Marta Vicente-Rodríguez

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.

Differential phosphoproteome of the striatum from pleiotrophin knockout and midkine knockout mice treated with amphetamine: correlations with amphetamine-induced neurotoxicity.

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) have been shown to modulate amphetamine-induced neurotoxicity. Accordingly, PTN-/- and MK-/- mice show an increased vulnerability to amphetamine-induced neurotoxic effects. In an effort to uncover new pharmacological targets to prevent amphetamine neurotoxic effects, 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-/-, MK-/- and wild type (WT) mice treated with amphetamine. We identified 13 differentially expressed phosphoproteins that are judged to be relevant in the neuroprotective roles of PTN and MK against amphetamine-induced neurotoxicity. It is very interesting to note that 4 of these phosphoproteins, annexin A7 (ANXA7), COP9 signalosome subunit 5 (COPS5), aldehyde dehydrogenase family 1 member A1 (ALDH1A1) and creatine kinase U-type (CKMT1), are known to be involved in Parkinsons disease, a result of significant importance since PTN and MK have been also demonstrated to limit Parkinsons disease (PD) progress and have been suggested to be among the important genetic factors possibly preventing the development of PD in methamphetamine abusers. The data identify phosphoproteins differentially regulated by amphetamine treatment and/or the presence of endogenous PTN/MK which may be relevant mediators of PTN/MK neuroprotective effects against amphetamine-induced neurotoxicity. The data support further studies to validate the phosphoproteins here identified as possible new pharmacological targets to prevent amphetamine neurotoxic effects.

Regulation of extinction of cocaine-induced place preference by midkine is related to a differential phosphorylation of peroxiredoxin 6 in dorsal striatum.

The neurotrophic factors Midkine (MK) and Pleiotrophin (PTN) have been suggested to modulate drugs of abuse-induced effects. To test this hypothesis, cocaine (10 and 15mg/kg)-induced conditioned place preference (CPP) was rendered in PTN knockout (PTN-/-), MK knockout (MK-/-) and wild type (WT+/+) mice, and then extinguished after repeated saline injections (distributed in 4 extinction sessions). Cocaine induced a similar CPP in all the three genotypes. We found a significantly increased percentage of MK-/- mice that did not extinguish cocaine CPP at the end of the extinction sessions. Particularly, 40% of MK-/- mice did not extinguish cocaine (15mg/kg)-induced CPP compared to WT+/+ and PTN-/- mice (∼0-6%). Interestingly, we found that a greater magnitude of extinction of CPP after the first extinction session (5 days after last administration of cocaine) correlates with increased tyrosine phosphorylation of the enzyme peroxiredoxin 6 in the dorsal striatum of MK-/- mice. On the other hand, a greater magnitude of CPP extinction correlates with increased tyrosine phosphorylation of aconitase 2 in the prefrontal cortex of WT+/+ mice. In contrast, a lower magnitude of CPP extinction correlates with increased phosphorylation of aconitase 2 in the prefrontal cortex of PTN-/- mice, suggesting that the correlation between the tyrosine phosphorylation levels of aconitase 2 and magnitude of CPP extinction depends on the genotype considered. The data demonstrate that MK is a novel genetic factor that plays a role in the extinction of cocaine-induced CPP by mechanisms that may involve specific phosphorylation of striatal peroxiredoxin 6.

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.

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 modulates morphine withdrawal but has no effects on morphine-conditioned place preference

Pleiotrophin (PTN) is a neurotrophic factor with important functions in addiction and neurodegenerative disorders. Morphine administration induces an increase in the expression of PTN and Midkine (MK), the only other member of this family of cytokines, in brain areas related with the addictive effects of drug of abuse, like the Ventral Tegmental Area or the hippocampus. In spite of previous studies showing that PTN modulates amphetamine and ethanol rewarding effects, and that PTN is involved in morphine-induced analgesia, it was still unknown if the rewarding effects of morphine may be regulated by endogenous PTN. Thus, we aim to study the role of PTN in the reward and physical dependence induced by morphine. We used the Conditioned Place Preference (CPP) paradigm in PTN genetically deficient (PTN-/-) and wild type (WT) mice to assess the rewarding effects of morphine in absence of endogenous PTN. Second, to study if PTN may be involved in morphine physical dependence, naloxone-precipitated withdrawal syndrome was induced in PTN-/- and WT morphine dependent mice. Although the increase in the time spent in the morphine-paired compartment after conditioning tended to be more pronounced in PTN-/- mice, statistical significance was not achieved. The data suggest that PTN does not exert an important role in morphine reward. However, our results clearly indicate that PTN-/- mice develop a more severe withdrawal syndrome than WT mice, characterized as a significant increase in the time standing and in the total incidences of forepaw licking, forepaw tremors, wet dog shake and writhing. The data presented here suggest that PTN is a novel genetic factor that plays a role in morphine withdrawal syndrome.

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.

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.

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 Parkinsons disease and Alzheimers disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations.

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 RPTPβ/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.