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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
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
(Lee et al., 2012)
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.
Goal
•
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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
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•
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.
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 predetected in the microarray strategy and
validated each one by Real-Time PCR.
Conclusions
-
-
-
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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
Laboratório de Genética Animal e Humana
Profa. Dra. Ana Lúcia Brunialti Godard
Thank you!
Meet the eminent gathering once again at
Addiction Therapy 2016
Miami, USA
October 06-08, 2016
Addiction Therapy 2016
Website: addictiontherapy.conferenceseries.com