Addiction Therapy 2015 Florida, USA August 03-08, 2015 Ana Lucia Brunialti Godard

“Analysis of the transcriptome in an animal model of alcohol addiction - evidence for a new pathway for the control and maintenance of the phenotype ” Ana Lúcia Brunialti Godard UFMG – ICB – PPG Genética

Cleary a Public Health Issue!!! Alcohol and Society Alcohol addiction associated with chronic excessive alcohol consumption is one of the largest health burdens on society (Cargiulo, 2007). . In Brazil it is estimated that 59% of the population make use of ethanol in an abusive manner (binge), and . Approximately 14% of them are dependents (INPAD, 2012); Cleary a Public Health Issue!!!

Definition Alcoholism, or alcohol addiction, is a complex multifactorial condition and can be defined as: relapsing disorder that is modulated by compulsion to take the drug; a loss of control in limiting intake; and emergence of a negative emotional state when access to the drug is interrupted. (Koob, Volkow, 2010)

Neurobiology of Addiction The Mesolimbic Dopaminergic System is implicated in the reinforcing effects of ethanol (Boileau et al., 2003); a brain circuit involved in associative learning and reward (Gonzales et al., 2004); and the drugs increase synaptic dopamine concentration in this system.

Mesolimbic Dopaminergic System Psychotropic drugs act in specific brain regions and integrative systems which include the amygdala, prefrontal cortex, hippocampus, nucleus and the striatum. The striatum is a brain region of integration among various neurobiological circuits; it plays a central role in reward; and neuro adaptations in this area are linked with the transition of the occasional use to drug habit. (Lee et al., 2012)

Goal

Search for genes that may be related to addict behavior, through global transcription analysis in mice exposed to ethanol; Identify changes in biochemical pathways related to the development or maintenance of ethanol consumption, through molecular analysis of the striatum transcriptome.

Methods

Do we have an animal model of addiction?

Intake Model for assessing loss of control over alcohol ingestion (Fachin-Scheit et al., 2006) Three bottles choice between: H2O, EtOH 5% and 10%.

Animal Model Records made during the animal experiment: Fluid intake was measured volumetrically every two days ; Animals were weighed once a week; The positions of the bottles were changed on alternate days when fluid intake was measured volumetrically ; Behavioral tests were made; Blood Ethanol Concentration (BEC) was determined at the end of the protocol for each animal.

CPF HIPOC ESTR

Results

Pathway Number of genes p value LRRK2 in neurons in Parkinson’s disease 10/33 1.534e-8 Signal transduction PKA signaling 11/51 1.405e-7 Neurophysiological process Glutamate regulation of Dopamine D1A receptor signaling 9/45 3.869e-6 Development Regulation of CDK5 in CNS 5/28 1.033e-3 Development_Delta- and kappa-type opioid receptors signaling via beta-arrestin 9/23 6.183e-9

The Pathways Analysis Lrrk2 Pathway This pathway, previously related with Parkinson’s disease, is centered in the LRRK2 or dardarin kinase; is involved with dendritic spine formation in striatal neurons; and with reuptake of protein synapsis components.

Lrrk2 Pathway After the release of the neurotransmitter in the synaptic gap, the vesicle and its membrane components are endocytosed and transported to the endosome, where it will be reloaded with new neurotransmitter molecules and reused in the next action potential.

Lrrk2 Pathway Considering: The Pathways Analysis Lrrk2 Pathway The dopamine transporter protein (DAT) is one of the proteins that are endocytosed by this pathway. Considering: (1) endocytosis plays a key role in recycling DAT by presynaptic neurons; (2) DAT controls the availability of dopamine in the synaptic gap; (3) and the dopaminergic tone (or availability of dopamine) is directly related to the genesis of the addition.

The Pathways Analysis Lrrk2 Pathway We decided to investigate if this pathway could be involved in addictive behavior evidenced in our animal model; Selected 9 genes of this pathway pre-detected in the microarray strategy and validated each one by Real-Time PCR.

Conclusions Our results suggest for the first time the involvement of the LRRK2 pathway over the uncontrolled behavior seen in the alcohol use disorders; and the overexpression of the Lrrk2 gene, and possibly its protein in the striatal neurons promote an imbalance in those processes; promoting an impairment in the dopaminergic system; potentially contributing for the loss of control over the alcohol intake.

Perspectives - Future functional experiments are needed in order to correlate the RNA alterations with the protein levels; - Efforts should be made to see the direct relationship between the Lrrk2 gene and the alcohol intake behavior through in vivo genetic manipulations like iRNA and transgenic animals.

Collaborators Profa. Dra. Roseli Boerngen de Lacerda Laboratório de Farmacologia Depto. Farmacologia Profa Rosana Camarini Departamento de Farmacologia - USP Laboratório de Genética e Cardiologia Molecular - InCor Prof. Dr. José Eduardo Krieger Profa. Dra. Silvana Chiavegatto Profa. Dra. Iscia Teresinha Lopes Mendes Profa. Dra. Cláudia Vianna Maurer Morelli Vinícius D´Ávila Bitencurt Pascoal Cristiane Rocha Laboratório de RNAi - Depto. Genética Médica Laboratório Nacional de Luz Síncrotron Dra. Maria Eugênia Ribeiro de Camargo

Thank you! Laboratório de Genética Animal e Humana Profa. Dra. Ana Lúcia Brunialti Godard Thank you!

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