Implication of the PTN/RPTPß/ζ Signaling Pathway in Acute Ethanol Neuroinflammation in Both Sexes: A Comparative Study with LPS.

Binge drinking during adolescence increases the risk of alcohol use disorder, possibly by involving alterations of neuroimmune responses. Pleiotrophin (PTN) is a cytokine that inhibits Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ. PTN and MY10, an RPTPß/ζ pharmacological inhibitor, modulate ethanol behavioral and microglial responses in adult mice. Now, to study the contribution of endogenous PTN and the implication of its receptor RPTPß/ζ in the neuroinflammatory response in the prefrontal cortex (PFC) after acute ethanol exposure in adolescence, we used MY10 (60 mg/kg) treatment and mice with transgenic PTN overexpression in the brain. Cytokine levels by X-MAP technology and gene expression of neuroinflammatory markers were determined 18 h after ethanol administration (6 g/kg) and compared with determinations performed 18 h after LPS administration (5 g/kg). Our data indicate that Ccl2, Il6, and Tnfa play important roles as mediators of PTN modulatory actions on the effects of ethanol in the adolescent PFC. The data suggest PTN and RPTPß/ζ as targets to differentially modulate neuroinflammation in different contexts. In this regard, we identified for the first time important sex differences that affect the ability of the PTN/RPTPß/ζ signaling pathway to modulate ethanol and LPS actions in the adolescent mouse brain.

Receptor protein tyrosine phosphatase ß/ζ regulates loss of neurogenesis in the mouse hippocampus following adolescent acute ethanol exposure.

Adolescence is a critical period for brain maturation in which this organ is more vulnerable to the damaging effects of ethanol. Administration of ethanol in mice induces a rapid cerebral upregulation of pleiotrophin (PTN), a cytokine that regulates the neuroinflammatory processes induced by different insults and the behavioral effects of ethanol. PTN binds Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ and inhibits its phosphatase activity, suggesting that RPTPß/ζ may be involved in the regulation of ethanol effects. To test this hypothesis, we have treated adolescent mice with the RPTPß/ζ inhibitor MY10 (60 mg/kg) before an acute ethanol (6 g/kg) administration. Treatment with MY10 completely prevented the ethanol-induced neurogenic loss in the hippocampus of both male and female mice. In flow cytometry studies, ethanol tended to increase the number of NeuN+/activated Caspase-3+ cells particularly in female mice, but no significant effects were found. Ethanol increased Iba1+ cell area and the total marked area in the hippocampus of female mice, suggesting sex differences in ethanol-induced microgliosis. In addition, ethanol reduced the circulating levels of IL-6 and IL-10 in both sexes, although this reduction was only found significant in males and not affected by MY10 treatment. Interestingly, MY10 alone increased the total marked area and the number of Iba1+ cells only in the female hippocampus, but tended to reduce the circulating levels of TNF-α only in male mice. In summary, the data identify a novel modulatory role of RPTPß/ζ on ethanol-induced loss of hippocampal neurogenesis, which seems unrelated to glial and inflammatory responses. The data also suggest sex differences in RPTPß/ζ function that may be relevant to immune responses and ethanol-induced microglial responses.

Genetic inactivation of midkine, not pleiotrophin, facilitates extinction of alcohol-induced conditioned place preference.

Pleiotrophin (PTN) and midkine (MK) are growth factors that modulate alcohol consumption and reward. Since both PTN and MK limit the rewarding effects of alcohol, pharmacological potentiation of the PTN and MK signaling pathways has been proposed for the treatment of alcohol use disorders (AUD). Although the use of this therapy in the prevention of alcohol relapse is important, the potential role of these cytokines in extinguishing alcohol-induced seeking behavior is a key question that remains unanswered. To fill this gap, we have now studied the extinction of the conditioned place preference (CPP) induced by different doses of alcohol in Ptn knockout (Ptn-/-) and Mk knockout (Mk-/-) mice. The data confirm a higher sensitivity of Ptn-/- mice to the conditioning effects of a low dose (1 g/kg) and a rewarding dose (2 g/kg) of alcohol, while Mk-/- mice are only more susceptible to the conditioning effects of the low dose of this drug. More importantly, the percentage of Mk-/- mice, not Ptn-/- mice, that efficiently extinguished alcohol-induced CPP was significantly higher than that of Wt mice. Taken together, the data presented here confirm that Ptn and Mk are genetic factors that determine the conditioning effects of alcohol in mice and that Mk is a novel factor that plays an important role in the extinction of alcohol-induced CPP.

Effects of Intermittent versus Chronic-Moderate Ethanol Administration during Adolescence in the Adult Hippocampal Phosphoproteome.

Alcohol consumption during adolescence is known to cause different impairments in the hippocampus that could lead to persistent deficits in adulthood. A common pattern of alcohol use in adolescents consists of excessive and intermittent alcohol consumption over a very short period of time (binge drinking). Protein phosphorylation is a mechanism underlying memory processes and we have previously demonstrated changes in the rat hippocampal phosphoproteome after a single dose of ethanol; however, studies showing the phosphoprotein alterations in the hippocampus after repeated exposition to alcohol are limited. This study focuses on the identification of the phosphoproteins differentially regulated in the adolescent rat hippocampus after repeated ethanol administration by comparing different patterns of alcohol treatments according to dose and frequency of administration ((i) moderate dose-chronic use, (ii) low dose-intermittent use, and (iii) high dose-intermittent use). We have used a proteomic approach, including phosphoprotein enrichment by immobilized metal affinity chromatography, which revealed 21 proteins differentially affected depending on the pattern of alcohol treatment used. Many of these proteins are included in glycolysis and glucagon signaling pathways and are also involved in neurodegeneration, which could reinforce the role of metabolic alterations in the neural damage induced by repeated alcohol exposure during adolescence.

Intermittent-Excessive and Chronic-Moderate Ethanol Intake during Adolescence Impair Spatial Learning, Memory and Cognitive Flexibility in the Adulthood.

Intermittent and excessive ethanol consumption over very short periods of time, known as binge drinking, is common in the adolescence, considered a vulnerable period to the effects of alcohol in terms of cognitive performance. One of the brain functions most drastically affected by ethanol in adolescent individuals seems to be spatial learning and memory dependent on the hippocampus. In the current study we have focused on the long-lasting effects on spatial learning and memory of intermittent and excessive alcohol consumption compared to chronic and moderate alcohol exposure during adolescence. Five-week old male Wistar rats consumed ethanol for 24 days following two different self-administration protocols that differed in the intake pattern. Spatial learning and memory were evaluated in the radial arm maze. Hippocampal synaptic plasticity was assessed by measuring field excitatory postsynaptic potentials. Hippocampal expression of AMPA and NMDA receptor subunits as well as levels of phosphorylated Ser9-GSK3ß (the inactive form of GSK3ß) were also quantified. Our results show that both patterns of ethanol intake during adolescence impair spatial learning, memory and cognitive flexibility in the adulthood in a dose-dependent way. Nevertheless, changes in synaptic plasticity, gene expression and levels of inactive GSK3ß depended on the pattern of ethanol intake.

Proteomic Identification of Biomarkers Associated with Eating Control and Bariatric Surgery Outcomes in Patients with Morbid Obesity.

BACKGROUND: The current therapeutics of morbid obesity could be significantly improved after the identification of novel biomarkers associated with the food addiction endophenotype of obesity and with bariatric surgery outcomes. METHODS: We applied differential expression proteomics and enzyme-linked immunosorbent confirmatory assays to identify (a) proteins that varied according to loss of control over eating in morbidly obese patients and (b) proteins that varied between normoweight controls and patients before and 1 year after bariatric surgery. RESULTS: Clusterin was the only protein that consistently varied according to eating control in patients. Patients showed increased levels of serum amyloid P protein, apolipoprotein A4, serotransferrin, complement factors B and C3 and haptoglobin with respect to controls; the levels of all these proteins tended to return to control values 1 year after surgery. In contrast, apolipoprotein A1 and transthyretin were initially downregulated in patients and were scarcely changed by surgery. Leucine-rich alpha-2-glycoprotein was markedly increased in patients only after surgery. CONCLUSIONS: Clusterin could be of interest as a putative biomarker for food addiction diagnosis in people with morbid obesity. In addition, postsurgical normalization of the proteins initially dysregulated in obese subjects might help monitor clinical improvements after surgery, while lasting or newly detected alterations (i.e., those affecting transthyretin and leucine-rich alpha-2-glycoprotein) could reflect partial refractoriness and/or contribute to the early prediction of clinical problems.

Pharmacological inhibition of Receptor Protein Tyrosine Phosphatase ß/ζ (PTPRZ1) modulates behavioral responses to ethanol.

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are the endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ (a.k.a. PTPRZ1, RPTPß, PTPζ), suggesting a potential role for this phosphatase in the regulation of alcohol effects. To determine if RPTPß/ζ regulates ethanol consumption, we treated mice with recently developed small-molecule inhibitors of RPTPß/ζ (MY10, MY33-3) before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with RPTPß/ζ inhibitors, particularly with MY10, drank less ethanol than controls. MY10 treatment blocked ethanol conditioned place preference, showed limited effects on ethanol-induced ataxia, and potentiated the sedative effects of ethanol. We also tested whether RPTPß/ζ is involved in ethanol signaling pathways. We found that ethanol treatment of neuroblastoma cells increased phosphorylation of anaplastic lymphoma kinase (ALK) and TrkA, known substrates of RPTPß/ζ. Treatment of neuroblastoma cells with MY10 or MY33-3 also increased levels of phosphorylated ALK and TrkA. However, concomitant treatment of neuroblastoma cells with ethanol and MY10 or MY33-3 prevented the increase in pTrkA and pALK. These results demonstrate for the first time that ethanol engages TrkA signaling and that RPTPß/ζ modulates signaling pathways activated by alcohol and behavioral responses to this drug. The data support the hypothesis that RPTPß/ζ might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Development of inhibitors of receptor protein tyrosine phosphatase ß/ζ (PTPRZ1) as candidates for CNS disorders.

A new series of blood-brain barrier permeable molecules designed to mimic the activity of Pleiotrophin in the CNS has been designed and synthesized. These compounds exert their action by interacting with the intracellular domain PD1 of the Protein Tyrosine-Phosphatase Receptor Z1 (PTPRZ1), and inhibiting its tyrosine phosphatase activity. The most potent compounds 10a and 12b (IC50 = 0,1 µM) significantly increase the phosphorylation of key tyrosine residues of PTPRZ1 substrates involved in neuronal survival and differentiation, and display protective effects against amphetamine-induced toxicity. Docking and molecular dynamics experiments have been used to analyze the binding mode and to explain the observed selectivity against PTP1B. An In vivo experiment has demonstrated that 10a can cross the BBB, thus promoting the possibility of moving forward these candidates for the development of drugs for the treatment of CNS disorders, such as drug addiction and neurodegenerative diseases.

Endogenous pleiotrophin and midkine regulate LPS-induced glial responses.

Pleiotrophin (PTN) and Midkine (MK) are two growth factors that modulate neuroinflammation. PTN overexpression in the brain prevents LPS-induced astrocytosis in mice but potentiates microglial activation. The modest astrocytic response caused by a low dose of LPS (0.5mg/kg) is blocked in the striatum of MK-/- mice whereas microglial response is unaffected. We have now tested the effects of an intermediate dose of LPS (7.5mg/kg) in glial response in PTN-/- and MK-/- mice. We found that LPS-induced astrocytosis is prevented in prefrontal cortex and striatum of both PTN-/- and MK-/- mice. Some of the morphological changes of microglia induced by LPS tended to increase in both genotypes, particularly in PTN-/- mice. Since we previously showed that PTN potentiates LPS-induced activation of BV2 microglial cells, we tested the activation of FYN kinase, a substrate of the PTN receptor RPTPß/ζ, and the subsequent ERK1/2 phosphorylation on LPS and PTN-treated BV2 cells. LPS effects on BV2 cells were not affected by the addition of PTN, suggesting that PTN does not recruit the FYN-MAP kinase signaling pathway in order to modulate LPS effects on microglial cells. Taking together, evidences demonstrate that regulation of astroglial responses to LPS administration are highly dependent on the levels of expression of PTN and MK. Further studies are needed to clarify the possible roles of endogenous expression of PTN and MK in LPS-induced microglial responses.

Age-Dependent Effects of Acute Alcohol Administration in the Hippocampal Phosphoproteome.

Alcohol consumption during adolescence is deleterious to the developing brain and leads to persistent deficits in adulthood. Several results provide strong evidence for ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the hippocampus. Protein phosphorylation is a well-known and well-documented mechanism in memory processes, but information on phosphoprotein alterations in hippocampus after ethanol exposure is limited. This study focuses on age-related changes in the hippocampal phosphoproteome after acute alcohol administration. We have compared the phosphoprotein expression in the hippocampus of adult and adolescent Wistar rats treated with a single dose of ethanol (5 g/kg i.p.), using a proteomic approach including phosphoprotein enrichment by immobilized metal affinity chromatography (IMAC). Our proteomic analysis revealed that 13 proteins were differentially affected by age, ethanol administration, or both. Most of these proteins are involved in neuroprotection and are expressed less in young rats treated with ethanol. We conclude that acute alcohol induces important changes in the expression of phosphoproteins in the hippocampus that could increase the risk of neurodegenerative disorders, especially when the alcohol exposure begins in adolescence.

Pleiotrophin regulates microglia-mediated neuroinflammation.

BACKGROUND: Pleiotrophin (PTN) is a cytokine found highly upregulated in the brain in different disorders characterized by overt neuroinflammation such as neurodegenerative diseases, drug addiction, traumatic injury, and ischemia. In the present work, we have explored whether PTN modulates neuroinflammation and if Toll-like receptor 4 (TLR4), crucial in the initiation of an immune response, is involved. METHODS: In immunohistochemistry assays, we studied lipopolysaccharide (LPS, 7.5 mg/kg i.p.)-induced changes in glial fibrillary acidic protein (GFAP, astrocyte marker) and ionized calcium-binding adaptor molecule 1 (Iba1, microglia marker) expression in the prefrontal cortex (PFC) and striatum of mice with transgenic PTN overexpression in the brain (PTN-Tg) and in wild-type (WT) mice. Cytokine protein levels were assessed in the PFC by X-MAP technology. The influence of TLR4 signaling in LPS effects in both genotypes was assessed by pretreatment with the TLR4 antagonist (TAK-242, 3.0 mg/kg i.p.). Murine BV2 microglial cells were treated with PTN (0.5 µg/ml) and LPS (1.0 µg/ml) and assessed for the release of nitric oxide (NO). RESULTS: We found that LPS-induced microglial activation is significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. The levels of TNF-α, IL-6, and MCP-1 in response to LPS were significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. Pretreatment with TAK-242 efficiently blocked increases in cytokine contents in a similar manner in both genotypes. Concomitant incubation of BV2 cells with LPS and PTN significantly potentiated the production of NO compared to cells only treated with LPS. CONCLUSIONS: Our findings identify for the first time that PTN is a novel and potent regulator of neuroinflammation. Pleiotrophin potentiates LPS-stimulated microglia activation. Our results suggest that regulation of the PTN signaling pathways may constitute new therapeutic opportunities particularly in those neurological disorders characterized by increased PTN cerebral levels and neuroinflammation.

Pleiotrophin overexpression regulates amphetamine-induced reward and striatal dopaminergic denervation without changing the expression of dopamine D1 and D2 receptors: Implications for neuroinflammation.

It was previously shown that mice with genetic deletion of the neurotrophic factor pleiotrophin (PTN-/-) show enhanced amphetamine neurotoxicity and impair extinction of amphetamine conditioned place preference (CPP), suggesting a modulatory role of PTN in amphetamine neurotoxicity and reward. We have now studied the effects of amphetamine (10mg/kg, 4 times, every 2h) in the striatum of mice with transgenic PTN overexpression (PTN-Tg) in the brain and in wild type (WT) mice. Amphetamine caused an enhanced loss of striatal dopaminergic terminals, together with a highly significant aggravation of amphetamine-induced increase in the number of GFAP-positive astrocytes, in the striatum of PTN-Tg mice compared to WT mice. Given the known contribution of D1 and D2 dopamine receptors to the neurotoxic effects of amphetamine, we also performed quantitative receptor autoradiography of both receptors in the brains of PTN-Tg and WT mice. D1 and D2 receptors binding in the striatum and other regions of interest was not altered by genotype or treatment. Finally, we found that amphetamine CPP was significantly reduced in PTN-Tg mice. The data demonstrate that PTN overexpression in the brain blocks the conditioning effects of amphetamine and enhances the characteristic striatal dopaminergic denervation caused by this drug. These results indicate for the first time deleterious effects of PTN in vivo by mechanisms that are probably independent of changes in the expression of D1 and D2 dopamine receptors. The data also suggest that PTN-induced neuroinflammation could be involved in the enhanced neurotoxic effects of amphetamine in the striatum of PTN-Tg mice.

Midkine Is a Novel Regulator of Amphetamine-Induced Striatal Gliosis and Cognitive Impairment: Evidence for a Stimulus-Dependent Regulation of Neuroinflammation by Midkine.

Midkine (MK) is a cytokine that modulates amphetamine-induced striatal astrogliosis, suggesting a possible role of MK in neuroinflammation induced by amphetamine. To test this hypothesis, we studied astrogliosis and microglial response induced by amphetamine (10 mg/kg i.p. four times, every 2 h) in different brain areas of MK-/- mice and wild type (WT) mice. We found that amphetamine-induced microgliosis and astrocytosis are enhanced in the striatum of MK-/- mice in a region-specific manner. Surprisingly, LPS-induced astrogliosis in the striatum was blocked in MK-/- mice. Since striatal neuroinflammation induced by amphetamine-type stimulants correlates with the cognitive deficits induced by these drugs, we also tested the long-term effects of periadolescent amphetamine treatment (3 mg/kg i.p. daily for 10 days) in a memory task in MK-/- and WT mice. Significant deficits in the Y-maze test were only observed in amphetamine-pretreated MK-/- mice. The data demonstrate for the first time that MK is a novel modulator of neuroinflammation depending on the inflammatory stimulus and the brain area considered. The data indicate that MK limits amphetamine-induced striatal neuroinflammation. In addition, our data demonstrate that periadolescent amphetamine treatment in mice results in transient disruption of learning and memory processes in absence of endogenous MK.

Chronic Cocaine Use Causes Changes in the Striatal Proteome Depending on the Endogenous Expression of Pleiotrophin.

The neurotrophic factor pleiotrophin (PTN) is upregulated in different brain areas after the administration of different drugs of abuse, including psychostimulants. PTN has been shown to prevent cocaine-induced cytotoxicity in NG108-15 and PC12 cells. We previously demonstrated that specific phosphoproteins related to neurodegeneration processes are differentially regulated in the mouse striatum by a single cocaine (15 mg/kg) administration depending on the endogenous expression of PTN. Since neurodegenerative processes are usually observed in patients exposed to toxicants for longer duration, we have now performed a striatal proteomic study using samples enriched in phosphorylated proteins from PTN knockout (PTN-/-) mice, from mice with transgenic PTN overexpression (PTN-Tg) in the brain, and from wild type (WT) mice after a chronic treatment with cocaine (15 mg/kg/day for 7 days). We have successfully identified 23 proteins significantly affected by chronic cocaine exposure, genotype, or both. Most of these proteins, including peroxiredoxin-6 (PRDX6), triosephosphate isomerase (TPI1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and annexins A5 (ANXA5) and A7 (ANXA7), may be of significant importance because they were previously identified in proteomic studies in animals treated with psychostimulants and/or because they are related to neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations.

Functional neuroimaging of amphetamine-induced striatal neurotoxicity in the pleiotrophin knockout mouse model.

Amphetamine-induced neurotoxic effects have traditionally been studied using immunohistochemistry and other post-mortem techniques, which have proven invaluable for the definition of amphetamine-induced dopaminergic damage in the nigrostriatal pathway. However, these approaches are limited in that they require large numbers of animals and do not provide the temporal data that can be collected in longitudinal studies using functional neuroimaging techniques. Unfortunately, functional imaging studies in rodent models of drug-induced neurotoxicity are lacking. The aim of this study was to evaluate in vivo the changes in brain glucose metabolism caused by amphetamine in the pleiotrophin knockout mouse (PTN-/-), a genetic model with increased vulnerability to amphetamine-induced neurotoxic effects. We showed that administration of amphetamine causes a significantly greater loss of striatal tyrosine hydroxylase content in PTN-/- mice than in wild-type (WT) mice. In addition, [(18)F]-FDG-PET shows that amphetamine produces a significant decrease in glucose metabolism in the striatum and prefrontal cortex in the PTN-/- mice, compared to WT mice. These findings suggest that [(18)F]-FDG uptake measured by PET is useful for detecting amphetamine-induced changes in glucose metabolism in vivo in specific brain areas, including the striatum, a key feature of amphetamine-induced neurotoxicity.

La práctica clínica enfermera en el abordaje de la violencia de género / Enfermeria prática clínica no tratamento de género da violencia / Clinical nurse practice approach to gender violence

La violencia contra las mujeres es un fenómeno que ocurre en todos los países, clases sociales y ámbitos de la sociedad. Según la definición de la ONU, la violencia de género (VG) es «cualquier acto o intención que origina daño o sufrimiento físico, sexual o psicológico a las mujeres, incluyendo las amenazas de dichos actos, la coerción o privación arbitraria de libertad, ya sea en la vida pública o privada» Las necesidades específicas de estas mujeres son multidimensionales, por lo que se requieren intervenciones sanitarias que tengan en cuenta los aspectos biológicos, psicológicos y sociales. Para ello es necesaria la implicación activa de todo el personal de los servicios sanitarios desde un modelo de atención integral. El estudio tiene como objetivo conocer la base de actuación enfermera para el abordaje de la violencia de género. La metodología usada para este estudio es de investigación cualitativo-descriptiva que se inicia con una búsqueda bibliográfica y continúa con el análisis hermenéutico de los documentos encontrados. Las conclusiones de este estudio versan sobre la formación de las enfermeras en el abordaje de la violencia de género, así como los puntos fuertes y debilidades al respecto (AU)

Violence against women is a phenomenon that occurs in all countries, social classes and sectors of society. According to the UN definition, gender-based violence (GBV) is «any act or intention that causes harm or physical, sexual or psychological suffering to women, including threats of such acts, coercion or arbitrary deprivation of liberty, and whether in public or private life» the specific needs of these women are multidimensional, so health interventions that take into account the biological, psychological and social aspects are required. This requires the active involvement of all staff in health services is required from a model of comprehensive care. The study aims to determine the basis of nurse for addressing GBV action. The methodology used for this study is qualitative-descriptive research begins with a literature search and continues with the hermeneutic analysis of the documents found. The findings of this study relate to the training of nurses in addressing gender-based violence, as well as the strengths and weaknesses in this regard (AU)

Violência contra a mulher é um fenômeno que ocorre em todos os países, classes sociais e setores da sociedade. De acordo com a definição da ONU, a violência baseada no género (VBG) é «qualquer ato ou intenção que causa dano ou física, sofrimento sexual ou psicológico à mulher, inclusive ameaças de tais atos, coerção ou privação arbitrária de liberdade, e seja na vida pública ou privada» as necessidades específicas dessas mulheres são, por assim intervenções multidimensionais de saúde que levem em conta os aspectos biológicos, psicológicos e sociais são necessários. Isso requer a participação activa de todos os funcionários em serviços de saúde é exigido de um modelo de atenção integral. O estudo tem como objetivo determinar a base de enfermeira para tratar de ação VBG. A metodologia utilizada para este estudo é uma pesquisa qualitativa-descritiva começa com uma pesquisa bibliográfica e continua com a análise hermenêutica dos documentos encontrados. As conclusões deste estudo referem-se à formação de enfermeiros no enfrentamento da violência de gênero, bem como os pontos fortes e fracos a este respeito (AU)

Genetic inactivation of midkine modulates behavioural responses to ethanol possibly by enhancing GABA(A) receptor sensitivity to GABA(A) acting drugs.

Midkine (MK) is a cytokine with important functions in dopaminergic neurons that is found upregulated in the prefrontal cortex of alcoholics. We have studied the behavioural effects of ethanol in MK genetically deficient (MK-/-) and wild type (MK+/+) mice. A low dose of ethanol (1.0g/kg), unable to cause conditioned place preference (CPP) in MK+/+ mice, induced a significant CPP in MK-/- mice, suggesting that MK prevents the rewarding effects of low doses of ethanol. However, this difference between genotypes is lost when a higher, rewarding, dose of ethanol (2.0g/kg) is used. Accordingly, the anxiolytic effects of 1.0mg/kg diazepam, other GABA(A) acting drug, were significantly enhanced in MK-/- mice compared to MK+/+ mice; however, 2.0mg/kg diazepam caused increased anxiolytic effects in MK+/+ mice. In addition, MK-/- mice showed a significant delayed recovery from ethanol (2.0g/kg)-induced ataxia whereas the sedative effects induced by ethanol (3.6g/kg), tested in a loss of righting reflex paradigm, were found to be similar in MK-/- and MK+/+ mice. The data indicate that MK differentially regulates the behavioural responses to ethanol. The results suggest that differences in the sensitivity of GABA(A) receptors to GABA(A) acting drugs caused by genetic inactivation of MK could underlie the different behavioural responses to ethanol in MK-/- mice. Overall, these results suggest that MK may be a novel genetic factor of importance in alcohol use disorders, and that potentiation of MK signalling pathway may be a promising therapeutic strategy in the treatment of these disorders.

Pleiotrophin differentially regulates the rewarding and sedative effects of ethanol.

Pleiotrophin (PTN) is a cytokine with important roles in dopaminergic neurons. We found that an acute ethanol (2.0 g/kg, i.p.) administration causes a significant up-regulation of PTN mRNA and protein levels in the mouse prefrontal cortex, suggesting that endogenous PTN could modulate behavioural responses to ethanol. To test this hypothesis, we studied the behavioural effects of ethanol in PTN knockout (PTN(-/-) ) mice and in mice with cortex- and hippocampus-specific transgenic PTN over-expression (PTN-Tg). Ethanol (1.0 and 2.0 g/kg) induced an enhanced conditioned place preference in PTN(-/-) compared to wild type mice, suggesting that PTN prevents ethanol rewarding effects. Accordingly, the conditioning effects of ethanol were completely abolished in PTN-Tg mice. The ataxic effects induced by ethanol (2.0 g/kg) were not affected by the genotype. However, the sedative effects of ethanol (3.6 g/kg) tested in a loss of righting reflex paradigm were significantly reduced in PTN-Tg mice, suggesting that up-regulation of PTN levels prevents the sedative effects of ethanol. These results indicate that PTN may be a novel genetic factor of importance in alcohol use disorders, and that potentiation of the PTN signalling pathway may be a promising therapeutic strategy in the treatment of these disorders.

Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine.