Nav channels, NF-186, NrCAM, βIV spectrin, and ankG are all highly enriched at the AIS in vivo (; NrCAM, NF-186, and ankG not depicted). We used cultured hippocampal neurons to determine the role these proteins play in AIS assembly. This well-characterized model (Banker and Goslin, 1998
) has been used to determine the mechanisms regulating protein sorting and localization in central nervous system (CNS) neurons (Lim et al., 2000
; Garrido et al., 2003
; Rivera et al., 2003
; Wisco et al., 2003
; Yang et al., 2007
). In the experiments described here, we define the AIS by the presence of high density, restricted clustering of at least one of the following five protein components: NF-186, NrCAM, βIV spectrin, Nav channels, and ankG. Both in vivo and in cultured hippocampal neurons, NrCAM, NF-186, ankG, βIV spectrin, and Nav channels all colocalize within the first 20–40 μm of the axon (). Further, these proteins are excluded from somatodendritic domains defined by microtubule-associated protein 2 (MAP2; , blue) immunoreactivity.
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 (more ...)
Which protein is the first to define the AIS? To answer this question, we immunostained hippocampal neurons at 2, 4, 6, 8, and 10 d in vitro (DIV) using antibodies against Nav channels, ankG, βIV spectrin, NrCAM, and NF (pan-NF). However, similar to Boiko et al. (2007)
, we were unable to clearly identify a single protein that clustered first at the AIS (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200705119/DC1
), as we observed only small differences in the time course of protein clustering. These results likely reflect differences in antibody affinity rather than provide an accurate picture of the temporal regulation of AIS protein localization. Thus, other methods are necessary to determine the molecular events leading to AIS formation.
Short hairpin RNA (shRNA)–mediated ablation of AIS proteins
To determine the mechanisms underlying AIS formation, we used RNAi methods to silence expression of AIS proteins. Compared with mice lacking these proteins altogether, this strategy has several advantages, including the following: (1) all experimental manipulations of protein expression occur at the same time and in similar neurons, (2) mouse strain differences are avoided, and (3) by working with cells rather than the whole organism, the role of proteins essential for survival (e.g., Nav channels) or that may serve diverse functions in different cellular contexts (e.g., ankG) can be investigated. Therefore, we used shRNA expression plasmids for gene silencing of NF-186, NrCAM, Nav channels (Nav1.x), βIV spectrin, and ankG; we also constructed a control shRNA expression plasmid using a sequence directed against a nonmammalian protein. Cotransfection of the control shRNA and EGFP (to identify transfected neurons) showed that the assembly of the AIS was unaffected by transfection at both 10 and 17 DIV (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200705119/DC1
; 10 DIV not depicted).
To confirm the specificity of each shRNA, we cotransfected COS-7 cells with an expression plasmid for the target protein together with each shRNA. In transfected COS-7 cells, each shRNA only reduced expression of the target protein and did not affect other AIS proteins (Fig. S2 B).
As a direct measure of each shRNA's efficacy, we examined their ability to eliminate the endogenous target protein in cultured hippocampal neurons. 3 h after plating neurons, each shRNA expression plasmid was cotransfected into hippocampal neurons together with GFP. At 10 or 17 DIV, the transfected neurons were then immunostained with antibodies to MAP2 (to define the somatodendritic domain) and the targeted protein. Immunostaining for the target protein revealed that untransfected neurons (, asterisk) had the target protein at the AIS, but transfected neurons (GFP+) had no AIS immunoreactivity. The shRNA expression plasmids were found to be effective at eliminating all AIS immunoreactivity for the target protein in 50–90% of transfected hippocampal neurons ( and ; note that when the immunoreactivity for the targeted protein was reduced in transfected neurons, but not completely eliminated, the neuron was still counted as having an AIS). Thus, the shRNA expression plasmids are both specific and effective for knockdown of the targeted protein.
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 (more ...)
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 (more ...)
Neither NF-186 nor NrCAM is required for Nav channel clustering at the AIS of CNS neurons
Because NF-186 is essential for Nav channel and ankG clustering at nodes of Ranvier (Sherman et al., 2005
), we tested whether the CAMs NF-186 or NrCAM are also necessary for the molecular assembly of the AIS (). We eliminated NF-186 using NF shRNA and then immunostained the cultures at 8–10 DIV with anti-MAP2 (, blue) and antibodies to Nav channels, ankG, βIV spectrin, or NrCAM (red). Cells transfected with the NrCAM shRNA were immunostained with the same set of antibodies, except anti–pan-NF was substituted for anti-NrCAM. In neurons transfected with NF or NrCAM shRNA expression plasmids, ~80% of neurons had no detectable staining for the targeted protein ( and ; transfected neurons are GFP+
). However, elimination of these CAMs did not affect Nav channel, ankG, or βIV spectrin (not depicted) localization and clustering at the AIS (, A and B; untransfected neurons are identified by an asterisk). We measured ~30% reduction in NrCAM+
AIS in neurons transfected with NF shRNA, and ~20% reduction in NF-186+
AIS when neurons were transfected with NrCAM shRNA (). This observation is interesting because Sherman et al. (2005)
reported that NFasc-null
mice lack NrCAM at PNS nodes of Ranvier, and NrCAM-null
mice have delayed PNS node formation (Custer et al., 2003
). Thus, although there may be some interdependence between NrCAM and NF-186, these results demonstrate that neither of these CAMs is required for the clustering of Nav channels at the AIS of CNS neurons.
NF-186 and NrCAM are not enriched at the AIS of dorsal root ganglion (DRG) neurons
As NF-186 has been shown to be essential for node formation in the PNS, we considered the possibility that NF-186 or NrCAM may contribute to AIS formation in the PNS rather than the CNS. To test this possibility, we used a DRG neuron–Schwann cell co-culture system, where Schwann cells make many myelin segments and form nodes of Ranvier. After 21 DIV and after induction of myelination by the addition of ascorbic acid, we determined that DRG neurons had ankG, βIV spectrin, and Nav channels at the AIS (; Nav channels not depicted). However, we were unable to detect any enrichment for NF-186 or NrCAM (, D and E, red). Even cultures that were 5 mo old did not have high densities of NrCAM or NF-186 at the DRG neuron AIS (unpublished data). In contrast, nodes of Ranvier along these same axons, defined by Caspr-labeled paranodes (, D and E, green), had high levels of clustered NF-186, NrCAM, Nav channels, ankG, and βIV spectrin (, red; and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200705119/DC1
). These results indicate that NF-186 and NrCAM are not required for Nav channel clustering at the AIS, and that the molecular requirements for AIS assembly are distinct from nodes of Ranvier. These results parallel those recently reported by Dzhashiashvili et al. (2007)
AnkG is required for Nav channel clustering and the molecular assembly of the AIS
If NrCAM and NF-186 are not required for AIS assembly, what protein is essential? As ankG, βIV spectrin, and Nav channels have each been reported to contribute to AIS formation (Zhou et al., 1998
; Komada and Soriano, 2002
; Xu and Shrager, 2005
), we introduced shRNA expression plasmids directed against these proteins into cultured hippocampal neurons. After 8–10 DIV, knockdown of Nav channels or βIV spectrin using the shRNA expression plasmids had no effect on the clustering of other AIS components (). However, when ankG shRNA expression plasmids were transfected into hippocampal neurons, ~80% of cells had no detectable clustering of any other AIS protein (, A and B, middle). Thus, ankG is required for the clustering of Nav channels and the molecular assembly of the AIS.
Because loss of βIV spectrin or Nav channels has also been reported to disrupt the AIS (Komada and Soriano, 2002
; Xu and Shrager, 2005
), we considered the possibility that NF-186, NrCAM, Nav channels, or βIV spectrin contribute to maintenance and long-term stability of the AIS. To test this, we transfected the various shRNA expression plasmids as described and then immunostained neurons at 17 DIV to determine whether loss of any one AIS component influenced the clustering of any other AIS protein at later time points. We found that at 17 DIV, the shRNA expression plasmids (with the exception of βIV spectrin shRNA) were effective at eliminating their target proteins in ~70% of neurons (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200705119/DC1
). However, only the ankG shRNA disrupted localization of the other AIS protein components.
To confirm further that ankG is essential for the molecular assembly of the AIS, we electroporated neurons with either NF-186 or ankG shRNA at embryonic day (E) 16 in utero. The shRNA expression plasmids also expressed a membrane-bound form of GFP to permit identification of transfected neurons and their axons. This method results in transfection of neurons that subsequently migrate into the cortex and become layer II–III pyramidal neurons. 6 wk after birth, we cut coronal brain sections and immunostained these sections to detect ankG, βIV spectrin, NF-186, and Nav channels. The axons of GFP+ neurons were easily identified (, arrowheads). 80% (n = 33) of NF-186 shRNA–transfected neurons lacked NF-186 at the AIS (, arrowheads), but untransfected neurons had robust NF-186 immunoreactivity (, arrows). Further, other components of the AIS, such as βIV spectrin (, arrowheads), were still appropriately clustered in neurons transfected with the NF-186 shRNA (22/22 NF-186 shRNA–transfected neurons had βIV spectrin immunoreactivity). In contrast, 70% (n = 27) of neurons electroporated with ankG shRNA lacked ankG at the AIS (, arrowheads; n = 27), but untransfected neurons had robust AIS ankG immunoreactivity (, arrows). As with cultured hippocampal neurons, silencing of ankG expression blocked the AIS clustering of βIV spectrin, NF-186, and Nav1.6 (, D and E; Nav1.6 not depicted); 1/21, 0/24, and 0/12 neurons had AIS immunoreactivity for βIV spectrin, NF-186, and Nav1.6, respectively. Together, these results confirm ankG's essential role in vivo for Nav channel clustering and AIS assembly.
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 (more ...)
Brevican, an ECM molecule, is enriched at the AIS and nodes of Ranvier
If NF-186 and NrCAM are not required for Nav channel clustering at the AIS, what are their functions? As both of these CAMs have large extracellular immunoglobulin domains, they might be important for mediating interactions with other cells or proteins. Consistent with this idea, John et al. (2006)
recently reported that the chondroitin sulfate proteoglycan brevican, mainly secreted by glia, is highly enriched at the AIS of cultured neurons. Brevican is a member of a family of ECM molecules called lecticans that assemble into perineuronal nets surrounding neurons (Yamaguchi, 2000
). Other lecticans include aggrecan and neurocan. Immunostaining of cultured hippocampal neurons with antibodies against aggrecan and neurocan at 16 DIV revealed prominent somatic staining (). In ~40% of neurons, there was punctate aggrecan and neurocan immunoreactivity along the AIS (, A and C, insets). In contrast, 93% of cultured hippocampal neurons had robust brevican immunoreactivity that was restricted to the AIS, where it colocalized with other AIS markers, such as βIV spectrin (). Brevican immunoreactivity was also enriched at the AIS of cortical neurons in vivo (), but neither aggrecan nor neurocan could be detected at these sites in vivo (not depicted). As the molecular composition of nodes is nearly identical to that at the AIS, we immunostained optic nerve with antibodies against aggrecan, neurocan, and brevican. Among these lecticans, only brevican could be detected at nodes (, arrowheads). These results indicate that brevican is highly enriched at sites where NF-186 and NrCAM are located.
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. (more ...)
The brevican-based ECM interacts with NF-186
To determine if NF-186 and/or NrCAM interact with brevican, we expressed each CAM in COS cells and then added soluble GFP-brevican to determine if it could bind to the transfected cells. We found punctate GFP-brevican bound to the surface of NF-186–transfected cells (), but not to untransfected () or NrCAM-transfected cells (), suggesting that brevican interacts with NF-186.
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) (more ...)
To further understand the mechanisms underlying recruitment of brevican to the AIS, we examined the effect of ectopically expressed NF-186 on brevican localization. We expressed an HA-tagged NF-186 in hippocampal neurons. Low levels of HA–NF-186 expression resulted in HA–NF-186 (Lemaillet et al., 2003
) and brevican being restricted to the AIS (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200705119/DC1
). In contrast, overexpressed HA–NF-186 was found both at the AIS and somatodendritic domains (). Overexpression did not interfere with the normal recruitment of brevican to the AIS (). However, whenever HA–NF-186 was ectopically localized to somatodendritic domains, brevican was recruited to these same sites (). Nontransfected neurons never had somatodendritic brevican (Fig. S5). The redistribution of brevican along dendrites of cells overexpressing HA–NF-186 can be seen in line scans through the AIS and dendrites (, I and J; these line scans correspond to the white lines crossing the AIS and a dendrite in ). Thus, ectopic expression of NF-186 is sufficient to recruit the brevican-based ECM to somatodendritic domains.
AIS clustering of brevican depends on NF-186
Because ankG knockdown eliminates clustering of all neuronal AIS proteins, including NF-186 and NrCAM (, , and Fig. S4), we first tested whether loss of ankG also blocked brevican from clustering at the AIS. Cotransfection of the ankG shRNA together with GFP in cultured hippocampal neurons showed that 84% of GFP+ neurons had no detectable brevican at the AIS (). However, untransfected neurons (, asterisk) had robust brevican immunoreactivity at the AIS. To directly determine whether AIS brevican clustering depends on the CAMs NF-186 and/or NrCAM, we silenced expression of these proteins in cultured hippocampal neurons using shRNA expression plasmids. Although untransfected neurons had robust brevican immunoreactivity at the AIS (, asterisk), knockdown of NF-186, but not NrCAM, completely blocked the AIS clustering of brevican ().
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 (more ...)
Finally, using the same in utero electroporation strategy as described, we knocked down expression of NF-186 in vivo using NF shRNA expression plasmids. We then immunolabeled postnatal day 10 brain sections using anti-brevican antibodies. The axons of electroporated neurons were easily identified by GFP fluorescence (, arrowheads). Although untransfected neurons had brevican immunoreactivity at the AIS (, E and F, arrows), we could not detect brevican immunoreactivity at the AIS of electroporated neurons (, arrowheads). Thus, NF-186 is required for the enrichment of brevican at the AIS. In contrast, NF-186 and ankG were both normally clustered at the AIS of brevican-null mice ().