Download Addictions, genomics and proteomics

Salud Mental 2012;35:129-137
Addictions, genomics and proteomics
Addictions, genomics and proteomics
Maura Epifanía Matus Ortega,1 Juan Carlos Calva Nieves,1 Anabel Flores Zamora,1
Philippe Leff Gelman,2 Benito Antón Palma1
Original artícle
SUMMARY
Drug addiction is a chronically relapsing disorder that has been characterized by (1) compulsion to seek and take the drug, (2) loss of
control in limiting intake, and (3) emergence of a negative emotional
state (e.g, dysphoria, anxiety, irritability) reflecting a motivational
withdrawal syndrome when access to the drug is prevented (defined
as Substance Dependence by the Diagnostic and Statistical Manual
of Mental Disorders [DSM] of the American Psychiatric Association).
Acute exposure to drugs of abuse initiates molecular and cellular alterations in the Central Nervous System that lead to an increased overall
vulnerability to addiction with subsequent drug exposures. These druginduced alterations employ changes in gene transcription that result
in the synthesis of new proteins. Therefore, one of the important goals
of addiction research is to identify the drug-induced gene expression
changes in the specific brain structures related to the addictive properties of various drugs.
The molecular and genomic mechanisms by which drugs of
abuse induce neuroplastic changes related to addiction remain largely unknown. Several studies have evaluated changes in gene and protein expression profiles in the brain after administration of drugs of
abuse. Exposure to psychostimulants induces the activity-dependent
gene expression of several transcription activators and repressors.
Genomic research strategies have recently transitioned from the
search for unknown genes to the identification and evaluation of coordinated gene networks and transcriptional signatures. New opportunities arising from the analysis of these networks include identifying
novel relationships between genes and signaling pathways, connecting biological processes with the regulation of gene transcription, and
associating genes and gene expression with diseases. The identification of gene networks requires large gene expression data sets with
multiple data points.
Functional genomics methods, studying the steady-state levels of
these mRNA species, such as quantitative RT-PCR (qRT-PCR), whole-genome microarray analysis, and next generation sequencing methods,
provide sensitive and high-throughput approaches to quantitatively
examining mRNA (and miRNA) species present within the cells of the
Nervous System.
Functional genomics studies can help to illuminate genes involved in the development of behaviors related to drug abuse and
relapse liability, but cannot provide insight into post-translational
modifications (e.g., phosphorylation and glycosylation of proteins after translation has occurred) or subcellular localization of the protein
product. Therefore, using proteomic techniques presents the opportunity to assess the totality of gene expression, translation, modification,
and localization. Unfortunately, the sensitivity of proteomic tools lags
behind those of functional genomics. Moreover, examining the mRNA
provides a restricted view of primarily the cell body. Indeed, from a
systems biology standpoint, analysis of both mRNA and protein levels
(as well as miRNA and epigenetic changes) will ultimately provide a
more integrated view of the molecular underpinnings of addiction.
When applying proteomic technologies to addiction research,
an understanding of the power of proteomic analysis is essential. After
genetic information is transcribed into mRNA, a template is provided
to the cell from which proteins will be synthesized.
Neuroproteomic studies offer great promise for increasing understanding of the biochemical basis of addiction. While proteomics
is still an evolving field, proteomic approaches have proven useful for
elucidating the molecular effects of several drugs of abuse. With a
number of ongoing research programs in addiction proteomics and
a growing number of investigators taking advantage of these tools,
the addiction research field will benefit from a consideration of the
capabilities and limitations of proteomic studies.
As with other biomedical research fields, drug abuse research is
making use of new proteomic capabilities to examine changes in protein expression and modification on a large scale. To obtain the maximum benefit and scientific advancement from these new technologies,
a clear understanding of the power and limitations of neuroproteomics
is necessary. With the main limitation of neuroproteomic studies being
the complexity of the proteome, approaches that focus these studies
need to be employed. The salient message is that there is not a single
best technical approach for all studies and that the main driver for the
choice of proteomic technology and experimental design should be
the advancement of the understanding and treatment of drug abuse.
An important area that has heretofore received limited attention is the experimental design and interpretation specific to neuroproteomic studies of drug abuse. These challenges include choice of
animal model, ensuring sample quality, the complexity of brain tissue,
confirming discovery findings, data analysis strategies, and integration of large data sets with the existing literature.
Epigenetics is the study of heritable changes other than those
in the DNA sequence and encompasses two major modifications of
DNA or chromatin: DNA methylation and post-translational modification of histones. In this context, now it is known that regulation of gene
expression contribute to the long-term adaptations underlying the effects of drugs of abuse. The precise molecular events that are required
Laboratory of Molecular Neurobiology and Neurochemistry of Addictions. Department of Clinical Research. Ramón de La Fuente Muñiz National Institute of
Psychiatry.
2
Department of Biomedical Research. Isidro Espinosa de los Reyes National Institute of Perinatology.
1
Correspondence: Benito Antón Palma. Laboratorio de Neurobiología Molecular y Neuroquímica de Adicciones, Instituto Nacional de Psiquiatría Ramón de la
Fuente Muñiz, Calz. México-Xochimilco 101, San Lorenzo Huipulco, Tlalpan, 14370, México, DF. E-mail: [email protected]
Received: July 28, 2011. Accepted: Noviembre 28, 2011.
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for modification of chromatin and that underlie gene repression or
activation have not been elucidated.
Recent reports have addressed this question and demonstrated
that drugs of abuse modify specific methyl-CpG-binding proteins that
control histone acetylation and gene expression. Further elucidation of
the wide-range of histone modifications and the ensuing consequences
on gene expression will be necessarily before the potential for drug
development can be realized. It is important to characterize the molecular alterations underlying chromatin remodeling and the regulation
of the epigenetics events by drugs of abuse. It is clear that modification
in gene expression by drugs of abuse promote cellular changes.
This review is intended to provide guidance on recent advances
in the field of drug addiction. This review also presents a number of
experimental design and sample approaches that have been applied
to genomic, proteomic and epigenetic studies of addiction. Coupled
with new technologies for data collection, analysis, and reporting,
these approaches represent the future of the addiction field and hold
the key to unlocking the complex of profile of drug abuse disorders.
Key words: Drug abuse, gene transcription, protein expression,
epigenetics.
RESUMEN
La adicción a las drogas es una enfermedad mental que se caracteriza
por ocasionar graves implicaciones sociales, económicas y de salud
de los individuos que la padecen. La exposición aguda a las drogas
de abuso provoca alteraciones moleculares y celulares en el Sistema
Nervioso Central que ocasionan una vulnerabilidad para sufrir adicción a subsecuentes exposiciones a sustancias de abuso diferentes. Las
alteraciones inducidas por las drogas producen cambios en la transcripción de genes que resultan en la síntesis de nuevas proteínas. Uno
de los objetivos importantes en la investigación en el campo de las
adicciones es identificar los cambios en la expresión de genes inducidos por las drogas en estructuras específicas del cerebro que están
relacionadas con las propiedades adictivas de diferentes sustancias.
El campo de la genómica y la proteómica, aplicada al estudio
de las adicciones, tiene como objetivo identificar a los genes y las proteínas candidatos involucrados en la regulación de los procesos adictivos. Se han logrado progresos considerables en la identificación de
genes y proteínas que regulan las conductas complejas presentes en
los procesos adictivos en modelos de animales y modelos de estudio
GENIC DISSECTION
OF COMPLEX BEHAVIORS
The field of genomics and proteomics, applied to the study
of psychiatric disorders, seeks to identify the potential genes
and proteins involved in regulating the processes of reinforcement and motivation and the cognitive functions of certain human behaviors. Genes and their respective proteins
have been identified as being involved in these behaviors.
For example, the role of specific receptors of neurotransmitters has been studied (such as dopamine, glutamate and serotonin), as well as the proteins participating in intracellular
signaling (kinase and arrestin proteins) and the transcription factors (such as CREB and cFosB proteins).1-3
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en humanos con material obtenido post-mortem. Estos descubrimientos se han sumado a los esfuerzos por identificar los circuitos neurales
implicados en las manifestaciones conductuales relacionadas con las
adicciones. También han permitido la identificación de genes candidatos que podrán ser blancos de futuras estrategias terapéuticas
desarrolladas para tratar los procesos adictivos.
Los estudios de genómica funcional han permitido identificar algunos de los genes involucrados en el desarrollo de las conductas
adictivas, pero no tienen la capacidad de proporcionar información
sobre las modificaciones post-traduccionales ni de la localización subcelular de las proteínas para las que codifican los genes. Por lo tanto,
la incorporación de estudios proteómicos ofrece la oportunidad de
lograr evaluar, en su totalidad, la expresión, la traducción, las modificaciones y la localización de los genes y sus productos de expresión.
Para obtener los máximos beneficios y avances con el empleo
de estas nuevas tecnologías, deben comprenderse en su totalidad los
alcances y limitaciones de la neuroproteómica. En este sentido, se
debe tener especial cuidado en la elección del modelo de estudio,
asegurar la calidad de la muestra, la complejidad de la estructura en
estudio, confirmar los resultados obtenidos, las estrategias de análisis
de resultados y la integración de los datos obtenidos con los ya reportados en la literatura científica.
Los estudios recientes sobre los mecanismos moleculares que
controlan los cambios inducidos por las drogas de abuso sobre la
función transcipcional, la conducta y la plasticidad sináptica han
identificado el importante papel que desempeña la remodelación de
cromatina en la regulación y estabilidad de los programas genéticos
neuronales mediados por las drogas y la subsecuente manifestación
de las conductas adictivas.
Se han identificado alteraciones epigenéticas sobre el genoma,
tales como metilación del DNA y modificaciones en la función de las
proteínas histonas. Estos importantes mecanismos se ven afectados
como una respuesta neurobiológica a la administración de sustancias
de abuso.
Esta revisión pretende mostrar algunos de los avances recientes
en el campo de las adicciones, presentando una breve descripción de
los hallazgos que emplean aproximaciones genómicas, proteómicas
y epigenéticas. Las implicaciones de estos estudios moleculares ponen de manifiesto nuevos conocimientos sobre el probable desarrollo
de intervenciones terapéuticas en el futuro.
Palabras clave: Abuso de drogas, transcripción genética, expresión de proteínas, epigenética.
In this review we will analyze representative examples
of current studies in this field, detailing the results obtained
by different researchers and the functional implications of
their findings.
GENOMIC ANALYSES
Lehrmann et al.4 conducted a microarray analysis to study
the gene expression in the aPFC from tissues obtained postmortem from 42 humans with a history of cocaine, marijuana
and/or phencyclidine abuse, comparing these to 30 control
subjects, in order to identify transcriptional modifications.
In the study, 39 transcripts were selected from genes whose
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Addictions, genomics and proteomics
change was highly significant. For analysis, they were divided according to their cellular function and location (Table 1).
Black et al.,5 however, evaluate the effect of chronic
administration of cocaine in an adolescent rodent population, to identify the effects caused by addictive substances
when ingested at early ages and their relationship with
neural plasticity. These authors identified groups of genes
which they classified based on their cellular functions. Of
the 56 genes whose expression decreased significantly, 34
transcripts were identified whose function is understood:
10 transcription factors and proteins that encode nuclear receptors; three transcripts that encode proteins related to the
functions of the cytoskeleton; while the remaining 22 transcripts encode proteins whose function is not understood.
145 transcripts of genes were identified whose expression
Table 1. Groups of genes altered by the administration of drugs of
abuse identified in the study by Lehrmann et al.4 The genes whose
expression was modified significantly were classified according to
their cellular function and location
Cellular Function/
Location
Proteín
Transcription calmodulin •Genes regulated by calcium-calmodulin
signaling
•Protein phosphatase of biosynthesis of
cyclic AMP and adenylate cyclase activity
Endoplasmic reticulum
and Golgi apparatus
•Genes involved in functions of the endoplasmic reticulum
•Genes involved in functions of the Golgi
apparatus
•Adaptin proteins and adaptin kinases
•Proteins involved in endocytosis of clathrin-coated vesicles in the plasma of the
cell membrane
•Proteins involved in the transport between
the Golgi apparatus and the endosomal
system
•Lysosomal proteins
•Proteins of vesicular trafficking in the synapsis
•Proteins involved in the transport of particles from the late endosoma to the lysosomes
•Marker protein of membrane microdomains
Metabolism of lipids
and cholesterol
•Proteins involved in biosynthesis and
transport of cholesterol
•Proteins involved in biosynthesis, metabolism and modifications of lipids
Other cellular
functions
•Proteins involved in signal transduction
processes
•Cytoskeleton proteins
•Proteins involved in ubiquitination processes
•Nuclear receptors
•Transcriptional regulation proteins
•Mitochondrial proteins: transporters,
energy metabolism, ribosomes
•Proteins with unknown function (31)
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increased significantly. Of these, 92 could be classified into
groups whose function is understood, 17 of which are for
proteins involved in functions of the extracellular matrix
and adhesion cells, and three of which encode proteins that
are related to the motor activity of the microfilaments. The
rest of the transcripts encode proteins whose function is not
yet understood (53 transcripts) (Table 2).
Hemby6 published a detailed review on the studies developed by several research groups to evaluate the effects of
the administration of cocaine on the coordinated expression
of genes from the brain regions involved in the mesocorticolimbic pathways, including the nucleus accumbens (NAc), the
prefrontal cortex, the hippocampus, the lateral hypothalamus
and the ventral tegmental area (VTA), in rodent models.
The studies showed that the administration of cocaine
induces changes in the expression of genes and proteins involved in the processes of neuroplasticity, particularly in
areas like the cortex and the hippocampus, as well as those
related to behavioral processes which involve the VTA, the
NAc, and the proteins involved in the regulatory function of
proliferation progenitor cells in the dentate gyrus.
The up-regulated transcripts include early genes, those
that encode effector and scaffold proteins, receptors, signaltransducing proteins, proteins associated with circadian
rhythms, transcriptional and translational proteins, signal
transduction of metabolic enzymes, and cytoskeletal proteins, while the down-regulated genes primarily comprise
transcripts related to mitochondrial function together with
transcripts encoding signal-transducing proteins. Likewise,
administration of cocaine induces changes in the gene expression of proteins involved in long-term potentiation, expressed in the hippocampus.
NEUROPROTEOMICS
To understand the intricate neuroadaptive machinery that
participates in addictive processes, gene expression analysis must be accompanied by studies that evaluate the corresponding proteomes.
With the advent of protein separation and analysis
strategies, based on high-performance mass spectrometry,
it is possible to encompass entire proteomes, allowing us to
delineate a multitude of neurobiological effects produced by
the consumption of drugs. Recent studies have used animal
models and samples of human tissues obtained postmortem, with emerging technologies to analyze protein expression and evaluate the changes produced in specific regions
of the brain as a result of chronic consumption of cocaine.6
THE PROTEOMIC STUDY
In a proteomic experiment, once must consider and optimize
multiple variables in each of the experimental stages: selec-
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Table 2. Groups of genes altered in the medial prefrontal cortex 22 hours after the last administration of cocaine, identified in the study by
Black et al. The groups were classified according to the modification of their expression
Down-regulated genes
•Transcription
•Cytoskeleton
EGR 1
TGFB-inducible EGR
Gene homolog to Notch-2
Nuclear receptor of subfamily 4, group A, member 2
Zinc finger and BTB 10 domain
Similar to BAF53a
Huntingtin-associated protein 1
EGR 2
homer 1
SMAD 5
Translocator similar to aryl-hydrocarbon
nuclear
Jumonji domain 1A
Protein 3 related to ARP3 actin
Up-regulated genes
•Extracellular matrix
and adhesion cells
•Microfilament motor activity
ADAM 17
CD36 antigen-like 2 (collagen receptor type I)
Dermatopontin
Glycoprotein nmb
Matrilin 2
Procollagen, typo IX, alpha 2
Sperm adhesion molecule
Transmembrane protein 8
Myosin, heavy 3
Tubulin, beta 3
Aggrecan 1
Protein rich in cysteine 61
Elastin
Integrin, alpha 6
Neurexin 3
External rod segment membrane protein 1
Tenascin XA
tion of the behavioral model, obtaining and preparation of
the sample, gathering and interpretation of data, with the
objective of establishing which are more significant and reliable.7 Figure 1 describes each of the main steps and the most
important variables.
PROTEOMIC PROFILE
The different research groups have used a wide variety of
proteomic techniques to analyze the proteome of the synapse and study the effects of drugs on the nervous system.
These methodologies are described in detail in the review
by Abul-Husn and Devi8 (Table 3).
THE PROTEOME OF THE SYNAPSE
The application of new proteomic techniques in neuroscience research has offered us a new understanding of the
structure and function of the nervous system. Subcellular
fractionation techniques have been essential for successful
proteomic analyses. Several groups of researchers have begun to identify the composition of the proteins in the synapse, with particular emphasis on the postsynaptic densities.
A lesser number of studies have focused on analyzing other
synaptic behaviors in mammals, such as synaptosomes,
synaptic membranes and synaptic and presynaptic vesicles.
In the review by Abul-Husn and Devi8 these studies are described (Table 4).
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Myosin heavy 7
NEUROPROTEOMICS AND DRUGS
In the field of drugs of abuse, these proteomic approaches
are useful in generating an objective and global vision of the
changes induced on a specific proteome after drug use. In
addition, it is possible to compare and characterize the effect
of the drugs, to better understand its mechanisms of action.
Given the hypothesis that drug abuse produces persistent and significant changes in the synapse, effects that
persist over the long term, several researchers have studied
the role of synaptic proteins in the addictive processes using
subcellular proteomic methodologies.
Prokai et al.9 reported that among the postsynaptic proteins whose expression was modified, they identified some
related to cellular adhesion, synaptic vesicular trafficking,
and endocytosis. The authors suggest that these findings
implicate changes in the synaptic structure induced by morphine, in addition to alterations in the release of neurotransmitters and/or in the trafficking of receptors.
In a different study, Lin et al.10 analyzed the modification
of the expression of proteins, based on total brain regions,
after chronic administration of morphine. The results of this
study show a modification in the expression of just three
synaptic proteins (synapsin, Lin-7, and beta-synuclein). The
fact so few synaptic proteins were identified suggests that
the proteins expressed in low abundance are lost in using
total brain regions.10 In a similar study,11,12 on the effect of
morphine and butorphanol (a mixture of opioid agonist and
antagonist) in rodents, significant changes were identified
in the expression of cytoskeleton proteins (tubulin and actin
isoforms), which suggests changes in neuronal morphology
and/or axonal transport. In addition, the expression of some
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Addictions, genomics and proteomics
Results of the behavior
Administration of the drug,
method and contingency
Abstinence from the drug
BEHAVIORAL MODEL
Consistent collection methods
OBTAINING
AND
STORING OF SAMPLE
Anatomical separation
Subcellular fractionation
Biochemical fractionation
Fresh and frozen tissue
Integrity and stabilization
of the sample
PREPARATION
OF THE SAMPLE
Identification of proteins
Confirmation
GATHERING
OF RESULTS
Statistical analysis
Ontogenic and pathway analysis
Compiling of data and biological
systems
Public databases
Confirmation based
on antibodies
Monitoring
of multiple reactions
INTERPRETATION
OF RESULTS
Figure 1. Experimental steps to be considered during the development of proteomic studies, given their direct influence on
the results obtained. Table modified from Lull et al.7
tyrosine phosphorylation proteins was modified, including
the proteins Gi and Go. This result suggests that there are
changes in the modulation of the transduction of signals
from opioid receptors during drug dependency.
FUNCTIONAL IMPLICATIONS
The findings described briefly in this review coincide in
their results. Modifications were identified in the expression
of transcripts that encode proteins related to calcium signaling. These results confirm prior observations of a decrease
in the transcripts of proteins involved in calcium signaling,
via cAMP, and in the expression of the transcript of adenylate cyclase I in the motor and frontal cortexes of alcoholic
patients.13,14
The administration of drugs of abuse induces the expression of calmodulin, a protein that modulates the activity
Vol. 35, No. 2, March-April 2012
and sensitivity of calcium-dependent signaling molecules,
thus affecting synaptic plasticity, memory, and the stabilization of the dendritic architecture.
The increase in the expression of most of the transcripts
that encode proteins relating to the metabolism of lipids
and cholesterol suggests changes in neuronal functioning,
in plasticity, and in the myelination of the CNS due to the
administration of drugs of abuse.15-17
The modifications in the expression of transcripts that
encode proteins relating to intracellular trafficking and
cell organelles, the Golgi apparatus, and the endoplasmic
reticulum, suggest effects on the development of neuronal
growth cones, axonal positioning, and modulation of the
dendrites of the apical cortex, growth and maturation of
dendritic spines, which ultimately imply consequences in
the synaptic transmission of the adult brain. The decrease
in the expression of transcripts related to vesicular synaptic
trafficking, clathrin-independent endocytosis and the trans-
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Table 3. Methodologies used in the neuroproteomic studies conducted by different research groups on the effect of drugs of abuse on the
synaptic proteome. Table modified from Abul-Husn and Devi.8
Technique
Uses
Limitations
2-DE (IEF/SDS-PAGE)
Separation of soluble proteins
Detection of post-translational modifications
and protein isoforms.
DIGE
16-BAC/SDS-PAGE
More quantitative than 2-DE.
Superior separation of membrane proteins, as
compared to 2-DE.
Identification of soluble and membrane proteins.
Quantitative analysis of soluble and membrane
proteins.
Poor detection of proteins with extreme pl or molecular
weight, of low abundance, or hydrophobic.
Quantification requires consistency and multiple replications due to the variability between each gel.
Only detects proteins that are sensitive to 2-DE.
Quantification requires consistency and multiple replications due to the variability between each gel.
Not quantitative, limited information on post-translational
modifications.
Detects only tryptic peptides that contain cysteine; limited
information on post-translation modifications.
Proteomic Profile
MudPIT
ICAT
Identification of proteins
MALDI-TOF MS
MS in tandem
Quickly obtains ‘fingerprint’ of the load-mass
of the peptides.
Ambiguous identification of proteins using a ‘peptide sequence tag,’ superior analysis of complex
mixtures of proteins.
Hinders analysis of complex mixtures of proteins.
Requires long amounts of time for sample analysis, interpretation of data, and identification of proteins.
Abbreviations: 2-DE: Two-dimensional gel electrophoresis; IEF: IsoElectric Focusing; SDS: Sodium Dodecyl Sulfate; PAGE: SDS-Polyacrylamide Gel Electrophoresis; pI: isoelectric Point; DIGE: Differential In-Gel Electrophoresis; 16-BAC: Benzildimethyl-n-hexadecylammonium chloride; MudPIT: Multidimensional Protein
Identification Tecnology; ICAT: Isotope-Coded Affinity Tag; MS: Mass Spectrometry; MALDI: Matrix-Aassisted Laser Desorption/Ionization; TOF: Time Of Flight.
Table modified from Abul-Husn and Devi, 2006.
Table 4. Studies of the synapse employ different proteomic methodologies in different combinations, according to the synaptic
region being studied. In this table, modified from Abul-Husn and Devi,8 the combinations used and the quantity of proteins
identified are detailed
Neuroproteomic analysis of the synapse
Proteomic methods and ms platform
Synaptic fraction
•Synaptosomes and synaptic membranes
•Rat forebrain synaptosomes
•Rat forebrain synaptosomes
•Rat forebrain synaptic membranes
•Mouse forebrain synaptic membranes
•Mouse hippocampus membranes
Number of proteins
identified
2-DE, MALDI-TOF and LC-MS/MS; LC-MS/MS
ICAT and LC-MS/MS
SDS-PAGE, RP-HPLC, SCX-LC, MALDI-TOF
and LC-MS/MS
LC-MS/MS
LC-MS/MS
Postsynaptic densities
•Rat forebrain
SDS-PAGE and MALDI-TOF
•Mouse forebrain
2-DE and LC-MS/MS
•Rat forebrain
2-DE and MALDI-TOF
ICAT and LC-MS/MS
•Rat forebrain
MudPITT
•Rat and mouse brain
SDS-PAGE and LC-MS/MS
•Rat forebrain
SDS-PAGE and LC-MS/MS
•Rat forebrain
MudPIT
•Rat hippocampus slices
MudPIT
•Presynapse
•Rat brain synaptic membranes
2-DE and LC-MS/MS, 16-BAC/SDS-PAGE
and LC-MS/MS
•Rat brain free synaptic membranes
16-BAC/SDS-PAGE and MALDI-TOF
•Rat brain synaptic vesicles associated with
presynaptic plasmatic membrane
16-BAC/SDS-PAGE and MALDI-TOF
•Rat forebrain postsynaptic membrane fraction
MudPIT
246
1131
107
862
1685
31
47
233
61
492
452
374
231
139
36
72
81
50
Abbreviations are the same as those in Table 1; RP-HPLC: Reverse Phasehigh-pressure liquid chromatography; SCX-LC: Strong Cation
eXchange – Liquid Chromatography; LC-MS/MS: Liquid chromatography-tandem MS; MS: Mass Spectrometry.
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port of late endosomes to the lysosomes, implies an effect of
drugs of abuse on the secretory pathways of neurons and
the transport of molecules to dendrites.
Modifications have been reported in the expression of
the proteins related to the motor activity of the microfilaments of the cytoskeleton, and in the proteins that make up
the actin and myosin microtubules involved in the transport
of molecules in the axons, which highlights the complexity
of the effects caused by drugs of abuse on the functions of
the cytoskeleton. This suggests a strong effect on the remodeling of the synapse.
In addition, an increase has been described in the expression of transcripts that encode proteins involved in cellular adhesion and the motor activity of microfilaments, and
a decrease in the transcripts that encode proteins that bind
to actin. It has been proposed that the increase in the expression of adhesion molecules impedes synaptic plasticity
through the forming of new, less adaptable synapses, which
thus inhibits the appropriate forming and strengthening of
specific learning connections.
In addition, the proteomic studies described allow us
to confirm that the chronic administration of drugs of abuse
significantly alters the levels and stages of phosphorylation
of the synaptic proteins, including those of proteins that
participate in signaling, vesicular trafficking, the endocytic
vesicles, the cytoskeleton, and cellular adhesion, coinciding
with the results of genetic studies. The administration of cocaine showed an effect on the expression of the early genes
in the mesocorticolimbic system, supporting the hypothesis
of the generating of a different form of neurobehavioral
plasticity that encourages the consolidation of the addictive process. It has been proposed that the rapid administration of drugs results in neuroadaptations that encourage
compulsive search and increase vulnerability to acquisition,
both behaviors characteristic of the addictive state.15
The study by Black et al.5 showed a high population of
genes whose function is not yet understood; in addition, the
expression was modified in many of the genes only temporarily, contrasting with the permanent physiological effects. These
significant physiological modifications may be irreversible.
post-translational modifications of histones, including methylation, acetylation, phosphorylation and sumoylation.18
In recent years the importance has been show of chromatin remodeling processes in the control of genetic transcription, revealing a complex network of mechanisms that
regulate the accessibility of the genes to the transcriptional
machinery responsible for RNA synthesis and the expression of genes. It has been reported that drugs of abuse interfere in the regulation of chromatin and in the machinery
associated with this process.18-21 In this regard, there is updated information on the modifications in chromatin that
affect gene expression, promoting or repressing it.
Similarly, Newton and Duman20 as well as Cassel et al.21
both published the effects caused by psychotropic drugs on
the process of chromatin remodeling and on the function
of specific proteins participating in the methylation of CpG
islands, an event that controls the acetylation of histones
and, ultimately, gene expression. The results of Cassel et
al.21 showed induction of the expression of proteins binding to methylated CpG islands, MeCP2 and MBD1, in the
striatum and with less intensity in the frontal cortex and the
dentate gyrus of the hippocampus, suggesting a repression
of gene expression in these structures caused by cocaine administration. In addition to these results, histone modification assays showed a decrease in acetylation of histone H3,
indicating a less relaxed state of the strand of DNA, which
impedes gene transcription. Additionally, the expression of
the mRNA of the protein histone deacetylase HDAC2 increased significantly, indicating that the regulation of histone deacetylation takes place through an increased expression of this protein family, which promotes gene expression
by preventing the winding of DNA.
The results of these studies allow us to conclude that
the function of the histones and the chromatin remodeling
event are modified by the administration of drugs of abuse.
This provides a starting line for future research that will allow us to establish the functional consequences of chromatin
modifications in response to the administration of drugs of
abuse, which ultimately will allow us to explain the modifications in gene expression.
EPIGENETIC IMPLICATIONS
THE FUTURE OF PSYCHOGENOMICS
AND PSYCHOPROTEOMICS
Most of the research on the issue of addiction has focused
on studying the intracellular cascades of signal transduction
and transcription factors that bind to promoter elements (sequences) of specific genes, but the molecular mechanisms
that control expression or repression of genes, known as epigenetic mechanisms, have not yet been sufficiently studied or
assessed. Epigenetics is defined as the study of the different
inherited changes which occur in the sequence of the DNA
and span two main modifications of DNA or chromatin:
DNA methylation, covalent modification of cytokines, and
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To relate genes to a complex behavior requires studies that
confirm the behavioral manifestations in appropriate animal
models, which can be extrapolated to diseases in humans.
There are many ways to evaluate behaviors in rodents,
for example, measurements of cognitive function, fear, and
reward. In the field of addictions, it is possible to measure
reinforcement and withdrawal in animal models.22,23
In most of the studies reported, rodent models have
been used to simulate the addictive processes relating to
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cocaine in humans. However, growing evidence indicates
the need to refine rodent and non-human primate models to
better recapitulate addiction in humans and link this condition to different neuropathologies.
It is hoped that in the future a patient with a behavioral
abnormality can have access to a battery of tests that include
genetic studies and brain imaging, which would allow us
to define the pathology from which he suffers and thus select the most effective treatment. Thus, it will be possible to
identify and educate individuals who are particularly vulnerable to abuse of a specific drug, and who, after a brief
exposure to said drug, run a significant risk of acquiring addiction. Thus, optimal programs could be selected for treatment of individual addicts.
CONCLUSIONS
It has been established that drugs of abuse provoke a modification in the neuronal architecture through the alteration of
the length of the dendrites and modify the neuronal physiology in general in the brain regions related to the processes
of acquisition and consolidation of addiction.
One of the areas the most important such regions is the
prefrontal cortex, due to its highly complex synaptic connectivity and dendritic arborization.22,23 For this reason, modifications in gene expression, specifically regarding important
processes such as calmodulin-transduced intracellular signaling, metabolism of cholesterol, and the functioning of the
Golgi apparatus and the endoplasmic reticulum, reflect the
changes in neuronal function and synaptic plasticity induced
by the administration of drugs of abuse. Likewise, initial epigenetic studies conducted to understand the implications regarding regulation of gene expression have shown that drugs
of abuse produce their effects because they cause modifications in the remodeling of chromatin.20 In this regard, we can
hope for an important development in the understanding of
this discipline, one which will be useful in understanding the
mechanisms of the processes of acquisition and relapse.
There is quite a lot of diversity in the results from proteomic studies carried out to characterize the expression
of proteins involved in the addictive processes of drugs
of abuse. To date, there have been relatively few studies
published in which self-administration studies are combined with proteomic approaches. A likely explanation is
that researchers in the areas of behavioral pharmacology
and molecular biology have been working separately from
one another. Other causes of the diversity in results are: the
improper use of experimental models, the dependency on
non-neuronal systems or neuronal tissues involved in the
reinforcement of the effects of the drug, and the lack of neural substrates defined on a cellular or biochemical level.
Other factors to consider, and which hinder the comparison between different studies, include the selection of
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the brain region, the subcellular fractionation, and/or the
protein subgroup being analyzed. Another relevant factor
is the selection of the proteomic techniques to be employed.
For example, the use of two separation techniques to analyze
the same sample of proteins produces drastically different results, due to the fact that the methods employed may be more
or less adequate for each specific protein subgroup (soluble
and membrane proteins). Generally, proteomic techniques
are implemented differently in each research laboratory.
In addition, there are multiple experimental factors
which must be considered: the type of drug, the method
and speed of administration, the age of the individuals, the
ethnic group to which they belong, and the importance of
identifying phenotypes.
Careful selection of these factors and a detailed study of
addictive processes, together with a combination of suitable
drug reinforcement behavioral models, specifically neurobiological systems, and the use of molecular techniques,
will expand our understanding toward developing objective diagnostic tests, improved treatments and preventive
measures, as well as effective cures.
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Declaration of conflict of interests: None
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