Epigenetic Effects Induced by Methamphetamine and Methamphetamine-Dependent Oxidative Stress

Abstract

Methamphetamine is a widely abused drug, which possesses neurotoxic activity and powerful addictive effects. Understanding methamphetamine toxicity is key beyond the field of drug abuse since it allows getting an insight into the molecular mechanisms which operate in a variety of neuropsychiatric disorders. In fact, key alterations produced by methamphetamine involve dopamine neurotransmission in a way, which is reminiscent of spontaneous neurodegeneration and psychiatric schizophrenia. Thus, understanding the molecular mechanisms operated by methamphetamine represents a wide window to understand both the addicted brain and a variety of neuropsychiatric disorders. This overlapping, which is already present when looking at the molecular and cellular events promoted immediately after methamphetamine intake, becomes impressive when plastic changes induced in the brain of methamphetamine-addicted patients are considered. Thus, the present manuscript is an attempt to encompass all the molecular events starting at the presynaptic dopamine terminals to reach the nucleus of postsynaptic neurons to explain how specific neurotransmitters and signaling cascades produce persistent genetic modifications, which shift neuronal phenotype and induce behavioral alterations. A special emphasis is posed on disclosing those early and delayed molecular events, which translate an altered neurotransmitter function into epigenetic events, which are derived from the translation of postsynaptic noncanonical signaling into altered gene regulation. All epigenetic effects are considered in light of their persistent changes induced in the postsynaptic neurons including sensitization and desensitization, priming, and shift of neuronal phenotype.

Figure 1

The effects of METH on DA-storing vesicles. METH enters into DA terminals either through the plasma membrane DAT or via passive diffusion. Within the axoplasm, it targets DA-storing vesicles to (1) disrupt their proton gradient, (2) inhibit and revert VMAT-2, and (3) displace VMAT-2 elsewhere (i.e., trans-Golgi network). These effects disrupt the physiological storage of DA, which diffuses from vesicles to the axoplasm and from the axoplasm to the extracellular space.

Figure 2

The effects of METH on DAT. METH impairs DAT activity either via direct inhibition or via reverting its direction. Such an effect potentiates the accumulation of freely diffusible DA in the extracellular space and prevents the main mechanisms of DA removal (reuptake within DA terminals).

Figure 3

The effects of METH on mitochondria. METH impairs the activity of complex II of the mitochondrial respiratory chain and directly inhibits MAO-A placed on the outer mitochondrial membrane within DA terminals. METH also inhibits MAO-B placed extracellularly at the level of glia. However, the affinity of METH for MAO-B is tenfold less when compared with MAO-A. Thus, MAO-B inhibition does not influence that much the amount of extracellular DA.

Figure 4

The effects of METH-induced MAO-A inhibition on DA metabolism. The loss of physiological DA deamination following MAO-A inhibition and its uncoupling with AD lead to the generation of highly reactive species including DOPALD (1), hydrogen peroxide (H₂O₂), and DA quinones (2).

Figure 5

METH induces oxidative stress within DA terminals. Toxic DA by-products (quinones and DOPALD) together with highly reactive species such as H₂O₂ and reactive oxygen species (ROS) react with sulfhydryl groups and promote structural modifications of proteins within the DA axon terminals. The enhanced redox imbalance also disrupts the homeostasis of endoplasmic reticulum (ER) and mitochondria, which further accelerates the production of ROS. Thus, an excessive amount of misfolded/insoluble proteins and damaged organelles occurs, which leads to an engulfment of autophagy (ATG) and ubiquitin proteasome (UP) cell-clearing systems. These events converge in producing neurotoxicity within DA terminals, which may either extend to DA cell bodies.

Figure 6

The effects of extracellular DA released following METH. Extracellular DA and DA-derived reactive species diffuse at considerable distance towards nonneuronal targets including the neurovascular unit (blood-brain barrier (BBB) and Glia), which is affected by METH (1). At short distance, METH produces an abnormal stimulation of postsynaptic neurons, mainly striatal MSNs. The pulsatile pattern of DA stimulation produces an abnormal pulsatile activation of postsynaptic DA D1 receptors (DRD1) (2). This leads to a series of noncanonical metabolic changes, which translate into activation of glutamate (GLUT) receptors N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (NMDAr and AMPAr, resp.) (3) potentiation of GLUT release and Ca2+ entry within postsynaptic neurons (4). This event triggers an enzymatic cascade further increasing reactive oxygen species (ROS) and nitrogen species (RNS) (5). Freely diffusible DA-derived free radicals together with GLUT-derived radical species synergize to produce detrimental effects on postsynaptic non-DA neurons. These consist in DNA instability, due to oxidative damage (fragmentation and/strand breaks) and alterations in gene expression (A), mitochondrial stress (B), and oxidation of organic substrates, mainly proteins, which are prone to misfold and produce insoluble aggregates leading to an impairment of cell-clearing systems (C).

Figure 7

An overview of canonical DA receptor signaling. During physiologic DA stimulation, AC activity is balanced by the excitatory and inhibitory effects of DRD1 and DRD2, respectively. Thus, there is a physiologic downregulation of cAMP and PKA activation. PKA has a broad array of targets such as the DA- and cAMP-regulated phosphoprotein (DARPP-32), voltage-gated ion channels, and GLUT receptors. PKA phosphorylates DARPP-32 at Thr34 (P-T34), but other proteins, such as cyclin-dependent kinase 5 (CDK5), counterbalance such an effect by phosphorylating DARPP-32 at a different site (P-T75). Thus, DARPP-32 can activate phosphatase protein 1 (PP1), which can surveil phosphorylation levels of all PKA targets. Likewise, canonical stimulation of DRD2, which are coupled with PLC, generates normal levels of inositol 1,4,5 trisphosphate (IP3) which induces Ca2+ release from the endoplasmic reticulum. Since ion channels and GLUT receptors are properly functioning, intracellular Ca2+ can be efficiently mobilized.

Figure 8

METH-induced noncanonical DRD1 signaling. Following METH, MSNs become supersensitive to pulsatile DA stimulation despite that the number of DA receptors is not increased. As a result, DRD1 move towards noncanonical signaling and the activity of DRD2 is enhanced. In these conditions, DRD1 overactivates AC, which enhances the production of cAMP and leads to abnormal activation of PKA. DRD1/PKA cascade turns out to increase the amount of DARPP-32 phosphorylated at Thr34, which inhibits PP1. Thus, all PKA targets, including voltage-gated ion channels and GLUT NMDAr and AMPAr, are abnormally phosphorylated and activated. In addition, DRD1/PKA leads to increased levels of MAPK and ERK1/2, which in turn phosphorylate several cytosolic and nuclear substrates. At the same time, DRD2-enhanced activity potentiates the increase of intracellular Ca2+ release, which cannot be properly mobilized, since ion channels and GLUT receptors are abnormally activated and potentiate the influx of Ca2+ within postsynaptic neurons. Such an event also promotes the activation of calmodulin-dependent kinase II (CaMKII), which can translocate into the nucleus to regulate gene expression.

Figure 9

The effects of DRD1/PKA pathway on CDK5 and DARPP-32. In physiologic conditions, CDK5 phosphorylates DARPP-32 at Th75, thus softening the effects of PKA on DARPP-32. However, the abnormal phosphorylation of Thr34 carried out by enhanced DRD1/PKA cannot be counterbalanced by CDK5. This occurs since DRD1/PKA activates phosphatase PP2A, which inhibits the effects of CDK5 and enhances those of PKA. As a result, DARPP-32 phosphorylated at Thr34 increases and potentiates the inhibition of PP1.

Figure 10

The nuclear effects of DRD1/PKA pathway and reactive species on postsynaptic neurons. The noncanonical DRD1 activation induced by METH produces an overactivation of several kinases, such as ERK1/2, DARPP-32-pT34, CREB, CDK5, and CaMKII. The latter, together with DA- and GLUT-derived reactive species, is shuttled into the nuclear compartment where they carry posttranslational modifications of histones and TFs. These events promote both a relaxation of chromatin structure (yielded by an increase of histone acetyltransferases (HAT)/decrease of histone deacetylases (HDAC)) and increased binding of TFs (such as Elk-1, AP-1, and CREB) at the level of their target gene sequences. These metabolic events eventually translate into an increase expression of IEGs.

Figure 11

Summarizing main epigenetic mechanisms. This cartoon roughly reports the main epigenetic enzymes carrying structural modifications of lysine (K) residues of histone tails and DNA promoter sequences at the level of CpG islands. HATs act by adding acetyl groups (Ac) which associates with increased gene expression; HDACs repress gene expression by removing Ac from K histone residues; KMT transfer methyl (Me) groups and KDMT remove Me groups from K histone residues; the effects of KMTs and KDMT on gene transcription depend on the specific histone K that is modified; DNMTs mediate increased methylation of cytosine (C) residues in CpG islands of gene promoters, which associates with repressed gene expression.

Figure 12

METH-induced gene desensitization. Exposure to chronic METH produces epigenetic effects, which repress further gene expression. This occurs mainly through increased activity of deacetylation enzymes (HDAC), increased methylation of lysine 9 and 27 (K9/K27) residues of histones (i.e., H3K9/27) by methyltransferases (KMTs) and hypermethylation of gene promoters by DNA methyltransferases (DNMTs), which produce a “closed chromatin” conformation. Me: methyl groups; Ac: acetyl groups.

Figure 13

METH-induced gene priming and sensitization. A single dose of METH may be sufficient to induce an epigenetic switch consisting in increased gene expression. Such an effect may also occur during chronic METH resulting in long-term sensitization. This occurs through increased histone acetylation and methylation at specific K residues (i.e., H3K4) joined with poor activity of DNMTs (hypomethylation of CpGs), which altogether produce an “open” chromatin conformation and allow the binding of TFs at the level of gene promoters.