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Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B.

Duflocq A, Chareyre F, Giovannini M, Couraud F, Davenne M - BMC Biol. (2011)

Bottom Line: We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins.We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS.Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM UMRS 952, 9 Quai St Bernard, F-75005, Paris, France.

ABSTRACT

Background: The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Results: We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.

Conclusions: α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

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Protein 4.1B is required to maintain a barrier at the para-axon initial segment (AIS) and to cluster voltage-gated potassium (Kv)1 channels. Triple immunostaining of ankyrin G (AnkG) (A), protein 4.1B (B) and Kv1.1 (C) in motor neurons (MNs) labeled with the anti-Peripherin antibody (data not shown) showing expression of protein 4.1B in the para-AIS, the juxtapara (JXP)-AIS and the internode. Triple immunostaining of AnkG (D, G, J), contactin-associated protein (Caspr) (E, H, K) and Kv1.1 (F, I, L) in MNs of wild-type (WT) (D-F) and 4.1B-/- (G-L) mice showing an abnormal expression of Caspr and Kv1.1 in the para-AIS of 4.1B-/- mice. Triple immunostaining of AnkG (M, P), PSD-93 (N) or Caspr2 (Q), and Kv1.1 (O, R) in MNs of 4.1B-/- mice showing a similar abnormal expression of PSD-93, Caspr2 and Kv1.1 in the para-AIS. Brackets indicate protein 4.1B+ (A-C) and Caspr+ (D-L) domains. Scale bar = 5 μm. (S, T) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG, Caspr and Kv1.1 in WT (S) and 4.1B-/- mice (T). Axon segments were aligned at the end of their AIS (dashed line). For each axon segment and each antibody, immunofluorescence intensities were normalized relative to its maximum intensity along that segment. (U) Mean (± SEM) length of AnkG and mean (± SEM) beginning and end positions of Caspr from n = 11 (soma-derived) AISs, aligned at the beginning of their AIS (dashed line), from WT and 4.1B-/- mice.
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Figure 10: Protein 4.1B is required to maintain a barrier at the para-axon initial segment (AIS) and to cluster voltage-gated potassium (Kv)1 channels. Triple immunostaining of ankyrin G (AnkG) (A), protein 4.1B (B) and Kv1.1 (C) in motor neurons (MNs) labeled with the anti-Peripherin antibody (data not shown) showing expression of protein 4.1B in the para-AIS, the juxtapara (JXP)-AIS and the internode. Triple immunostaining of AnkG (D, G, J), contactin-associated protein (Caspr) (E, H, K) and Kv1.1 (F, I, L) in MNs of wild-type (WT) (D-F) and 4.1B-/- (G-L) mice showing an abnormal expression of Caspr and Kv1.1 in the para-AIS of 4.1B-/- mice. Triple immunostaining of AnkG (M, P), PSD-93 (N) or Caspr2 (Q), and Kv1.1 (O, R) in MNs of 4.1B-/- mice showing a similar abnormal expression of PSD-93, Caspr2 and Kv1.1 in the para-AIS. Brackets indicate protein 4.1B+ (A-C) and Caspr+ (D-L) domains. Scale bar = 5 μm. (S, T) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG, Caspr and Kv1.1 in WT (S) and 4.1B-/- mice (T). Axon segments were aligned at the end of their AIS (dashed line). For each axon segment and each antibody, immunofluorescence intensities were normalized relative to its maximum intensity along that segment. (U) Mean (± SEM) length of AnkG and mean (± SEM) beginning and end positions of Caspr from n = 11 (soma-derived) AISs, aligned at the beginning of their AIS (dashed line), from WT and 4.1B-/- mice.

Mentions: We found protein 4.1B expression in MN axons, starting immediately after the AnkG+/Kv1+ distal AIS, and covering the para-AIS, the Kv1+ JXP-AIS, and extending beyond, within the internode (Figure 10A-F). Protein 4.1B could thus play an important role in stabilizing the molecular organization of the para-AIS and JXP-AIS. We therefore analyzed 4.1B- mice. We found that Caspr distribution was dramatically altered at the para-AIS of 4.1B-/- mice: it was not confined to a well delimited compartment between the AIS and the JXP-AIS, as in WT mice (Figure 10D-F); instead, it was extended along the axon, occupying either a single long Caspr+ segment (Figure 10H) or dispersed Caspr+ aggregates (Figure 10K; respectively 46 and 109 MNs analyzed from 3 4.1B-/- and 12 WT mice), suggesting that the para-AIS was disrupted. In addition, Kv1.1 and AnkG distributions were not segregated from the Caspr+ domain, as in WT mice (bracket, Figure 10D-F); instead AnkG (and Nav1.6) overlapped along varying distances with Caspr distribution (bracket, Figure 10G-H, 10J-K; see also Additional file 4, Figure S4A) and Kv1.1 distribution was continuous from the distal AIS to the JXP-AIS, covering completely the Caspr+ area (bracket, Figure 10H-I, 10K, L). Analysis of AnkG, Caspr and Kv1.1 immunofluorescence intensity profiles from WT and 4.1B-/- AISs, aligned at the end of their AnkG staining, confirmed these observations (Figure 10S, T; n = 6 AISs from three WT mice and n = 6 AISs from three 4.1B-/- mice). These results suggest that protein 4.1B is required to maintain a normal distribution of Caspr and thereby an efficient barrier at the MN para-AIS: when disturbed, Kv1 channels invade the MN para-AIS. In order to analyze whether the overlap between Caspr and AnkG in 4.1B-/- mice corresponds to AnkG invading the para-AIS, or to Caspr invading the AIS, we analyzed the length of AnkG, and the position of Caspr relative to the beginning of the AIS (Figure 10U). The changes observed for the mean AIS length and the mean beginning of Caspr between WT and 4.1B-/- mice was not statistically significant (n = 9 AISs from three WT mice and n = 11 AISs from four 4.1B-/- mice). We can therefore only conclude that in 4.1B-/- mice AnkG and Caspr do not segregate properly from one another.


Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B.

Duflocq A, Chareyre F, Giovannini M, Couraud F, Davenne M - BMC Biol. (2011)

Protein 4.1B is required to maintain a barrier at the para-axon initial segment (AIS) and to cluster voltage-gated potassium (Kv)1 channels. Triple immunostaining of ankyrin G (AnkG) (A), protein 4.1B (B) and Kv1.1 (C) in motor neurons (MNs) labeled with the anti-Peripherin antibody (data not shown) showing expression of protein 4.1B in the para-AIS, the juxtapara (JXP)-AIS and the internode. Triple immunostaining of AnkG (D, G, J), contactin-associated protein (Caspr) (E, H, K) and Kv1.1 (F, I, L) in MNs of wild-type (WT) (D-F) and 4.1B-/- (G-L) mice showing an abnormal expression of Caspr and Kv1.1 in the para-AIS of 4.1B-/- mice. Triple immunostaining of AnkG (M, P), PSD-93 (N) or Caspr2 (Q), and Kv1.1 (O, R) in MNs of 4.1B-/- mice showing a similar abnormal expression of PSD-93, Caspr2 and Kv1.1 in the para-AIS. Brackets indicate protein 4.1B+ (A-C) and Caspr+ (D-L) domains. Scale bar = 5 μm. (S, T) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG, Caspr and Kv1.1 in WT (S) and 4.1B-/- mice (T). Axon segments were aligned at the end of their AIS (dashed line). For each axon segment and each antibody, immunofluorescence intensities were normalized relative to its maximum intensity along that segment. (U) Mean (± SEM) length of AnkG and mean (± SEM) beginning and end positions of Caspr from n = 11 (soma-derived) AISs, aligned at the beginning of their AIS (dashed line), from WT and 4.1B-/- mice.
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Figure 10: Protein 4.1B is required to maintain a barrier at the para-axon initial segment (AIS) and to cluster voltage-gated potassium (Kv)1 channels. Triple immunostaining of ankyrin G (AnkG) (A), protein 4.1B (B) and Kv1.1 (C) in motor neurons (MNs) labeled with the anti-Peripherin antibody (data not shown) showing expression of protein 4.1B in the para-AIS, the juxtapara (JXP)-AIS and the internode. Triple immunostaining of AnkG (D, G, J), contactin-associated protein (Caspr) (E, H, K) and Kv1.1 (F, I, L) in MNs of wild-type (WT) (D-F) and 4.1B-/- (G-L) mice showing an abnormal expression of Caspr and Kv1.1 in the para-AIS of 4.1B-/- mice. Triple immunostaining of AnkG (M, P), PSD-93 (N) or Caspr2 (Q), and Kv1.1 (O, R) in MNs of 4.1B-/- mice showing a similar abnormal expression of PSD-93, Caspr2 and Kv1.1 in the para-AIS. Brackets indicate protein 4.1B+ (A-C) and Caspr+ (D-L) domains. Scale bar = 5 μm. (S, T) The mean immunofluorescence intensity profile (shown by the line) ± SEM from n = 6 AISs is shown for AnkG, Caspr and Kv1.1 in WT (S) and 4.1B-/- mice (T). Axon segments were aligned at the end of their AIS (dashed line). For each axon segment and each antibody, immunofluorescence intensities were normalized relative to its maximum intensity along that segment. (U) Mean (± SEM) length of AnkG and mean (± SEM) beginning and end positions of Caspr from n = 11 (soma-derived) AISs, aligned at the beginning of their AIS (dashed line), from WT and 4.1B-/- mice.
Mentions: We found protein 4.1B expression in MN axons, starting immediately after the AnkG+/Kv1+ distal AIS, and covering the para-AIS, the Kv1+ JXP-AIS, and extending beyond, within the internode (Figure 10A-F). Protein 4.1B could thus play an important role in stabilizing the molecular organization of the para-AIS and JXP-AIS. We therefore analyzed 4.1B- mice. We found that Caspr distribution was dramatically altered at the para-AIS of 4.1B-/- mice: it was not confined to a well delimited compartment between the AIS and the JXP-AIS, as in WT mice (Figure 10D-F); instead, it was extended along the axon, occupying either a single long Caspr+ segment (Figure 10H) or dispersed Caspr+ aggregates (Figure 10K; respectively 46 and 109 MNs analyzed from 3 4.1B-/- and 12 WT mice), suggesting that the para-AIS was disrupted. In addition, Kv1.1 and AnkG distributions were not segregated from the Caspr+ domain, as in WT mice (bracket, Figure 10D-F); instead AnkG (and Nav1.6) overlapped along varying distances with Caspr distribution (bracket, Figure 10G-H, 10J-K; see also Additional file 4, Figure S4A) and Kv1.1 distribution was continuous from the distal AIS to the JXP-AIS, covering completely the Caspr+ area (bracket, Figure 10H-I, 10K, L). Analysis of AnkG, Caspr and Kv1.1 immunofluorescence intensity profiles from WT and 4.1B-/- AISs, aligned at the end of their AnkG staining, confirmed these observations (Figure 10S, T; n = 6 AISs from three WT mice and n = 6 AISs from three 4.1B-/- mice). These results suggest that protein 4.1B is required to maintain a normal distribution of Caspr and thereby an efficient barrier at the MN para-AIS: when disturbed, Kv1 channels invade the MN para-AIS. In order to analyze whether the overlap between Caspr and AnkG in 4.1B-/- mice corresponds to AnkG invading the para-AIS, or to Caspr invading the AIS, we analyzed the length of AnkG, and the position of Caspr relative to the beginning of the AIS (Figure 10U). The changes observed for the mean AIS length and the mean beginning of Caspr between WT and 4.1B-/- mice was not statistically significant (n = 9 AISs from three WT mice and n = 11 AISs from four 4.1B-/- mice). We can therefore only conclude that in 4.1B-/- mice AnkG and Caspr do not segregate properly from one another.

Bottom Line: We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins.We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS.Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM UMRS 952, 9 Quai St Bernard, F-75005, Paris, France.

ABSTRACT

Background: The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Results: We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.

Conclusions: α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

Show MeSH
Related in: MedlinePlus