APOE in the bullseye of neurodegenerative diseases: impact of the APOE genotype in Alzheimer's disease pathology and brain diseases.

ApoE is the major lipid and cholesterol carrier in the CNS. There are three major human polymorphisms, apoE2, apoE3, and apoE4, and the genetic expression of APOE4 is one of the most influential risk factors for the development of late-onset Alzheimer's disease (AD). Neuroinflammation has become the third hallmark of AD, together with Amyloid-ß plaques and neurofibrillary tangles of hyperphosphorylated aggregated tau protein. This review aims to broadly and extensively describe the differential aspects concerning apoE. Starting from the evolution of apoE to how APOE's single-nucleotide polymorphisms affect its structure, function, and involvement during health and disease. This review reflects on how APOE's polymorphisms impact critical aspects of AD pathology, such as the neuroinflammatory response, particularly the effect of APOE on astrocytic and microglial function and microglial dynamics, synaptic function, amyloid-ß load, tau pathology, autophagy, and cell-cell communication. We discuss influential factors affecting AD pathology combined with the APOE genotype, such as sex, age, diet, physical exercise, current therapies and clinical trials in the AD field. The impact of the APOE genotype in other neurodegenerative diseases characterized by overt inflammation, e.g., alpha- synucleinopathies and Parkinson's disease, traumatic brain injury, stroke, amyotrophic lateral sclerosis, and multiple sclerosis, is also addressed. Therefore, this review gathers the most relevant findings related to the APOE genotype up to date and its implications on AD and CNS pathologies to provide a deeper understanding of the knowledge in the APOE field.

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.

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.

Role of RPTPß/ζ in neuroinflammation and microglia-neuron communication.

Pleiotrophin (PTN) is a cytokine that is upregulated in different neuroinflammatory disorders. Using mice with transgenic PTN overexpression in the brain (Ptn-Tg), we have found a positive correlation between iNos and Tnfα mRNA and Ptn mRNA levels in the prefrontal cortex (PFC) of LPS-treated mice. PTN is an inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ, which is mainly expressed in the central nervous system. We aimed to test if RPTPß/ζ is involved in the modulation of neuroinflammatory responses using specific inhibitors of RPTPß/ζ (MY10 and MY33-3). Treatment with MY10 potentiated LPS-induced microglial responses in the mouse PFC. Surprisingly, MY10 caused a decrease in LPS-induced NF-κB p65 expression, suggesting that RPTPß/ζ may be involved in a novel mechanism of potentiation of microglial activation independent of the NF-κB p65 pathway. MY33-3 and MY10 limited LPS-induced nitrites production and iNos increases in BV2 microglial cells. SH-SY5Y neuronal cells were treated with the conditioned media from MY10/LPS-treated BV2 cells. Conditioned media from non-stimulated and from LPS-stimulated BV2 cells increased the viability of SH-SY5Y cultures. RPTPß/ζ inhibition in microglial cells disrupted this neurotrophic effect of microglia, suggesting that RPTPß/ζ plays a role in the neurotrophic phenotype of microglia and in microglia-neuron communication.

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.

Inhibition of RPTPß/ζ blocks ethanol-induced conditioned place preference in pleiotrophin knockout mice.

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 endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ. Interestingly, pharmacological inhibition of RPTPß/ζ reduces ethanol consumption and blocks ethanol-induced conditioned place preference (CPP) in wild type mice. Since PTN-knockout (Ptn-/-) mice are more sensitive to the conditioning effects of alcohol, we aimed to test the effects of MY10, a small-molecule inhibitor of RPTPß/ζ, on ethanol-induced CPP in Ptn-/- mice. The data presented here demonstrate for the first time that a regular dose of MY10, known to block ethanol consumption and reward in wild type mice, also blocks the rewarding effects of ethanol in the more vulnerable individuals lacking PTN, the endogenous inhibitor of RPTPß/ζ. In addition, since MY10 readily penetrates the blood brain barrier (BBB), we tested its effects in a series of behavioural tests in Ptn+/+ and Ptn-/- mice. The data indicate that MY10 does not cause gross behavioural effects in wild type mice. However, MY10 tended to induce anxiolytic effects in Ptn-/- mice in the elevated plus maze paradigm. Overall, the data indicate that MY10 rescues Ptn-/- mice from their increased susceptibility to the conditioning effects of ethanol and may induce anxiolytic effects in individuals with reduced or absent PTN functions. Further studies are needed to confirm the potential of pharmacological inhibition of RPTPß/ζ as a new therapeutic strategy in the treatment of anxiety-related disorders.

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.

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.