Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Ma, J; Wan, J; Meng, J; Banerjee, S; Ramakrishnan, S; Roy, S

METH is an illicit recreational drug known to have effects on multiple organ systems. Exposure to METH leads to neurotoxic effects, which include damage to dopaminergic and serotonergic terminals, dopamine release, endoplasmic reticulum stress, activation of mitochondrial death cascades, glutamate release and hyperthermia. 28 In an attempt to better clarify the molecular and cellular mechanisms responsible for neurotoxicity, most studies have focused on oxidative stress, activation of transcription factors, DNA damage, excitotoxicity, microglial activation and various apoptotic pathways. 29, 30 More recent studies suggested that METH-induced neurotoxicity may also result from its ability to induce impairment of BBB function. The BBB has a predominant role in actively transporting nutrients to the brain and filtering harmful compounds from the brain back to the bloodstream. This process guarantees the homeostasis of the brain microenvironment, necessary for neuronal activity and proper functioning of the CNS. 31 METH induces a transient increase in the permeability of the BBB in the hippocampus comparing with the frontal cortex and striatum, which can be explained by alterations in the tight junction proteins and matrix metalloproteinase-9 (MMP-9). 32 However, the direct effects of low-dose METH in modulating BBB breakdown are not fully understood. human neuroblastoma SH-SY5Y) resulted in induction of autophagy and suppression of autophagy precipitated neuronal cell death. 1 In addition, another study showed that METH triggers an autophagic sequestering process in the dopaminergic SK-N-SH cell line by inducing the expression of LC3-II in a dose-dependent manner. 33 However, no studies to date have investigated the effects of METH at doses seen in METH addicts in BBB endothelial cells. We therefore investigated the functional role of autophagy in low-dose METH on endothelial cells. LC3 and Beclin1 are two key proteins for autophagy function. In this study, we first demonstrate that METH induced higher levels of LC3-II. LC3-II formation is regarded as a reliable biochemical evidence of autophagy as the amount of LC3-II usually correlates well with the number of autophagosomes. 34 In our study, Beclin1 expression was also increased in a time-dependent manner. Beclin1 has a role in the initiation step and is essential for the formation of the autophagosome. The anti-apoptotic protein Bcl-2 has a negative regulatory role in autophagy by blocking an essential protein in the signaling pathway, Beclin1. 15 METH induces autophagy in the human neuroblastoma SK-N-SH cell line by inhibiting the dissociation of Bcl-2/Beclin1 complex and its upstream pathway that leads to cell death. 35 Our data revealed that METH downregulated Bcl-2, which released Beclin1 from its asscociation with Bcl-2 thereby promoting autophagy ( Figure 6 ). Beclin1 interacts with Bcl-2, suggesting cross-talk between two potential programmed cell death pathways. It has been reported that activation of MEK/ ERK downstream of AMPK leads to an increase in Beclin1 expression. 36 To examine if the activation of the ERK pathway is directly related to the increase of Beclin1, we found that MEK1 inhibitor PD98059 prevented METH-induced Beclin1 mRNA and protein expressions in the endothelial cells (Figures 4e and f) .