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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. Vol. 35, No. 2, March-April 2012 129 Matus Ortega et al. 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).1-3 130 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 Vol. 35, No. 2, March-April 2012 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) Vol. 35, No. 2, March-April 2012 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- 131 Matus Ortega et al. 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). 132 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 Vol. 35, No. 2, March-April 2012 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- 133 Matus Ortega et al. 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. 134 Vol. 35, No. 2, March-April 2012 Addictions, genomics and proteomics 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 Vol. 35, No. 2, March-April 2012 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 135 Matus Ortega et al. 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 136 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. 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