Cellular and molecular responses to acute cocaine treatment in neuronal-like N2a cells: potential mechanism for its resistance in cell death

Ramesh B Badisa,Lekan M Latinwo,Zachary Jones,Elizabeth Mazzio,Carl B Goodman,Jens T Rosenberg,Samuel C Grant,Sungsool Wi,Yi Zhou

doi:10.1038/s41420-018-0078-x

Abstract

Cocaine is a highly abused drug that causes psychiatric and neurological problems. Its entry into neurons could alter cell-biochemistry and contribute in the manifestation of early pathological symptoms. We have previously shown the acute cocaine effects in rat C6 astroglia-like cells and found that these cells were highly sensitive to cocaine in terms of manifesting certain pathologies known to underlie psychological disorders. The present study was aimed to discern acute cocaine effects on the early onset of various changes in Neuro-2a (N2a) cells. Whole-cell patch-clamp recording of differentiated cells displayed the functional voltage-gated Na+ and K+ channels, which demonstrated the neuronal characteristics of the cells. Treatment of these cells with acute cocaine (1 h) at in vivo (nM to μM) and in vitro (mM) concentrations revealed that the cells remained almost 100% viable. Cocaine administration at 6.25 μM or 4 mM doses significantly reduced the inward currents but had no significant effect on outward currents, indicating the Na+ channel-blocking activity of cocaine. While no morphological change was observed at in vivo doses, treatment at in vitro doses altered the morphology, damaged the neurites, and induced cytoplasmic vacuoles; furthermore, general mitochondrial activity and membrane potential were significantly decreased. Mitochondrial dysfunction enabled the cells switch to anaerobic glycolysis, evidenced by dose-dependent increases in lactate and H2S, resulting unaltered ATP level in the cells. Further investigation on the mechanism of action unfolded that the cell’s resistance to cocaine was through the activation of nuclear factor E2-related factor-2 (Nrf-2) gene and subsequent increase of antioxidants (glutathione [GSH], catalase and GSH peroxidase [GPx]). The data clearly indicate that the cells employed a detoxifying strategy against cocaine. On a broader perspective, we envision that extrapolating the knowledge of neuronal resistance to central nervous system (CNS) diseases could delay their onset or progression.

Highlights

The abuse of pharmacological substances in modern society has increased exponentially during the last decade sets off profound psychostimulatory impact on its users.In addition, cocaine exerts toxic effects in differentCNS cells, such as astrocytes, under in vivo and in vitroOfficial journal of the Cell Death Differentiation AssociationBadisa et al Cell Death Discovery (2018)4:76 situations with regards to altering the morphology or cell size[5] or manifesting certain pathology underlying psychological disorders[6]
N2a cells (Supplementary Figures S1, 2), we assessed the effect of cocaine on neurite-connections and cell viability at pharmacologically relevant in vivo doses, which usually range from nano to lower micro molar in drug addicts[13]
Compared to the control, neurite structures were damaged with treatment, causing loss in the inter-neuronal connections (Fig. 2, black arrows in 4 mM); yet, there was no change in cell viability at any treatment (Supplementary Figure S3B; n = 12, P > 0.05)

Summary

Introduction

The abuse of pharmacological substances in modern society has increased exponentially during the last decade sets off profound psychostimulatory impact on its users.In addition, cocaine exerts toxic effects in differentCNS cells, such as astrocytes, under in vivo and in vitroOfficial journal of the Cell Death Differentiation AssociationBadisa et al Cell Death Discovery (2018)4:76 situations with regards to altering the morphology or cell size[5] or manifesting certain pathology underlying psychological disorders[6]. Autophagy[7] and oxidative stress arising from dopamine degradation at the synaptic cleft[8] have been cited as the common causes of death. Cocaine administration at 50–450 mg/kg/day in animal models neither caused cell death nor induced neurodegenerative changes[10,11] which raised doubt on the role of oxidative stress in neuronal death. Consistent with these reports, in vitro studies in mouse neuro-2a (N2a) cells treated with milli molar doses of cocaine did not show cell death (unpublished results). Post-mortem examination of several long-term cocaine users revealed only a 16% neuronal loss in the striatum and mid-brain[12]

Methods

Results

Conclusion