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Link to original content: https://pubmed.ncbi.nlm.nih.gov/18354028/
Voltage-gated Na+ channel beta1 subunit-mediated neurite outgrowth requires Fyn kinase and contributes to postnatal CNS development in vivo - PubMed Skip to main page content
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. 2008 Mar 19;28(12):3246-56.
doi: 10.1523/JNEUROSCI.5446-07.2008.

Voltage-gated Na+ channel beta1 subunit-mediated neurite outgrowth requires Fyn kinase and contributes to postnatal CNS development in vivo

William J Brackenbury  1 , Tigwa H Davis, Chunling Chen, Emily A Slat, Matthew J Detrow, Travis L Dickendesher, Barbara Ranscht, Lori L Isom
Affiliations

Voltage-gated Na+ channel beta1 subunit-mediated neurite outgrowth requires Fyn kinase and contributes to postnatal CNS development in vivo

William J Brackenbury et al. J Neurosci. .

Abstract

Voltage-gated Na(+) channel beta1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that beta1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that beta1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. beta1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on beta1-mediated neurite extension. These results suggest that beta1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that beta1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that beta1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for beta1 in vivo. We conclude that voltage-gated Na(+) channel beta1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

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Figures

Figure 1.
Figure 1.
β1-Mediated neurite outgrowth is impaired in acutely dissociated CGNs from Fyn null mice. A, Neurite lengths of CGNs isolated from Fyn wild-type and null mice grown on CHL or CHL-β1 monolayers. Data are presented as mean and SEM (n = 250). Significance: **p < 0.01, ANOVA with Tukey's post hoc test. B, Neurite distribution (in percentage) plotted against neurite length for Fyn wild-type and null CGNs grown on CHL or CHL-β1 monolayers. C, Typical Western blot of brain membrane protein prepared from Fyn wild-type and null mice, using anti-β1 antibody. Anti-α-tubulin antibody was used as a control for protein loading. D, Sucrose step gradient separation of protein extracted from wild-type mouse brain tissue. Blots were probed with anti-fyn kinase antibody, anti-β1 antibody, or anti-transferrin receptor (TfR) antibody. The numbers at top are gradient fractions of low-high density. Numbers at left are marker band sizes (kDa). E, Images of an acutely dissociated wild-type CGN labeled with anti-β1ex (green) and GAP-43 (red) antibodies, showing β1 expression at growth cone. The bottom panels are a 3× zoom of growth cone of CGN shown in top panels. Scale bars, 5 μm.
Figure 2.
Figure 2.
β1-Mediated neurite outgrowth is impaired in acutely dissociated CGNs from Cntn null mice. A, Neurite lengths of CGNs isolated from Cntn wild-type and null mice grown on CHL or CHL-β1 monolayers. Data are presented as mean and SEM (n = 200). Significance: ***p < 0.001, ANOVA with Tukey's post hoc test. B, Neurite distribution (in percentage) plotted against neurite length for Cntn wild-type and null CGNs grown on CHL or CHL-β1 monolayers. C, Typical Western blot of brain membrane protein prepared from Cntn wild-type and null mice, using an anti-β1 antibody. Anti-α-tubulin antibody was used as a control for protein loading.
Figure 3.
Figure 3.
β1-Mediated neurite outgrowth in acutely dissociated CGNs is not affected by inhibition of EGF or FGF signaling. A, Neurite length of wild-type C57BL/6 CGNs grown on CHL monolayers and treated with FGF (20 ng/ml) and/or PD173074 (50 nm) for 48 h (n = 200). B, Neurite length of wild-type C57BL/6 CGNs grown on CHL or CHL-β1 monolayers and treated with/without PD173074 (50 nm) for 48 h (n = 200). C, Dose-dependent inhibition of proliferation of N/TERT keratinocytes by AG1478 (2 nm to 20 μm). The line represents fit to sigmoidal function (n = 16). D, Neurite length of wild-type C57BL/6 CGNs grown on CHL or CHL-β1 monolayers and treated with/without AG1478 (2 μm) for 48 h (n = 250). Data are presented as mean and SEM. Significance: ***p < 0.001, ANOVA with Tukey's post hoc test.
Figure 4.
Figure 4.
β1 modulates axonal migration in the postnatal developing cerebellum in vivo. A, Schematic outline of left side of cerebellum in the coronal plane. The black boxes labeled “B–D” show locations of high-magnification images in B–D. CENT6–9, Central lobe, lobules 6–9; COPY, copula pyramidis; PRM, paramedian lobule; sec, secondary fissure; prepyramidal fissure. Inset, Parasagittal diagram indicating rostrocaudal location of coronal section. i, ii, Right-hand plates, Low-magnification images (10×) of TAG-1 immunolabeling (green) on coronal cerebellar sections from Scn1b wild-type and Scn1b null P14 littermates, respectively. Scale bar, 100 μm. B–D, High-magnification (100×) Z-series projections of the same Scn1b wild-type (i) and Scn1b null (ii) cerebellar sections, at the locations defined in A. Scale bar, 20 μm. The schematic outline in A was adapted with permission (Hof et al., 2000). Three mice of each genotype were examined, with similar results.
Figure 5.
Figure 5.
The external germinal layer is thicker in Scn1b null mice compared with wild type. A, High-magnification (100×) Z-series projections of coronal cerebellar sections from Scn1b wild-type and null P14 littermates, labeled with anti-BrdU antibody (red) and DAPI (blue). Scale bar, 20 μm. B, Thickness (in micrometers) of BrdU-positive zone (left-hand bars), and total EGL (right-hand bars), for Scn1b wild-type and null P14 littermates (n = 90 measurements taken from 3 mice of each genotype). C, Number of BrdU-positive cells (left-hand bars), and total number of cells (right-hand bars) per 60 μm-long section of EGL, for Scn1b wild-type and null P14 littermates (n = 30 measurements taken from 3 mice of each genotype). Data are presented as mean and SEM. Significance: ***p < 0.001, ANOVA with Tukey's post hoc test.
Figure 6.
Figure 6.
Loss of β1 results in axonal pathfinding abnormalities in the corticospinal tract. a–g, Consecutive coronal sections across the pyramidal decussation in a Scn1b wild-type brain. h–n, Consecutive coronal sections across the pyramidal decussation in a Scn1b null brain. o–r, Example sections from additional Scn1b null mice. h–l, o–r, Arrows, Defasciculation across pyramidal decussation. i–k, o–r, Arrowheads, Mislocalization of axons lateral to dorsal column. m, n, Arrowheads, Axons deviating from dorsal column after pyramidal decussation. v, Ventral pyramid; d, dorsal column. Scale bar, 250 μm. Six Scn1b null mice were examined. All six showed similar CST abnormalities compared with seven wild-type mice.
Figure 7.
Figure 7.
Schematic representation of a possible signaling mechanism underlying β1-mediated neurite outgrowth in CGNs. β1 from an adjacent cell interacts with a multiprotein complex of β1 and contactin in the CGN, initiating a signaling cascade through fyn leading to neurite outgrowth. Homophilic adhesion between β1 expressed by an adjacent cell and β1 in the CGN, and/or heterophilic adhesion between β1 expressed by an adjacent cell and contactin in the CGN may occur. Ig, Immunoglobulin domain; FN III, fibronectin type III domain. This figure was produced using Science Slides 2006 software.
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