The axon initial segment (AIS) functions as both a physiological and physical bridge between somatodendritic and axonal domains. Given its unique molecular composition, location, and physiology, the AIS is thought to maintain neuronal polarity. To identify the molecular basis of this AIS property, we used adenovirus-mediated RNA interference to silence AIS protein expression in polarized neurons. Some AIS proteins are remarkably stable with half-lives of at least 2 wk. However, silencing the expression of the cytoskeletal scaffold ankyrinG (ankG) dismantles the AIS and causes axons to acquire the molecular characteristics of dendrites. Both cytoplasmic- and membrane-associated proteins, which are normally restricted to somatodendritic domains, redistribute into the former axon. Furthermore, spines and postsynaptic densities of excitatory synapses assemble on former axons. Our results demonstrate that the loss of ankG causes axons to acquire the molecular characteristics of dendrites; thus, ankG is required for the maintenance of neuronal polarity and molecular organization of the AIS.

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.

SUMMARY AnkyrinG (ankG) is highly enriched in neurons at axon initial segments (AIS) where it clusters Na+ and K+ channels and maintains neuronal polarity. How ankG becomes concentrated at the AIS is unknown. Here, we show that as neurons break symmetry, they assemble a distal axonal submembranous cytoskeleton comprised of ankyrinB (ankB), αII spectrin, and βII spectrin that defines a boundary limiting ankG to the proximal axon. Experimentally moving this boundary altered the length of ankG staining in the proximal axon, whereas disruption of the boundary through silencing of ankB, αII spectrin, or βII spectrin expression blocked AIS assembly and permitted ankG to redistribute throughout the distal axon. In support of an essential role for the distal cytoskeleton in ankG clustering, we also found that αII and βII spectrin -deficient mice had disrupted AIS. Thus, the distal axonal cytoskeleton functions as an intra-axonal boundary restricting ankG to the AIS.

Summary Rapid action potential propagation in myelinated axons requires Na+ channel clustering at nodes of Ranvier. However, the mechanism of clustering at CNS nodes remains poorly understood. Here, we show that the assembly of nodes of Ranvier in the CNS involves three mechanisms: a glia-derived extracellular matrix (ECM) complex containing proteoglycans and adhesion molecules that cluster NF186, paranodal axoglial junctions that function as barriers to restrict the position of nodal proteins, and axonal cytoskeletal scaffolds (CSs) that stabilize nodal Na+ channels. We show that while mice with a single disrupted mechanism had mostly normal nodes, disruptions of the ECM and paranodal barrier, the ECM and CS, or the paranodal barrier and CS all lead to juvenile lethality, profound motor dysfunction, and significantly reduced Na+ channel clustering. Our results demonstrate that ECM, paranodal, and axonal cytoskeletal mechanisms ensure robust CNS nodal Na+ channel clustering.

High densities of ion channels at axon initial segments (AISs) and nodes of Ranvier are required for initiation, propagation, and modulation of action potentials in axons. The organization of these membrane domains depends on a specialized cytoskeleton consisting of two submembranous cytoskeletal and scaffolding proteins, ankyrinG (ankG) and βIV spectrin. However, it is not known which of these proteins is the principal organizer, or if the mechanisms governing formation of the cytoskeleton at the AIS also apply to nodes. We identify a distinct protein domain in βIV spectrin required for its localization to the AIS, and show that this domain mediates βIV spectrin's interaction with ankG. Dominant-negative ankG disrupts βIV spectrin localization, but does not alter endogenous ankG or Na+ channel clustering at the AIS. Finally, using adenovirus for transgene delivery into myelinated neurons, we demonstrate that βIV spectrin recruitment to nodes of Ranvier also depends on binding to ankG.

Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, ␣II spectrin, and ␤II spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein ␤IV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.

Motoneurons and oligodendrocytes in the embryonic spinal cord are produced from a restricted domain of the ventral ventricular zone, termed the pMN domain. The pMN domain is the site of expression of two basic helix-loop-helix transcription factors, Olig1 and Olig2, which are essential for motoneuron and oligodendrocyte development. Previous lineage-tracing experiments using Olig1-Cre and Olig2-GFP mice suggested that motoneurons and oligodendrocytes, but not astrocytes, are produced from the pMN domain. However, important questions remain, including the fate of neuroepithelial cells in the pMN domain, and specifically whether motoneurons and oligodendrocytes are the only types of cells produced in the pMN domain. We performed lineage-tracing experiments using a tamoxifen-inducible Cre-recombinase inserted into the Olig2 locus. We demonstrated that motoneurons and oligodendrocyte progenitors are derived from the Olig2+ progenitors in the pMN domain, and also found that a subset of astrocytes at the ventral surface of the spinal cord and ependymal cells at the ventricular surface are also produced from the pMN domain. These findings demonstrate that motoneurons and oligodendrocytes are not the only cell types originating from this domain.

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