ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury

Abstract

Axon properties, including action potential initiation and modulation, depend on both AIS integrity and the regulation of ion channel expression in the AIS. Alteration of the axon initial segment (AIS) has been implicated in neurodegenerative, psychiatric, and brain trauma diseases, thus identification of the physiological mechanisms that regulate the AIS is required to understand and circumvent AIS alterations in pathological conditions. Here, we show that the purinergic P2X7 receptor and its agonist, adenosine triphosphate (ATP), modulate both structural proteins and ion channel density at the AIS in cultured neurons and brain slices. In cultured hippocampal neurons, an increment of extracellular ATP concentration or P2X7-green fluorescent protein (GFP) expression reduced the density of ankyrin G and voltage-gated sodium channels at the AIS. This effect is mediated by P2X7-regulated calcium influx and calpain activation, and impaired by P2X7 inhibition with Brilliant Blue G (BBG), or P2X7 suppression. Electrophysiological studies in brain slices showed that P2X7-GFP transfection decreased both sodium current amplitude and intrinsic neuronal excitability, while P2X7 inhibition had the opposite effect. Finally, inhibition of P2X7 with BBG prevented AIS disruption after ischemia/reperfusion in rats. In conclusion, our study demonstrates an involvement of P2X7 receptors in the regulation of AIS mediated neuronal excitability in physiological and pathological conditions.

Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment

“…The construct uses the same mechanism as Na V channels for AnkG binding and AIS localization, so, under the assumption that Na V binding sites on AnkG are normally saturated in DGCs at 10–12 DIV, it might be expected to at least partially out-compete native channels. The resulting reduction in AIS Na V density would then be predicted, on the basis of previous experimental and theoretical work, to impact on action potential initiation (Khaliq and Raman, 2006; Laezza et al, 2007; Kole et al, 2008; Kress et al, 2010; Tapia et al, 2013; Del Puerto et al, 2015). A reduction in whole-cell Na V current was indeed reported with overexpression of GFP-Na V II–III in hippocampal neurons of similar maturational status (Garrido et al, 2003), although that study did not assess the construct’s impact on spike firing.…”

Functional implications of axon initial segment cytoskeletal disruption in stroke

“…P2X7 channels are considered to be activated in neuronal injury due to high extracellular concentrations of ATP and its metabolites under ischemic conditions [99] , but the activation is not sufficient to cause neuronal death alone [101] . A recently published study found that P2X7 receptor activation reduced the density of AnkG and Na v channels in the AIS, whereas inhibition of these receptors resulted in the opposite [102] . Interestingly, in a MCA occlusion model similar to that in Schafer et al [3] , inhibition of P2X7 receptors prevented the AIS disruption.…”

Cannabinoid Receptors Modulate Neuronal Morphology and AnkyrinG Density at the Axon Initial Segment

“…Mouse hippocampal neurons were prepared as previously described (del Puerto et al, 2012, 2015). Neurons were obtained from E17 mouse hippocampi, which were incubated in a 0.25% trypsin solution in Ca 2+ /Mg 2+ free Hank’s buffered salt solution (HBSS) and dissociated using fire-polished Pasteur pipettes.…”

“…Furthermore, the discovery of AIS structural plasticity (changes in position or length) and its capacity to adapt in response to different physiological and pathological conditions in order to maintain neuronal survival and excitability shed light on the dynamical complexity of the AIS (Schafer et al, 2009; Grubb and Burrone, 2010; Kuba et al, 2010). Besides AIS structural plasticity, AIS proteins density also can change in response to physiological or pathological stimuli (Kuba et al, 2015; del Puerto et al, 2015). …”

“…Moreover, the mechanisms involved in the targeting and modulation of AIS proteins during the very early stages of neuronal development remain elusive. Recent studies have shown that the purinergic system (del Puerto et al, 2015), serotonin receptors (Ko et al, 2016) or GABAergic innervation (Muir and Kittler, 2014) influence and modulate AIS proteins and neuronal excitability. However, the role of another important neuromodulatory system, the cannabinoid system, has not been analyzed in the context of AIS development and maturation.…”