Psychostimulant induce neuronal plasticity by cytoskeleton remodeling via cdc42 pathway

The development of addiction is known to depend on synaptic and morphological adaptations in the brain. The hippocampus displays high levels of synaptic plasticity, with a key role in maladaptive drug-context associations,related to drug use cessation and relapse. Previous works addressed the effects of psychostimulants in synaptic remodeling in the hippocampus, but how these mechanisms are regulated is largely unknown. Methamphetamine (Meth),a highly neurotoxic psychostimulant, leads to long-term neuronal dysfunction.Acutely, Meth increases neurite growth and dendritic spine density in several brain regions, which is likely associated to RhoGTPases (e.g. rhoA, rac1, cdc42), key regulators of actin cytoskeleton dynamics. Cdc42 and rac1 promote neurite growth and the formation/maintenance of dendritic spines, whereas rhoA negatively regulate these mechanisms. Here, we hypothesized that modulation of RhoGTPases, particularly cdc42, may impact Meth-induced morphological changes in hippocampal neurons and play a role in drug related behavior. To test this hypothesis, primary hippocampal neuronal cultures were exposed to Meth for 24h, showing increased neurite growth and dendritic spine density, concomitant with decreased spine head size, potentially affecting synapse number and function.Using specific Förster resonance energy transfer (FRET) reporter probes, we specifically determined that cdc42 is activated at dendritic spines in hippocampal neurons following Meth exposure. When neurons were manipulated to express an inactive form of cdc42, Meth-induced effects in neurite outgrowth and dendritic spines were prevented. Moreover, Meth induced an increase in the expression of genes and proteins that regulate cdc42 activity, particularly intersectin1, a cdc42 specific activator. We explored the role of intersectin1 in METH-induced effects in hippocampal neurons, using an intersectin1/cdc42 interaction inhibitor (ZCL278) and a siRNA silencing approach to reduce intersectin 1 expression. Both approaches prevented METH effects at the neurite outgrowth and dendritic spine level. Consequently, we explored the involvement of the signaling pathway Intersectin1/cdc42/N-WASP/Arp2/3, which regulates actin cytoskeleton. To confirm the relevance of our findings in vivo, we exposed wildtype mice to a binge Meth regimen (5 mg/kg intraperitoneal administration x4,at 2h intervals),in the presence or absence of the inhibitor ZCL. Meth-induced behavioral effects were analyzed 24 hours after the first Meth administration using the elevated plus maze (EPM) test (we have previously verified that under this Meth regimen risk evaluation was compromised). Immediately after the test, mice were perfused and neuronal morphology and molecular alterations were analyzed. For that, 21 days before Meth administration, mice were intravenously injected with adenovirus (AAV9) to allow GFP expression specifically in neurons. Collectively, our data shows a relevant role for cdc42 and its regulators in the context of addiction. We believe that our work provides cutting edge advances regarding neuronal drug effects, which will be of major interest to understand the addictive process to psychostimulants, and potentiate the development of novel therapies for this complex pathology.