Abstract
The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing βIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on βIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in βIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.
Highlights
The axon initial segment (AIS) is a subcellular compartment that exhibits dynamic plasticity of protein interactions which contribute to functional modulation of neuronal excitability (Grubb and Burrone, 2010a; Grubb et al, 2011; Evans et al, 2015)
Analysis by Support Vector Machines (SVM) was able to detect whether a cell was treated with an AKT inhibitor or Wee1 inhibitor with greater than 50% accuracy, but is able to do so significantly more accurately for the AKT inhibitor treated cells (Figure 4H)
Voltage threshold (mV) Current threshold (pA) Input resistance (M ) Tau (ms) Capacitance (pF) Upstroke velocity (dV/dt, mV/ms rise) Downstroke velocity (dV/dt, mV/ms decay) Latency to first peak (ms)
Summary
The axon initial segment (AIS) is a subcellular compartment that exhibits dynamic plasticity of protein interactions which contribute to functional modulation of neuronal excitability (Grubb and Burrone, 2010a; Grubb et al, 2011; Evans et al, 2015). Crucial to Nav channel clustering to the AIS is a complex nexus of protein:protein interactions (PPI) that incorporates intracellular scaffolding and signaling molecules (Grubb and Burrone, 2010b; Grubb et al, 2011; Jones and Svitkina, 2016; Yamada and Kuba, 2016; Leterrier, 2018) that serve as building blocks anchoring these channels to the local cytoskeleton (Xu and Shrager, 2005; Fréal et al, 2019), an integral step in establishing neuronal polarity and ensuring directional firing (Grubb and Burrone, 2010a, b; Yamada and Kuba, 2016; Huang and Rasband, 2018; Salzer, 2019). Nav channel clustering is dependent upon the initial localization of other scaffolding proteins, especially ankyrin G (Kordeli et al, 1995; Grubb and Burrone, 2010b; Jenkins et al, 2015; Yang et al, 2019) and βIV spectrin (Xu and Shrager, 2005; Yang et al, 2007; Leterrier, 2018; Liu et al, 2020), which are both necessary for the AIS proper formation, and neurofascin, a member of the L1 cell-adhesion molecule family (Boiko et al, 2007; Zonta et al, 2011; Kriebel et al, 2012; Hamdan et al, 2020)
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