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Cited by 268Ion channel regulation and functionNeuroscience and Neuropharmacology ResearchIon Transport and Channel Regulation

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Neurofascin assembles a specialized extracellular matrix at the axon initial segment

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Hedstrom, Kristian L., Xu, Xiaorong, Ogawa, Yasuhiro, Frischknecht, Renato, Seidenbecher, Constanze I., Shrager, Peter, Rasband, Matthew N.·2007·The Journal of Cell Biology
DOI 10.1083/jcb.200705119PMC PMC2064550
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Abstract

Action potential initiation and propagation requires clustered Na+ (voltage-gated Na+ [Nav]) channels at axon initial segments (AIS) and nodes of Ranvier. In addition to ion channels, these domains are characterized by cell adhesion molecules (CAMs; neurofascin-186 [NF-186] and neuron glia–related CAM [NrCAM]), cytoskeletal proteins (ankyrinG and βIV spectrin), and the extracellular chondroitin-sulfate proteoglycan brevican. Schwann cells initiate peripheral nervous system node formation by clustering NF-186, which then recruits ankyrinG and Nav channels. However, AIS assembly of this protein complex does not require glial contact. To determine the AIS assembly mechanism, we silenced expression of AIS proteins by RNA interference. AnkyrinG knockdown prevented AIS localization of all other AIS proteins. Loss of NF-186, NrCAM, Nav channels, or βIV spectrin did not affect other neuronal AIS proteins. However, loss of NF-186 blocked assembly of the brevican-based AIS extracellular matrix, and NF-186 overexpression caused somatodendritic brevican clustering. Thus, NF-186 assembles and links the specialized brevican-containing AIS extracellular matrix to the intracellular cytoskeleton.

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Figures

Figure 1. The molecular composition of AIS in vitro is identical to those in vivo, and shRNA expression plasmids effectively knock down their target proteins in hippocampal neurons. (A) Immunolabeling of cortical brain sections for Nav channels (green) and βIV spectrin (red). (B–D) Cultured hippocampal neurons labeled with antibodies against Nav channels (B; green), NrCAM (B; red), ankG (C; red), NF (D; green), and βIV spectrin (D; red). In B–D, somatodendritic domains were immunolabeled with antibodies against MAP2 (blue). (E) Hippocampal neurons were cotransfected with one shRNA and EGFP (GFP) 3 h after plating. After 17 DIV, neurons were immunolabeled for MAP2 (blue) to mark the somatodendritic domain and the targeted AIS protein (red). Control, untransfected cells are indicated by an asterisk. GFP fluorescence identifies cells that were transfected with the shRNA. Bars, 10 μm.
Figure 1. The molecular composition of AIS in vitro is identical to those in vivo, and shRNA expression plasmids effectively knock down their target proteins in hippocampal neurons. (A) Immunolabeling of cortical brain sections for Nav channels (green) and βIV spectrin (red). (B–D) Cultured hippocampal neurons labeled with antibodies against Nav channels (B; green), NrCAM (B; red), ankG (C; red), NF (D; green), and βIV spectrin (D; red). In B–D, somatodendritic domains were immunolabeled with antibodies against MAP2 (blue). (E) Hippocampal neurons were cotransfected with one shRNA and EGFP (GFP) 3 h after plating. After 17 DIV, neurons were immunolabeled for MAP2 (blue) to mark the somatodendritic domain and the targeted AIS protein (red). Control, untransfected cells are indicated by an asterisk. GFP fluorescence identifies cells that were transfected with the shRNA. Bars, 10 μm.CC BY-NC-SA · PubMed Central
Figure 2. NF-186 is not required for Nav channel clustering or the molecular assembly of the AIS. (A) Immunolabeling of cultured hippocampal neurons transfected with either NF shRNA or NrCAM shRNA. At 10 DIV, cells were labeled for the nontargeted AIS proteins (red): Nav channels (pan-Nav), ankG, βIV spectrin, NF (pan-NF), or NrCAM (βIV spectrin, pan-NF, and NrCAM not depicted). GFP fluorescence indicates transfected cells, whereas MAP2 (blue) marks the somatodendritic domain. Asterisks indicate untransfected neurons. (B) The ratio of GFP+ neurons labeled at the AIS (for the protein indicated) to the total number of GFP+ neurons. In a minimum of three independent experiments, 25–30 GFP+ positive neurons were counted per shRNA. Error bars indicate ± SD. (C–E) Cultured DRG neurons in DRG/Schwann cell co-cultures immunolabeled for βIV spectrin (C [red] and D [green]), ankG (C and E, green), NF-186 (D, red), and NrCAM (E, red). MAP2 (blue) marks the soma in these neurons. (F) Nodes of Ranvier in PNS myelinated cultures have high densities of Nav channels, ankG, βIV spectrin, NF-186, and NrCAM (red). Paranodes are labeled with anti-Caspr (green). Myelin is labeled with anti-MBP (blue). Bars: (A–E) 10 μm; (F) 5 μm.
Figure 2. NF-186 is not required for Nav channel clustering or the molecular assembly of the AIS. (A) Immunolabeling of cultured hippocampal neurons transfected with either NF shRNA or NrCAM shRNA. At 10 DIV, cells were labeled for the nontargeted AIS proteins (red): Nav channels (pan-Nav), ankG, βIV spectrin, NF (pan-NF), or NrCAM (βIV spectrin, pan-NF, and NrCAM not depicted). GFP fluorescence indicates transfected cells, whereas MAP2 (blue) marks the somatodendritic domain. Asterisks indicate untransfected neurons. (B) The ratio of GFP+ neurons labeled at the AIS (for the protein indicated) to the total number of GFP+ neurons. In a minimum of three independent experiments, 25–30 GFP+ positive neurons were counted per shRNA. Error bars indicate ± SD. (C–E) Cultured DRG neurons in DRG/Schwann cell co-cultures immunolabeled for βIV spectrin (C [red] and D [green]), ankG (C and E, green), NF-186 (D, red), and NrCAM (E, red). MAP2 (blue) marks the soma in these neurons. (F) Nodes of Ranvier in PNS myelinated cultures have high densities of Nav channels, ankG, βIV spectrin, NF-186, and NrCAM (red). Paranodes are labeled with anti-Caspr (green). Myelin is labeled with anti-MBP (blue). Bars: (A–E) 10 μm; (F) 5 μm.CC BY-NC-SA · PubMed Central
Figure 3. AnkG is required for AIS formation. (A) 10-DIV hippocampal neurons transfected with Nav channel shRNA (Nav1.x shRNA), ankG shRNA, or βIV spectrin shRNA, and immunolabeled for Nav channels (pan-Nav), ankG, βIV spectrin, NF (pan-NF), or NrCAM (red), and MAP2 (blue). GFP fluorescence indicates transfected neurons. Asterisks indicate untransfected neurons. (B) The ratio of GFP+ neurons labeled at the AIS (for the protein indicated) to the total number of GFP+ neurons. In a minimum of three independent experiments, 25–30 GFP+ positive neurons were counted per shRNA. Error bars indicate ± SD. Bar, 10 μm.
Figure 3. AnkG is required for AIS formation. (A) 10-DIV hippocampal neurons transfected with Nav channel shRNA (Nav1.x shRNA), ankG shRNA, or βIV spectrin shRNA, and immunolabeled for Nav channels (pan-Nav), ankG, βIV spectrin, NF (pan-NF), or NrCAM (red), and MAP2 (blue). GFP fluorescence indicates transfected neurons. Asterisks indicate untransfected neurons. (B) The ratio of GFP+ neurons labeled at the AIS (for the protein indicated) to the total number of GFP+ neurons. In a minimum of three independent experiments, 25–30 GFP+ positive neurons were counted per shRNA. Error bars indicate ± SD. Bar, 10 μm.CC BY-NC-SA · PubMed Central
Figure 4. ShRNA knockdown of ankG in vivo, but not NF-186, inhibits AIS assembly. (A and B) NF shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Transfected cortical neurons were immunolabeled with anti–pan-NF (A) or βIV spectrin (B). (C–E) AnkG shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Layer II–III cortical neurons immunolabeled with antibodies against ankG (C), pan-NF (D), and βIV spectrin (E). Arrowheads indicate axons of each transfected (GFP+) neuron. Arrows indicate the AIS of nontransfected neurons. Bar, 10 μm.
Figure 4. ShRNA knockdown of ankG in vivo, but not NF-186, inhibits AIS assembly. (A and B) NF shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Transfected cortical neurons were immunolabeled with anti–pan-NF (A) or βIV spectrin (B). (C–E) AnkG shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Layer II–III cortical neurons immunolabeled with antibodies against ankG (C), pan-NF (D), and βIV spectrin (E). Arrowheads indicate axons of each transfected (GFP+) neuron. Arrows indicate the AIS of nontransfected neurons. Bar, 10 μm.CC BY-NC-SA · PubMed Central
Figure 5. Brevican is enriched at the AIS and node of Ranvier. (A–D) Double immunostaining for aggrecan or neurocan (red) and pan-NF (green) in cultured hippocampal neurons. Insets show punctate aggrecan (A) or neurocan (C) staining at the AIS of some neurons. (E) Brevican (red) and βIV spectrin (green) immunostaining of cultured hippocampal neurons. Brevican is enriched only at the AIS (see inset). In A–E, MAP2 (blue) defines the somatodendritic domain. (F) Double immunostaining for brevican (red) and βIV spectrin (green) in vivo; brevican immunoreactivity colocalizes with βIV spectrin. Inset shows brevican immunoreactivity alone. The soma of the neuron is indicated by an asterisk. (G–I) Immunostaining of optic nerve using antibodies against neurocan (G, red), aggrecan (H, green), or brevican (I, green). Nodes of Ranvier were identified by anti–βIV spectrin (G [green] and I [blue]) or anti-Caspr to label paranodes (H and I; red). Arrowheads indicate nodes. Bars: (A–F) 10 μm; (G–I) 5 μm.
Figure 5. Brevican is enriched at the AIS and node of Ranvier. (A–D) Double immunostaining for aggrecan or neurocan (red) and pan-NF (green) in cultured hippocampal neurons. Insets show punctate aggrecan (A) or neurocan (C) staining at the AIS of some neurons. (E) Brevican (red) and βIV spectrin (green) immunostaining of cultured hippocampal neurons. Brevican is enriched only at the AIS (see inset). In A–E, MAP2 (blue) defines the somatodendritic domain. (F) Double immunostaining for brevican (red) and βIV spectrin (green) in vivo; brevican immunoreactivity colocalizes with βIV spectrin. Inset shows brevican immunoreactivity alone. The soma of the neuron is indicated by an asterisk. (G–I) Immunostaining of optic nerve using antibodies against neurocan (G, red), aggrecan (H, green), or brevican (I, green). Nodes of Ranvier were identified by anti–βIV spectrin (G [green] and I [blue]) or anti-Caspr to label paranodes (H and I; red). Arrowheads indicate nodes. Bars: (A–F) 10 μm; (G–I) 5 μm.CC BY-NC-SA · PubMed Central
Figure 6. Brevican interacts with NF-186. (A–D) Soluble GFP-brevican (green) binds to COS-7 cells transfected with HA-tagged NF-186 (A [red] and B) but not cells transfected with NrCAM (C [red] and D). Nuclei are labeled using Hoechst (blue). (E–H) Transfected neurons overexpressing HA-tagged NF-186 labeled with anti-HA (green; E and G) and anti-brevican (red; E and H). MAP2 (blue; E and F) was used to mark the somatodendritic domain. (I and J) Intensity plots measuring fluorescence along the white lines shown in E for the AIS (I) and a dendrite (J). Bar, 10 μm.
Figure 6. Brevican interacts with NF-186. (A–D) Soluble GFP-brevican (green) binds to COS-7 cells transfected with HA-tagged NF-186 (A [red] and B) but not cells transfected with NrCAM (C [red] and D). Nuclei are labeled using Hoechst (blue). (E–H) Transfected neurons overexpressing HA-tagged NF-186 labeled with anti-HA (green; E and G) and anti-brevican (red; E and H). MAP2 (blue; E and F) was used to mark the somatodendritic domain. (I and J) Intensity plots measuring fluorescence along the white lines shown in E for the AIS (I) and a dendrite (J). Bar, 10 μm.CC BY-NC-SA · PubMed Central
Figure 7. NF-186, but not NrCAM, is required for brevican clustering at the AIS. (A and B) Knockdown of ankG or NF-186 via shRNA plasmids in hippocampal neurons (GFP+) blocks AIS localization of brevican (red). (C) Hippocampal neurons lacking NrCAM still cluster brevican (red) at the AIS. MAP2 immunoreactivity (blue) marks the somatodendritic domain. Asterisks indicate untransfected neurons. (D) The ratio of GFP+ neurons labeled at the AIS (for the protein indicated) to the total number of GFP+ neurons. In a minimum of three independent experiments, 25–30 GFP+ positive neurons were counted per shRNA. (E and F) NF shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Transfected cortical neurons were visualized by GFP fluorescence (green), and sections were immunostained with anti-brevican (E [red] and F). The axon of the transfected neuron is indicated by the arrowheads, and the anti-brevican–labeled initial segments of untransfected neurons are indicated by the arrow. (G and H) NF-186 and ankG are both normally clustered at the AIS in brevican−/− brain. Note the neurons shown in G and H are not the same neurons but come from different brain sections, and NF-186 immunostaining was performed using a rabbit polyclonal antibody. Error bars indicate ± SD. Bars, 10 μm.
Figure 7. NF-186, but not NrCAM, is required for brevican clustering at the AIS. (A and B) Knockdown of ankG or NF-186 via shRNA plasmids in hippocampal neurons (GFP+) blocks AIS localization of brevican (red). (C) Hippocampal neurons lacking NrCAM still cluster brevican (red) at the AIS. MAP2 immunoreactivity (blue) marks the somatodendritic domain. Asterisks indicate untransfected neurons. (D) The ratio of GFP+ neurons labeled at the AIS (for the protein indicated) to the total number of GFP+ neurons. In a minimum of three independent experiments, 25–30 GFP+ positive neurons were counted per shRNA. (E and F) NF shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Transfected cortical neurons were visualized by GFP fluorescence (green), and sections were immunostained with anti-brevican (E [red] and F). The axon of the transfected neuron is indicated by the arrowheads, and the anti-brevican–labeled initial segments of untransfected neurons are indicated by the arrow. (G and H) NF-186 and ankG are both normally clustered at the AIS in brevican−/− brain. Note the neurons shown in G and H are not the same neurons but come from different brain sections, and NF-186 immunostaining was performed using a rabbit polyclonal antibody. Error bars indicate ± SD. Bars, 10 μm.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Hedstrom, K. L., Xu, X., Ogawa, Y., Frischknecht, R., Seidenbecher, C. I., Shrager, P., & Rasband, M. N.
Affiliations
  • UConn Health (US)
  • University of Rochester Medicine (US)
  • Leibniz Institute for Neurobiology (DE)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2007-08-20
Volume
178
Issue
5
Pages
875-886
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.200705119
PMC
PMC2064550
OpenAlex
W2159315879
Cited by
268
References
45
Citations by year
2026: 4 · 2025: 8 · 2024: 12 · 2023: 10 · 2022: 11 · 2021: 12 · 2020: 15 · 2019: 18 · 2018: 15 · 2017: 15 · 2016: 10 · 2015: 20 · 2014: 17 · 2013: 21 · 2012: 20
FWCI
5.96
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
National Institute of Neurological Disorders and Stroke (R01NS017965, NS 044916, NS 17965, R01NS044916, R37NS044916)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
Keywords
neurofascin 186, axon initial segment, extracellular matrix assembly, brevican, chondroitin sulfate proteoglycans, ankyrin-G, βIV-spectrin, nodes of Ranvier, Schwann cells, adhesion molecules, cytoskeletal anchoring, action potential generation, RNAi knockdown
MeSH terms
Animals, Axons, Cells, Cultured, Cytoskeleton, Extracellular Matrix, Hippocampus, Nerve Growth Factors, Nerve Tissue Proteins, Chondroitin Sulfate Proteoglycans, Ranvier's Nodes, Recombinant Fusion Proteins, RNA, Spectrin, Sodium Channels, Cell Adhesion Molecules, Ankyrins, RNA Interference, Lectins, C-Type, Rats, Brevican

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