Genetic deletion or tyrosine phosphatase inhibition of PTPRZ1 activates c-Met to up-regulate angiogenesis and lung adenocarcinoma growth.

Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) expressed in endothelial cells and required for stimulation of cell migration by vascular endothelial growth factor A165 (VEGFA165 ) and pleiotrophin (PTN). It is also over or under-expressed in various tumor types. In this study, we used genetically engineered Ptprz1-/- and Ptprz1+/+ mice to study mechanistic aspects of PTPRZ1 involvement in angiogenesis and investigate its role in lung adenocarcinoma (LUAD) growth. Ptprz1-/- lung microvascular endothelial cells (LMVEC) have increased angiogenic features compared with Ptprz1+/+ LMVEC, in line with the increased lung angiogenesis and the enhanced chemically induced LUAD growth in Ptprz1-/- compared with Ptprz1+/+ mice. In LUAD cells isolated from the lungs of urethane-treated mice, PTPRZ1 TP inhibition also enhanced proliferation and migration. Expression of beta 3 (ß3 ) integrin is decreased in Ptprz1-/- LMVEC, linked to enhanced VEGF receptor 2 (VEGFR2), c-Met tyrosine kinase (TK) and Akt kinase activities. However, only c-Met and Akt seem responsible for the enhanced endothelial cell activation in vitro and LUAD growth and angiogenesis in vivo in Ptprz1-/- mice. A selective PTPRZ1 TP inhibitor, VEGFA165 and PTN also activate c-Met and Akt in a PTPRZ1-dependent manner in endothelial cells, and their stimulatory effects are abolished by the c-Met TK inhibitor (TKI) crizotinib. Altogether, our data suggest that low PTPRZ1 expression is linked to worse LUAD prognosis and response to c-Met TKIs and uncover for the first time the role of PTPRZ1 in mediating c-Met activation by VEGFA and PTN.

Protein tyrosine phosphatase receptor-ζ1 deletion triggers defective heart morphogenesis in mice and zebrafish.

Protein tyrosine phosphatase receptor-ζ1 (PTPRZ1) is a transmembrane tyrosine phosphatase receptor highly expressed in embryonic stem cells. In the present work, gene expression analyses of Ptprz1-/- and Ptprz1+/+ mice endothelial cells and hearts pointed to an unidentified role of PTPRZ1 in heart development through the regulation of heart-specific transcription factor genes. Echocardiography analysis in mice identified that both systolic and diastolic functions are affected in Ptprz1-/- compared with Ptprz1+/+ hearts, based on a dilated left ventricular (LV) cavity, decreased ejection fraction and fraction shortening, and increased angiogenesis in Ptprz1-/- hearts, with no signs of cardiac hypertrophy. A zebrafish ptprz1-/- knockout was also generated and exhibited misregulated expression of developmental cardiac markers, bradycardia, and defective heart morphogenesis characterized by enlarged ventricles and defected contractility. A selective PTPRZ1 tyrosine phosphatase inhibitor affected zebrafish heart development and function in a way like what is observed in the ptprz1-/- zebrafish. The same inhibitor had no effect in the function of the adult zebrafish heart, suggesting that PTPRZ1 is not important for the adult heart function, in line with data from the human cell atlas showing very low to negligible PTPRZ1 expression in the adult human heart. However, in line with the animal models, Ptprz1 was expressed in many different cell types in the human fetal heart, such as valvar, fibroblast-like, cardiomyocytes, and endothelial cells. Collectively, these data suggest that PTPRZ1 regulates cardiac morphogenesis in a way that subsequently affects heart function and warrant further studies for the involvement of PTPRZ1 in idiopathic congenital cardiac pathologies.NEW & NOTEWORTHY Protein tyrosine phosphatase receptor ζ1 (PTPRZ1) is expressed in fetal but not adult heart and seems to affect heart development. In both mouse and zebrafish animal models, loss of PTPRZ1 results in dilated left ventricle cavity, decreased ejection fraction, and fraction shortening, with no signs of cardiac hypertrophy. PTPRZ1 also seems to be involved in atrioventricular canal specification, outflow tract morphogenesis, and heart angiogenesis. These results suggest that PTPRZ1 plays a role in heart development and support the hypothesis that it may be involved in congenital cardiac pathologies.

Deletion of pleiotrophin impairs glucose tolerance and liver metabolism in pregnant mice: Moonlighting role of glycerol kinase.

Pleiotrophin is a pleiotropic cytokine that has been demonstrated to have a critical role in regulating energy metabolism, lipid turnover and plasticity of adipose tissue. Here, we hypothesize that this cytokine can be involved in regulatory processes of glucose and lipid homeostasis in the liver during pregnancy. Using 18-days pregnant Ptn-deficient mice, we evaluated the biochemical profile (circulating variables), tissue mRNA expression (qPCR) and protein levels of key enzymes and transcription factors involved in main metabolic pathways. Ptn deletion was associated with a reduction in body weight gain, hyperglycemia and glucose intolerance. Moreover, we observed an impairment in glucose synthesis and degradation during late pregnancy in Ptn-/- mice. Hepatic lipid content was significantly lower (73.6%) in Ptn-/- mice and was associated with a clear reduction in fatty acid, triacylglycerides and cholesterol synthesis. Ptn deletion was accompanying with a diabetogenic state in the mother and a decreased expression of key proteins involved in glucose and lipid uptake and metabolism. Moreover, Ptn-/- pregnant mice have a decreased expression of transcription factors, such as PPAR-α, regulating lipid uptake and glucose and lipid utilization. Furthermore, the augmented expression and nuclear translocation of glycerol kinase, and the decrease in NUR77 protein levels in the knock-out animals can further explain the alterations observed in hepatic glucose metabolism. Our results point out for the first time that pleiotrophin is an important player in maintaining hepatic metabolic homeostasis during late gestation, and further highlighted the moonlighting role of glycerol kinase in the regulation of maternal glucose homeostasis during pregnancy.

Role of Receptor Protein Tyrosine Phosphatase ß/ζ in Neuron-Microglia Communication in a Cellular Model of Parkinson's Disease.

Pleiotrophin (PTN) is a neurotrophic factor that regulates glial responses in animal models of different types of central nervous system (CNS) injuries. PTN is upregulated in the brain in different pathologies characterized by exacerbated neuroinflammation, including Parkinson's disease. PTN is an endogenous inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ, which is abundantly expressed in the CNS. Using a specific inhibitor of RPTPß/ζ (MY10), we aimed to assess whether the PTN/RPTPß/ζ axis is involved in neuronal and glial injury induced by the toxin MPP+. Treatment with the RPTPß/ζ inhibitor MY10 alone decreased the viability of both SH-SY5Y neuroblastoma cells and BV2 microglial cultures, suggesting that normal RPTPß/ζ function is involved in neuronal and microglial viability. We observed that PTN partially decreased the cytotoxicity induced by MPP+ in SH-SY5Y cells underpinning the neuroprotective function of PTN. However, MY10 did not seem to modulate the SH-SY5Y cell loss induced by MPP+. Interestingly, we observed that media from SH-SY5Y cells treated with MPP+ and MY10 decreases microglial viability but may elicit a neuroprotective response of microglia by upregulating Ptn expression. The data suggest a neurotrophic role of microglia in response to neuronal injury through upregulation of Ptn levels.

Pleiotrophin Deficiency Induces Browning of Periovarian Adipose Tissue and Protects against High-Fat Diet-Induced Hepatic Steatosis.

(1) Background: Pleiotrophin preserves insulin sensitivity, regulates adipose tissue lipid turnover and plasticity, energy metabolism and thermogenesis. The aim of this study was to determine the role of pleiotrophin in hepatic lipid metabolism and in the metabolic crosstalk between the liver and brown and white adipose tissue (AT) in a high-fat diet-induced (HFD) obesity mice model. (2) Methods: We analyzed circulating variables, lipid metabolism (hepatic lipid content and mRNA expression), brown AT thermogenesis (UCP-1 expression) and periovarian AT browning (brown adipocyte markers mRNA and immunodetection) in Ptn-/- mice either fed with standard-chow diet or with HFD and in their corresponding Ptn+/+ counterparts. (3) Results: HFD-Ptn-/- mice are protected against the development of HFD-induced insulin resistance, had lower liver lipid content and lower expression of the key enzymes involved in triacylglycerides and fatty acid synthesis in liver. HFD-Ptn-/- mice showed higher UCP-1 expression in brown AT. Moreover, Ptn deletion increased the expression of specific markers of brown/beige adipocytes and was associated with the immunodetection of UCP-1 enriched multilocular adipocytes in periovarian AT. (4) Conclusions: Ptn deletion protects against the development of HFD-induced insulin resistance and liver steatosis, by increasing UCP-1 expression in brown AT and promoting periovarian AT browning.

Inhibition of Receptor Protein Tyrosine Phosphatase ß/ζ Reduces Alcohol Intake in Rats.

BACKGROUND: Pleiotrophin (PTN) and midkine (MK) are cytokines that are up-regulated in the prefrontal cortex (PFC) after alcohol administration and have been shown to reduce alcohol intake and reward. Both cytokines are endogenous inhibitors of receptor protein tyrosine phosphatase (RPTP) ß/ζ (a.k.a. PTPRZ1). Recently, a new compound named MY10 was designed with the aim of mimicking the activity of PTN and MK. MY10 has already shown promising results regulating alcohol-related behaviors in mice. METHODS: We have now tested the effects of MY10 on alcohol operant self-administration and Drinking In the Dark-Multiple Scheduled Access (DID-MSA) paradigms in rats. Gene expression of relevant genes in the PTN/MK signaling pathway in the PFC was analyzed by real-time PCR. RESULTS: MY10, at the highest dose tested (100 mg/kg), reduced alcohol consumption in the alcohol operant self-administration paradigm (p = 0.040). In the DID-MSA paradigm, rats drank significantly less alcohol (p = 0.019) and showed a significant decrease in alcohol preference (p = 0.002). We observed that the longer the exposure to alcohol, the greater the suppressing effects of MY10 on alcohol consumption. It was demonstrated that the effects of MY10 were specific to alcohol since saccharin intake was not affected by MY10 (p = 0.804). MY10 prevented the alcohol-induced down-regulation of Ptprz1 (p = 0.004) and anaplastic lymphoma kinase (Alk; p = 0.013) expression. CONCLUSIONS: Our results support and provide further evidence regarding the efficacy of MY10 on alcohol-related behaviors and suggest the consideration of the blockade of RPTPß/ζ as a target for reducing excessive alcohol consumption.

Pleiotrophin deletion alters glucose homeostasis, energy metabolism and brown fat thermogenic function in mice.

AIMS/HYPOTHESIS: Pleiotrophin, a developmentally regulated and highly conserved cytokine, exerts different functions including regulation of cell growth and survival. Here, we hypothesise that this cytokine can play a regulatory role in glucose and lipid homeostasis. METHODS: To test this hypothesis, we performed a longitudinal study characterising the metabolic profile (circulating variables and tissue mRNA expression) of gene-targeted Ptn-deficient female mice and their corresponding wild-type counterparts at different ages from young adulthood (3 months) to older age (15 months). Metabolic cages were used to investigate the respiratory exchange ratio and energy expenditure, at both 24°C and 30°C. Undifferentiated immortalised mouse brown adipocytes (mBAs) were treated with 0.1 µg/ml pleiotrophin until day 6 of differentiation, and markers of mBA differentiation were analysed by quantitative real-time PCR (qPCR). RESULTS: Ptn deletion was associated with a reduction in total body fat (20.2% in Ptn+/+ vs 13.9% in Ptn-/- mice) and an enhanced lipolytic response to isoprenaline in isolated adipocytes from 15-month-old mice (189% in Ptn+/+ vs 273% in Ptn-/- mice). We found that Ptn-/- mice exhibited a significantly lower QUICKI value and an altered lipid profile; plasma triacylglycerols and NEFA did not increase with age, as happens in Ptn+/+ mice. Furthermore, the contribution of cold-induced thermogenesis to energy expenditure was greater in Ptn-/- than Ptn+/+ mice (42.6% and 33.6%, respectively). Body temperature and the activity and expression of deiodinase, T3 and mitochondrial uncoupling protein-1 in the brown adipose tissue of Ptn-/- mice were higher than in wild-type controls. Finally, supplementing brown pre-adipocytes with pleiotrophin decreased the expression of the brown adipocyte markers Cidea (20% reduction), Prdm16 (21% reduction), and Pgc1-α (also known as Ppargc1a, 11% reduction). CONCLUSIONS/INTERPRETATION: Our results reveal for the first time that pleiotrophin is a key player in preserving insulin sensitivity, driving the dynamics of adipose tissue lipid turnover and plasticity, and regulating energy metabolism and thermogenesis. These findings open therapeutic avenues for the treatment of metabolic disorders by targeting pleiotrophin in the crosstalk between white and brown adipose tissue.

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.

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.

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.

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.

Adolescent intermittent ethanol exposure decreases perineuronal nets in the hippocampus in a sex dependent manner: Modulation through pharmacological inhibition of RPTPß/ζ.

Adolescence is a critical period for brain maturation in which this organ undergoes critical plasticity mechanisms that increase its vulnerability to the effects of alcohol. Significantly, ethanol-induced disruption of hippocampal neurogenesis has been related to cognitive decline in adulthood. During adolescence, the maturation of perineuronal nets (PNNs), extracellular matrix structures highly affected by ethanol consumption, plays a fundamental role in neurogenesis and plasticity in the hippocampus. Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ is a critical anchor point for PNNs on the cell surface. Using the adolescent intermittent access to ethanol (IAE) model, we previously showed that MY10, a small-molecule inhibitor of RPTPß/ζ, reduces chronic ethanol consumption in adolescent male mice but not in females and prevents IAE-induced neurogenic loss in the male hippocampus. We have now tested if these effects of MY10 are related to sex-dependent modulatory actions on ethanol-induced effects in PNNs. Our findings suggest a complex interplay between alcohol exposure, neural structures, and sex-related differences in the modulation of PNNs and parvalbumin (PV)-positive cells in the hippocampus. In general, IAE increased the number of PV + cells in the female hippocampus and reduced PNNs intensity in different hippocampal regions, particularly in male mice. Notably, we found that pharmacological inhibition of RPTPß/ζ with MY10 regulates ethanol-induced alterations of PNNs intensity, which correlates with the protection of hippocampal neurogenesis from ethanol neurotoxic effects and may be related to the capacity of MY10 to increase the gene expression of key components of PNNs.

Periadolescent amphetamine treatment causes transient cognitive disruptions and long-term changes in hippocampal LTP depending on the endogenous expression of pleiotrophin.

Amphetamine treatment during adolescence causes long-term cognitive deficits in rats. Pleiotrophin (PTN) is a cytokine with important roles in the modulation of synaptic plasticity, whose levels of expression are significantly regulated by amphetamine administration. To test the possibility that the long-term consequences of periadolescent amphetamine treatment cross species and, furthermore, to test the hypothesis that PTN could be one of the factors involved in the adult cognitive deficits observed after periadolescent amphetamine administrations, we comparatively studied the long-term consequences of periadolescent amphetamine treatment (3 mg/kg intraperitoneal, daily during 10 days) in normal wild-type (PTN+/+) and in PTN genetically deficient (PTN-/-) mice. Within the first week after cessation of treatment, significant deficits in the passive avoidance and Y-maze tests were only observed in amphetamine-pretreated PTN-/- mice. However, 13 and 26 days after the last administration, we did not find significant differences in Y-maze between amphetamine- and saline-pretreated PTN-/- mice. In addition, we did not find any genotype- or treatment-related anxiogenic- or depressive-like behaviour in adult mice. Furthermore, we observed a significantly enhanced long-term potentiation (LTP) in CA1 hippocampal slices from saline-pretreated PTN-/- mice compared with saline-pretreated PTN+/+ mice. Interestingly, amphetamine pre-treatment during adolescence significantly enhanced LTP in adult PTN+/+ mice but did not cause any effect in PTN-/- mice, suggesting LTP mechanisms saturation in naïve PTN-/- mice. The data demonstrate that periadolescent amphetamine treatment causes transient cognitive deficits and long-term alterations of hippocampal LTP depending on the endogenous expression of PTN.

Pleiotrophin deficiency protects against high-fat diet-induced neuroinflammation: Implications for brain mitochondrial dysfunction and aberrant protein aggregation.

Metabolic Syndrome (MetS) is a risk factor for the development of neurodegenerative diseases. Neuroinflammation associated with MetS may contribute significantly to neurodegeneration. Pleiotrophin (PTN) is a neurotrophic factor that modulates neuroinflammation and is a key player in regulating energy metabolism and thermogenesis, suggesting that PTN could be important in the connection between MetS and neuroinflammation. We have now used a high-fat diet (HFD)-induced obesity model in Ptn-/- mice. HFD and Ptn deletion caused alterations in circulating hormones including GIP, leptin and resistin. HFD produced in Ptn+/+ mice a neuroinflammatory state as observed in cerebral quantifications of proinflammatory markers, including Il1ß, Tnfα and Ccl2. The upregulation of neuroinflammatory markers was prevented in Ptn-/- mice. Changes induced by HFD in genes related to mitochondrial biogenesis and dynamics were less pronounced in the brain of Ptn-/- mice and were accompanied by significant increases in the protein expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I and IV. HFD-induced changes in genes related to the elimination of protein aggregates were also less pronounced in the brain of Ptn-/- mice. This study provides substantial evidence that Ptn deletion protects against HFD-induced neuroinflammation, mitochondrial dysfunction, and aberrant protein aggregation, prominent features in neurodegenerative diseases.

Inhibition of RPTPß/ζ reduces chronic ethanol intake in adolescent mice and modulates ethanol effects on hippocampal neurogenesis and glial responses in a sex-dependent manner.

Pleiotrophin (PTN) is a cytokine that modulates ethanol drinking and reward and regulates glial responses in different contexts. PTN is an inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ. Inhibition of RPTPß/ζ reduces binge-like drinking in adult male mice. Whether inhibition of RPTPß/ζ is effective in reducing ethanol consumption during adolescence and in both sexes remained to be studied. In this work, male and female adolescent mice underwent an intermittent access to ethanol (IAE) 2-bottle choice protocol. Treatment with MY10 (60 mg/kg, i.g.), a small-molecule RPTPß/ζ inhibitor, reduced chronic 3-week ethanol consumption only in male mice. We detected an ethanol-induced overall decrease in hippocampal GFAPir and Iba1ir, independently of the treatment received, suggesting that RPTPß/ζ is not key in the regulation of IAE-induced glial responses. However, we found a significant negative correlation between the size of microglial cells and the number of hippocampal neuronal progenitors only in male mice after IAE. This correlation was disrupted by treatment with MY10 before each drinking session, which may be related to the ability of MY10 to regulate the intensity of the perineuronal nets (PNNs) in the hippocampus in a sex-dependent manner. The data show for the first time that inhibition of RPTPß/ζ reduces chronic voluntary ethanol consumption in adolescent mice in a sex-dependent manner. In addition, we show evidence for sex-specific differences in the effects of IAE on glial responses and hippocampal neurogenesis, which may be related to different actions of the RPTPß/ζ signalling pathway in the brains of male and female mice.

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.

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.

Peripheral modulation of antidepressant targets MAO-B and GABAAR by harmol induces mitohormesis and delays aging in preclinical models.

Reversible and sub-lethal stresses to the mitochondria elicit a program of compensatory responses that ultimately improve mitochondrial function, a conserved anti-aging mechanism termed mitohormesis. Here, we show that harmol, a member of the beta-carbolines family with anti-depressant properties, improves mitochondrial function and metabolic parameters, and extends healthspan. Treatment with harmol induces a transient mitochondrial depolarization, a strong mitophagy response, and the AMPK compensatory pathway both in cultured C2C12 myotubes and in male mouse liver, brown adipose tissue and muscle, even though harmol crosses poorly the blood-brain barrier. Mechanistically, simultaneous modulation of the targets of harmol monoamine-oxidase B and GABA-A receptor reproduces harmol-induced mitochondrial improvements. Diet-induced pre-diabetic male mice improve their glucose tolerance, liver steatosis and insulin sensitivity after treatment with harmol. Harmol or a combination of monoamine oxidase B and GABA-A receptor modulators extend the lifespan of hermaphrodite Caenorhabditis elegans or female Drosophila melanogaster. Finally, two-year-old male and female mice treated with harmol exhibit delayed frailty onset with improved glycemia, exercise performance and strength. Our results reveal that peripheral targeting of monoamine oxidase B and GABA-A receptor, common antidepressant targets, extends healthspan through mitohormesis.

Metabolomics and biochemical alterations caused by pleiotrophin in the 6-hydroxydopamine mouse model of Parkinson's disease.

Pleiotrophin (PTN) is a cytokine involved in nerve tissue repair processes, neuroinflammation and neuronal survival. PTN expression levels are upregulated in the nigrostriatal pathway of Parkinson's Disease (PD) patients. We aimed to characterize the dopaminergic injury and glial responses in the nigrostriatal pathway of mice with transgenic Ptn overexpression in the brain (Ptn-Tg) after intrastriatal injection of the catecholaminergic toxic 6-hydroxydopamine (6-OHDA) at a low dose (5 µg). Ten days after surgery, the injection of 6-OHDA induced a significant decrease of the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and of the striatal TH contents in Wild type (Wt) mice. In contrast, these effects of 6-OHDA were absent in Ptn-Tg mice. When the striatal Iba1 and GFAP immunoreactivity was studied, no statistical differences were found between vehicle-injected Wt and Ptn-Tg mice. Furthermore, 6-OHDA did not cause robust glial responses neither on Wt or Ptn-Tg mice 10 days after injections. In metabolomics studies, we detected interesting metabolites that significantly discriminate the more injured 6-OHDA-injected Wt striatum and the more protected 6-OHDA-injected Ptn-Tg striatum. Particularly, we detected groups of metabolites, mostly corresponding to phospholipids, whose trends were opposite in both groups. In summary, the data confirm lower 6-OHDA-induced decreases of TH contents in the nigrostriatal pathway of Ptn-Tg mice, suggesting a neuroprotective effect of brain PTN overexpression in this mouse model of PD. New lipid-related PD drug candidates emerge from this study and the data presented here support the increasingly recognized "lipid cascade" in PD.