−/−) neurons and of wt neurons treated during 7DIV with the PC-Ectodomain have a decreased number of synaptic appositions compared to controls. (L-M) Examples of electron micrographs illustrating hippocampal synaptic contacts (arrows) in wt and podxl (−/−) E18 hippocampi. (N) Density of synaptic contacts in the stratum radiatum and the stratum lacunosum moleculare, showing decreased number of synapses in podxl (−/−) E18 embryos. (O,P) Electron micrographs showing synaptic contacts (arrowheads) in wt and podxl (−/−) E18 hippocampal slice cultures, cultured for 7 additional DIV. (Q) Density of synaptic contacts in hippocampal organotypic cultures, showing decreased number of synapses in podxl (−/−) slices. UT, untreated control; Fc, rabbit Fc containing medium; PC-Ecto, PC ectodomain-containing medium; SR, stratum radiatum; SLM, stratum lacunosum moleculare. Mann-Whitney test (J,K) or Student's t test (N,Q) were used; statistical significance, ** P<0.01, *** P<0.001. Data are expressed as mean ± s.e.m. Scale bars = 10 µm (A–I); 0.5 µm (L,M,O,P).
Figure 7
Figure 7. podxl (−/−) embryos show a reduction in the density of AChR clusters in the neuromuscular system.
(A, B) Traversal sections of the spinal cord (E18) showing PC-immunoreactivity in motorneurons. Sections counterstained with bisbenzimide. (C–G) Longitudinal slices of soleus muscles from podxl (−/−) and wt littermates at E18, stained with Alexa488-α-bungarotoxin to visualize postsynaptic AChRs (C, D) and antibodies against neurofilament 200 and synaptophysin (E, F) to visualize motor axons and nerve terminals. The merged images are shown in (G, H). Open squares in G and H illustrate sample areas used for the quantitative determination of synaptic junctions. (I) Bar graph showing that podxl (−/−) soleus muscles have a decreased density of AChR clusters compared to controls. The Student's t test was used; statistical significance, ** P<0.01. Scale bars = 150 µm (A); 50 µm (C–H); 40 µm (B).
Figure 8
Figure 8. PSA-dependent and PSA-independent functions of PC.
(A–C) Examples of hippocampal neurons cultured in control medium (A), or in the presence of PC-Ectodomain (B) or PC-Ectodomain incubated with neuraminidase (C). Neurons were labeled with the TUJ-1 antibody. (D) Histograms showing that whereas the effects on neurite length are blocked by Neuraminidase treatment (left histogram), the effects on branching remain unchanged after Neuraminidase treatment (right histogram). (E–G) Hippocampal neuronal cultures immunolabelled for Synapsin I/II and the specific dendritic marker MAP2, showing synaptic appositions on MAP2-positive dendrites, in control conditions (E), and after treatment with PC-Ectodomain(F) or with PC-Ectodomain incubated with Neuraminidase (G). (I–K) illustrate high-power confocal micrographs. (H) Histogram showing that cultures of hippocampal neurons show decreased number of synaptic appositions after incubation with either PC-ectodomain or PC-Ectodomain treated with Neuraminidase. UT, untreated control; Fc, control substrate with rabbit Fc; PCc-Ecto, PC-ectodomain substrate; PCc-Ecto/EndoN, PC-Ectodomain substrate incubated with EndoN; PC-Ecto/Neuraminidase, PC-Ectodomain substrate incubated with Neuraminidase. The Student's t test was used; statistical significance, * P<0.05, ** P<0.01 and *** P<0.001. Scale bars = 15 µm (A–C), 10 µm (W–F), 30 µm (L–N).
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References

    1. Mann F, Chauvet S, Rougon G. Semaphorins in development and adult brain: Implication for neurological diseases. Prog Neurobiol. 2007;82:57–79. - PubMed
    1. Round J, Stein E. Netrin signaling leading to directed growth cone steering. Curr Opin Neurobiol. 2007;17:15–21. - PubMed
    1. Tessier-Lavigne M, Goodman CS. The molecular biology of axon guidance. Science. 1996;274:1123–1133. - PubMed
    1. Holmberg J, Clarke DL, Frisen J. Regulation of repulsion versus adhesion by different splice forms of an Eph receptor. Nature. 2000;408:203–206. - PubMed
    1. Wilkinson DG. Multiple roles of EPH receptors and ephrins in neural development. Nat Rev Neurosci. 2001;2:155–164. - PubMed

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