Neurodevelopmental mutation of giant ankyrin-G disrupts a core mechanism for axon initial segment assembly

Abstract

Giant ankyrin-G (gAnkG) coordinates assembly of axon initial segments (AISs), which are sites of action potential generation located in proximal axons of most vertebrate neurons. Here, we identify a mechanism required for normal neural development in humans that ensures ordered recruitment of gAnkG and β4-spectrin to the AIS. We identified 3 human neurodevelopmental missense mutations located in the neurospecific domain of gAnkG that prevent recruitment of β4-spectrin, resulting in a lower density and more elongated pattern for gAnkG and its partners than in the mature AIS. We found that these mutations inhibit transition of gAnkG from a closed configuration with close apposition of N- and C-terminal domains to an extended state that is required for binding and recruitment of β4-spectrin, and normally occurs early in development of the AIS. We further found that the neurospecific domain is highly phosphorylated in mouse brain, and that phosphorylation at 2 sites (S1982 and S2619) is required for the conformational change and for recruitment of β4-spectrin. Together, these findings resolve a discrete intermediate stage in formation of the AIS that is regulated through phosphorylation of the neurospecific domain of gAnkG.

Cargo hold and delivery: Ankyrins, spectrins, and their functional patterning of neurons

“…In contrast, staining of hippocampal sections of βII‐spectrin KO mice of unreported age with a βIV‐spectrin‐specific antibody, also an AIS marker, detected significant AIS fragmentation and increased area of immunoreactivity (Galiano et al, ). It is worth noting that loss of βII‐spectrin, both in vivo and in vitro, results in over a 40% increase in βIV‐spectrin‐∑VI (Lorenzo et al, ), which is critical for the maintenance of the AIS (discussed below; Yang, Walder‐Christensen, Lalani, et al, ), and may explain some of the observed differences between the studies. Given the importance and multifaceted roles of βII‐spectrin, it will important to further assess how it might impact AIS morphology, composition, and function.…”

“…However, the DAR999AAA mutation in 480‐kDa ankyrin‐G, which eliminates the canonical binding of ankyrin‐G to βII‐spectrin (He et al, ; Kizhatil et al, ), has no effect on 480 kDa ankyrin‐G or βIV‐spectrin clustering at the AIS (Jenkins et al, ), suggesting that either the site for βIV‐spectrin binding is not conserved in 480‐kDa ankyrin‐G and/or additional coupling mechanisms between these two partners exist (Jenkins et al, ). On the other hand, introduction of the Y1901A mutation (corresponding to the Y1874A mutation in βII‐spectrin that disrupts binding to ankyrin‐G) in βIV‐spectrin‐∑I, the full‐length βIV‐spectrin splice variant, fails to restore AIS morphology and the clustering of 480‐kDa ankyrin‐G, and its AIS partners, in βIV‐spectrin null neurons (Yang, Walder‐Christensen, Lalani, et al, ). Surprisingly, deficits in AIS morphology and molecular composition of βIV‐spectrin null neurons can be rescued in vitro with βIV‐spectrin‐∑VI, the other βIV‐spectrin isoform present in the AIS, which lacks the actin binding site plus the first nine spectrin repeats, but retains the ankyrin‐G binding site and a PH domain (Yang, Walder‐Christensen, Lalani, et al, ).…”

“…On the other hand, introduction of the Y1901A mutation (corresponding to the Y1874A mutation in βII‐spectrin that disrupts binding to ankyrin‐G) in βIV‐spectrin‐∑I, the full‐length βIV‐spectrin splice variant, fails to restore AIS morphology and the clustering of 480‐kDa ankyrin‐G, and its AIS partners, in βIV‐spectrin null neurons (Yang, Walder‐Christensen, Lalani, et al, ). Surprisingly, deficits in AIS morphology and molecular composition of βIV‐spectrin null neurons can be rescued in vitro with βIV‐spectrin‐∑VI, the other βIV‐spectrin isoform present in the AIS, which lacks the actin binding site plus the first nine spectrin repeats, but retains the ankyrin‐G binding site and a PH domain (Yang, Walder‐Christensen, Lalani, et al, ). This is consistent with the actin skeleton not being required for proper βIV‐spectrin organization and function in the AIS, as mentioned above (Leterrier et al, ).…”

“…This is consistent with the actin skeleton not being required for proper βIV‐spectrin organization and function in the AIS, as mentioned above (Leterrier et al, ). Targeting of βIV‐spectrin to the AIS can be further modified by conformational changes in 480 kDa ankyrin‐G, which, in turn, are modulated by phosphorylation of specific residues (S1982A, S2619A, and S2417A) or by human neurodevelopmental mutations (T1861M, K2864N, and P2490L), all within the inserted region unique to this giant isoform (Jenkins et al, ; Yang, Walder‐Christensen, Lalani, et al, ). 480 kDa ankyrin‐G can expand 150 nm in its extended conformation in the mature AIS, as revealed by immunogold label platinum replica EM imaging (Jenkins et al, ; Jones, Korobova, & Svitkina, ), and inferred from proximity ligation signaling with antibodies against its N‐ and C‐terminal ends (Yang, Walder‐Christensen, Lalani, et al, ).…”

“…Targeting of βIV‐spectrin to the AIS can be further modified by conformational changes in 480 kDa ankyrin‐G, which, in turn, are modulated by phosphorylation of specific residues (S1982A, S2619A, and S2417A) or by human neurodevelopmental mutations (T1861M, K2864N, and P2490L), all within the inserted region unique to this giant isoform (Jenkins et al, ; Yang, Walder‐Christensen, Lalani, et al, ). 480 kDa ankyrin‐G can expand 150 nm in its extended conformation in the mature AIS, as revealed by immunogold label platinum replica EM imaging (Jenkins et al, ; Jones, Korobova, & Svitkina, ), and inferred from proximity ligation signaling with antibodies against its N‐ and C‐terminal ends (Yang, Walder‐Christensen, Lalani, et al, ). Note that 3D‐STORM imaging suggests an average radial conformation of ~32 nm of the inserted and C‐terminus region of 480 kDa ankyrin‐G, which might reflect a more relaxed conformation of the polypeptide, although it could also underestimate the true length of the protein depending on its orientation relative to the imaging direction (Leterrier et al, ).…”