Ca2+-dependent recruitment of voltage-gated sodium channels underlies bilirubin-induced overexcitation and neurotoxicity

Hao-Song Shi,Xin-Lu Yin,Min Liang,Min Liang,Hai-Bo Ye,Hai-Bo Ye,Shan-Kai Yin,Shan-Kai Yin,Hai-Bo Shi,Hai-Bo Shi,Lu-Yang Wang,Lu-Yang Wang,Ke Lai,Ke Lai

doi:10.1038/s41419-019-1979-1

Abstract

Neonatal jaundice is prevalent among newborns and can lead to severe neurological deficits, particularly sensorimotor dysfunction. Previous studies have shown that bilirubin (BIL) enhances the intrinsic excitability of central neurons and this can potentially contribute to their overexcitation, Ca2+ overload, and neurotoxicity. However, the cellular mechanisms underlying elevated neuronal excitability remain unknown. By performing patch-clamp recordings from neonatal neurons in the rat medial vestibular nucleus (MVN), a crucial relay station for locomotor and balance control, we found that BIL (3 μM) drastically increases the spontaneous firing rates by upregulating the current-mediated voltage-gated sodium channels (VGSCs), while shifting their voltage-dependent activation toward more hyperpolarized potentials. Immunofluorescence labeling and western immunoblotting with an anti-NaV1.1 antibody, revealed that BIL elevates the expression of VGSCs by promoting their recruitment to the membrane. Furthermore, we found that this VGSC-trafficking process is Ca2+ dependent because preloading MVN neurons with the Ca2+ buffer BAPTA-AM, or exocytosis inhibitor TAT-NSF700, prevents the effects of BIL, indicating the upregulated activity and density of functional VGSCs as the core mechanism accountable for the BIL-induced overexcitation of neonatal neurons. Most importantly, rectification of such overexcitation with a low dose of VGSC blocker lidocaine significantly attenuates BIL-induced cell death. We suggest that this enhancement of VGSC currents directly contributes to the vulnerability of neonatal brain to hyperbilirubinemia, implicating the activity and trafficking of NaV1.1 channels as a potential target for neuroprotection in cases of severe jaundice.

Highlights

85% of newborns are affected with neonatal hyperbilirubinemia and clinical jaundice
We suggest that elevated activity and expression of voltage-gated sodium channels (VGSCs) by BIL underlie increased intrinsic excitability of neonatal neurons and neurotoxicity seen in the medial vestibular nucleus (MVN), and potentially other BIL-vulnerable brain regions
These results demonstrated that BIL facilitates spontaneous firings of MVN neurons by enhancing Iinward, implicating VGSCs as one of the main driving forces

Summary

Introduction

85% of newborns are affected with neonatal hyperbilirubinemia and clinical jaundice. Hyperbilirubinemia-induced neurotoxicity of MVN neurons has been linked to motor disorders observed during kernicterus, such as strabismus and gaze palsies, hypotonia, and delays in vestibular-evoked myogenic potentials (VEMP)[8,9,10]. Neuropathological changes such as yellow staining in vestibular nuclei are notable in Gunn rat jaundice model[11], and in the brainstem tissue from an infant with hyperbilirubinemia-induced kernicterus[12]. Shi et al Cell Death and Disease (2019)10:774 concentration ([Ca2+]i) and transmitter release from presynaptic terminals, as well as the excitability of postsynaptic neurons[13,14,15]. Neither the identity of ion channels, nor the mechanisms underlying elevated intrinsic excitability of neonatal neurons and neurotoxicity by BIL, are known

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