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.

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 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.

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.

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.

The use of gene array technology and proteomics in the search of new targets of diseases for therapeutics.

The advent of functional genomics has been greatly broadening our view and accelerating our way in numerous medical research fields. The complete genomic data acquired from the human genome project and the desperate clinical need of comprehensive analytical tools to study complex diseases, has allowed rapid evolution of genomic and proteomic technologies, speeding the rate and number of discoveries in new biomarkers. By jointly using genomics, proteomics and bioinformatics there is a great potential to make considerable contribution to biomarker identification and to revolutionize both the development of new therapies and drug development process.

Grafted neural stem cells increase the life span and protect motoneurons in pmn mice.

In this study, we have grafted neural stem cells (NSCs) into the lumbar spinal cord of a mouse mutant that has a specific loss of motoneurons (progressive motor neuronopathy/pmn). A small number of grafted cells ( approximately 3000) increased the life span of the mice by 56%. The improved survival was accompanied by a rescue of host motoneurons, a stabilization in the weight and an increase in the size of the muscle fibers. The grafted NSCs were small and round and exhibited no neural markers, suggesting that they remained in an undifferentiated state. Thus grafting of NSCs in a mouse model with motoneuron degeneration exerts a neuroprotective effect.

Deglycosylation of Fas receptor and chronic morphine treatment up-regulate high molecular mass Fas aggregates in the rat brain.

This study was designed to immunodetect and characterize Fas receptor aggregates (oligomerization) in the brain and to assess its possible modulation in opiate addiction. High molecular mass, sodium dodecyl sulfate (SDS)- and beta-mercaptoethanol-resistant Fas aggregates (approximately 110/120 and approximately 203 kDa specific peptides) were immunodetected with a cytoplasmic domain-specific antibody in brain tissue (rat, mouse and human) and SH-SY5Y cells by Western blot analysis. Preincubation of rat cortical membranes with N-ethylmaleimide (NEM; 1 mM for 1 h at 37 degrees C) reduced the immunodensity of approximately 203 kDa Fas aggregates (51%) and increased that of 35 kDa native Fas (172%) and 51/48 kDa glycosylated Fas (47%), indicating that disulfide bonds are involved in Fas dimerization. Enzymatic N-deglycosylation of Fas receptor increased the content of Fas aggregates (approximately 110/120 kDa: five- to sixfold, and approximately 203 kDa: two- to threefold), suggesting that Fas glycosylation is involved in regulating receptor dimerization. Chronic (10-100 mg/kg for 5 days), but not acute (30 mg/kg for 2 h), treatment with morphine (a micro-opioid peptide receptor agonist) induced up-regulation of Fas aggregates in the brain (approximately 110/120 kDa: 39%, and approximately 203 kDa: 89%). The acute and/or chronic treatments with delta- and kappa-opioid peptide receptor agonists and with a sigma1-receptor agonist did not readily alter the content of Fas aggregates in the rat brain. The results indicate that Fas aggregates are natively expressed in the brain and that its density is regulated by the state of Fas glycosylation. These forms of Fas (receptor homodimerization) are functionally relevant because they were up-regulated in the brain of morphine-dependent rats.

Motoneuron resistance to apoptotic cell death in vivo correlates with the ratio between X-linked inhibitor of apoptosis proteins (XIAPs) and its inhibitor, XIAP-associated factor 1.

Apoptotic cell death occurs in motoneurons in the neonate but not in the adult after a lesion of a peripheral nerve. To investigate the molecular basis for this difference, we have analyzed the expression and localization of inhibitors of apoptosis proteins (IAPs) and their inhibitors X-linked IAP (XIAP)-associated factor 1 (XAF1), Smac/DIABLO, and Omi/HtrA2 in motoneurons at both ages. Quantitative immunohistochemical and immunoblotting analysis of these proteins in motoneurons revealed an increase in IAP expression [XIAP, neuronal apoptosis inhibitory protein, human IAP1 (HIAP1), and HIAP2] during postnatal development as opposed to XAF1, which decreased during the same period; there was no significant alteration in either Smac/DIABO or Omi/HtrA2. The regulation of IAPs and XAF1 varied after axotomy of the sciatic nerve; in the neonate, there was a significant loss of IAP in the injured motoneurons as opposed to the adult, in which there was only a moderate decrease. By overexpressing exogenous IAPs in neonatal axotomized motoneurons, it was possible to delay motoneuron cell death (Perrelet et al., 2000, 2002). In opposition, the overexpression of exogenous XAF1 in adult motoneurons totally abrogated the natural resistance of these cells to axotomy. The degradation in the adult, induced by XAF1, could be overcome by simultaneously expressing high levels of exogenous XIAP in adult motoneurons. These experiments suggest that it may be the ratio between XAF1 and XIAP that confers the resistance of adult motoneurons to axotomy. In addition, the regulation in the levels of IAPs and XAF1 may be essential in the cell death mechanism of injured motoneurons.

Decreased immunodensities of micro-opioid receptors, receptor kinases GRK 2/6 and beta-arrestin-2 in postmortem brains of opiate addicts.

The homologous regulation of opioid receptors, through G protein-coupled receptor kinases (GRKs) and beta-arrestins, is an initial step in the complex molecular mechanisms leading to opiate tolerance and dependence. This study was designed to evaluate in parallel the contents of immunolabeled micro-opioid receptors (glycosylated proteins), two representative GRKs (GRK 2 and GRK 6) and beta-arrestin-2 in brains of opiate addicts who had died of an opiate overdose (heroin or methadone). The immunodensities of micro-opioid receptors were decreased (66 kDa protein: 24%, n=24, P<0.0001; 85 kDa protein: 16%, n=24, P<0.05) in the prefrontal cortex of opiate addicts compared with sex-, age-, and PMD-matched controls. This down-regulation of brain micro-opioid receptors was more pronounced in opiate addicts dying of a heroin overdose (27-30%, n=13) than in those who died of a methadone overdose (5-16%, n=11). In the same brains, significant decreases in the immunodensities of GRK 2 (19%, n=24, P<0.05), GRK 6 (25%, n=24, P<0.002) and beta-arrestin-2 (22%, n=24, P< 0.0005) were also quantitated. In contrast, the content of alpha-internexin (a neuronal marker used as a negative control) was not changed in brains of opiate addicts. In these subjects, there was a significant correlation between the densities of GRK 6 and beta-arrestin-2 (r=0.63, n=24, P=0.001), suggesting that both proteins are regulated in a coordinated manner by opiate drugs in the brain. The results indicate that opiate addiction in humans (tolerant state) is associated with down-regulation of brain micro-opioid receptors and regulatory GRK 2/6 and beta-arrestin-2 proteins. These molecular adaptations may be relevant mechanisms for the induction of opiate tolerance in brains of opiate addicts.

Regulation of platelet alpha 2A-adrenoceptors, Gi proteins and receptor kinases in major depression: effects of mirtazapine treatment.

Major depression is associated with the upregulation of alpha(2A)-adrenoceptors in brain tissue and blood platelets. The homologous regulation of these receptors by G-protein-coupled receptor kinases (GRKs) might play a relevant role in the pathogenesis and treatment of depression. This study was designed to assess the status of the complex alpha(2A)-adrenoceptor/Galphai/GRK 2 in the platelets of depressed patients (n=22) before and after treatment with the antidepressant mirtazapine, an antagonist at alpha(2A)-adrenoceptors (30-45 mg/day for up to 6 months). A second series of depressed suicide attempters (n=32) were also investigated to further assess the status of platelet GRK 2 and GRK 6. Platelet alpha(2A)-adrenoceptors and Galphai protein immunoreactivities were increased in depressed patients (49 and 35%) compared with matched controls. In contrast, GRK 2 content was decreased in the two series of depressed patients (27 and 28%). GRK 6 (a GRK with different properties) was found unchanged. In drug-free depressed patients, the severity of depression (behavioral ratings with two different instruments) correlated inversely with the content of platelet GRK 2 (r=-0.46, n=22, p=0.032, and r=-0.55, n=22, p=0.009). After 4-24 weeks of treatment, mirtazapine induced downregulation of platelet alpha(2A)-adrenoceptors (up to 34%) and Galphai proteins (up to 28%), and the upregulation of GRK 2 (up to 30%). The results indicate that major depression is associated with reduced platelet GRK 2, suggesting that a defect of this kinase may contribute to the observed upregulation of alpha(2A)-adrenoceptors. Moreover, treatment with mirtazapine reversed this abnormality and induced downregulation of alpha(2A)-adrenoceptor/Galphai complex. The results support a role of supersensitive alpha(2A)-adrenoceptors in the pathogenesis and treatment of major depression.

Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain.

The Fas receptor is involved in the regulation of apoptosis but also can function as a non-apoptotic signal transducer. This study was mainly designed to quantitate Fas proteins in rat brain during heroin addiction and opiate withdrawal. In rat, mouse and human brains, and in SH-SY5Y cells, similar forms of Fas were immunodetected with different antibodies (i.e., 35 kDa native Fas and 48- and 51-kDa glycosylated Fas). Acute (2 h) treatments with the micro-opioid receptor agonists heroin (10 mg/kg) and morphine (30 mg/kg) increased the immunodensity of native Fas (124% and 36%) but not that of glycosylated Fas in the cerebral cortex. Chronic (5 days) heroin (5-30 mg/kg) and morphine (10-100 mg/kg) were also associated with increased native Fas (76% and 45%) and with different expressions of glycosylated Fas. In heroin-dependent rats, opiate withdrawal (48 h) resulted in a sustained increase in native Fas (107%) and in up-regulation of 51 kDa glycosylated Fas (51%). Acute treatments with selective delta-receptor (SNC-80, 10 mg/kg) or kappa-receptor (U 50488-H, 10 mg/kg) agonists did not alter the content of native or glycosylated Fas. Chronic pentazocine (10-80 mg/kg, 5 days), a mixed opiate drug and sigma(1) receptor agonist, decreased native (48%) and glycosylated (38-82%) Fas proteins. Similarly, the selective sigma(1) agonist (+)-SKF 10047 also decreased native Fas (37%) and the effect was blocked by the sigma(1) antagonist BD 1063. Brain dynamin was up-regulated by acute and/or chronic heroin (30-39%), morphine (47-85%), pentazocine (51%) and heroin withdrawal (74%). The main results indicate that chronic heroin/morphine treatment and heroin withdrawal are associated with up-regulation of 35 kDa native Fas (and with different expressions of glycosylated Fas), and also with concomitant increases of dynamin in rat brain.

Regulation of GRK 2 and 6, beta-arrestin-2 and associated proteins in the prefrontal cortex of drug-free and antidepressant drug-treated subjects with major depression.

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK 2, GRK 6, beta-arrestin-2 and associated proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in parallel (immunodensity with specific antibodies) in brains of depressed subjects (drug-free and antidepressant-treated) to investigate the effect of major depression and antidepressant drugs on these receptor regulatory proteins. Specimens of the prefrontal cortex (Brodmann's area 9) were collected from 19 suicide and non-suicide depressed subjects and 13 control subjects. In drug-free (n=9), but not in antidepressant-treated (n=10), depressed subjects an increase in the density of membrane-associated GRK 2 (30%, n=9, P=0.005) was found compared with that in sex-, age-, and PMD-matched controls. Comparison between drug-free and antidepressant-treated depressed subjects showed that GRK 2 was reduced in membrane (39%, n=10, P=0.008) and cytosolic (44%, n=10, P=0.09) preparations after antidepressant drug treatment. In contrast, membrane-associated GRK 6 (drug-free and antidepressant-treated depressed subjects) was found unchanged when compared with that in matched controls. Similarly, the densities of beta-arrestin-2, PP-2A, and Gbeta proteins were not significantly different from those in matched controls. There was a positive correlation between the immunodensities of GRK 2 and beta-arrestin-2 in membrane preparations (r=0.48, n=19, P=0.04), suggesting that both proteins are regulated in a coordinated manner in brains of depressed subjects. The results of this study indicate that major depression is associated with upregulation of brain GRK 2, but not GRK 6, and that antidepressant drug treatment appears to induce downregulation of GRK 2 protein.

Downregulation of neuronal cdk5/p35 in opioid addicts and opiate-treated rats: relation to neurofilament phosphorylation.

Neuronal cyclin-dependent kinase-5 (Cdk5) and its neuron-specific activator p35 play a major role in regulating the cytoskeleton dynamics. Since opioid addiction was associated with hyperphosphorylation of neurofilament (NF) in postmortem human brains, this study was undertaken to assess the status of the cdk5/p35 complex and its relation with NF-H phosphorylation in brains of chronic opioid abusers. Decreased immunodensities of cdk5 (18%) and p35 (26-44%) were found in the prefrontal cortex of opioid addicts compared with matched controls. In the same brains, the densities of p25 (a truncated neurotoxic form of p35), phosphatase PP2Ac and mu-calpain were found unaltered. Acute treatment of rats with morphine (30 mg/kg, 2 h) increased the density of cdk5 (35%), but not that of p35, in the cerebral cortex. In contrast, chronic morphine (10-100 mg/kg for 5 days) induced marked decreases in cdk5 (40%) and p35 (47%) in rat brain. In brains of opioid addicts, the density of phosphorylated NF-H was increased (43%) as well as the ratio of phosphorylated to nonphosphorylated NF-H forms (two-fold). In these brains, phosphorylated NF-H significantly correlated with p35 (r=0.58) but not with cdk5 (r=0.03). The results suggest that opiate addiction is associated with downregulation of cdk5/p35 levels in the brain. This downregulation and the aberrant hyperphosphorylation of NF-H proteins might have important consequences in the development of neural plasticity associated with opiate addiction in humans.

In vivo effects of the I(2)-alkylating agent BU99006 on the immunodensity of imidazoline receptor proteins in the mouse brain.

The binding sites for imidazol(ine)/guanidine drugs (identified inter alia with [(3)H]-clonidine and [(3)H]-idazoxan) are heterogeneous in nature, and various pharmacologic types of imidazoline receptors (IRs) have been characterized (I(1)R, I(2A)R, I(2B)R, and I(3)R). IR-receptor proteins have also been immunodetected using an antibody raised against an approximately 70-kD idazoxan/clonidine binding protein, which probably recognizes all types of IRs. In this study, the in vivo effects of the selective I(2)-alkylating agent BU99006 (5-isothiocyanato-2-benzofuranyl-2-imidazoline) on immunoreactive IR proteins were assessed in the mouse brain to unravel the molecular nature of the I(2)R subtypes. In mouse tissues (cerebral cortex, liver, testis, and kidney) this antibody revealed the presence of 30-, 43-, 45-, 66-, and/or 85-kD IR proteins. Treatment with BU99006 (20 mg/kg, intraperitoneally, for 4 hours) significantly decreased the immunodensity of specific IR proteins in the brain (30 kD: 249%; 45 kD: 237%; 66 kD: 218%). In contrast, the immunoreactivities of 43-kD and 85-kD IR proteins were not altered after I(2)R alkylation. Prolonged treatment with BU99006 (20 mg/kg, intraperitoneally. for 8 hours) resulted in modest but significant increases in the expression of all the immunodetected IR proteins in the mouse brain (20%-36%), suggesting compensatory increases in IR protein synthesis after alkylation of I(2) sites. The results indicate that the 30-kD and 45-kD proteins, but not the 43-kD and 85-kD proteins, immunodetected in the mouse brain are related to the I(2)R.

G protein-coupled receptor kinases, beta-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution.

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK2, GRK6, beta-arrestin-2 and associated regulatory proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in human brains (immunodensity with specific antibodies) to assess for postmortem changes (pattern of protein degradation) and to investigate the effect of aging on these regulatory proteins as well as their subcellular distribution (cytosol and membrane fractions). In brain (prefrontal cortex, total homogenate) of healthy subjects (n=14) the immunodensities of GRK2 (r=-0.76), GRK6 (r=-0.64), beta-arrestin-2 (r=-0.57), Gbeta proteins (r=-0.59) and neurofilament (NF)-L (r=-0.64), but not PP-2A, declined markedly with the length of postmortem delay (PMD, 3-81 h). With these linear decay models, the average decreases per 12 h of PMD (from 12 to 72 h) were 7-11% for the various proteins. The immunodensities of GRK2 (r=-0.71), GRK6 (r=-0.61), and beta-arrestin-2 (r=-0.54) in human brain (n=12) also declined with aging (16 to 87 years) and the average decreases per decade (from 20 to 80 years) were 3-5%. In contrast, the immunodensities of PP-2A, Gbeta and NF-L in brain did not correlate significantly with the age of the subject at death (16-87 years). The immunodensities of GRK2/6 and beta-arrestin-2 showed marked individual variations and were strongly reduced after several freeze/thaw cycles. In the prefrontal cortex the subcellular distribution (cytosol/membrane) of the two GRKs differed markedly (GRK2: 60%/40%; GRK6: 5%/95%), and that of beta-arrestin-2 was as expected for a soluble protein (60%/40%). In brains of healthy subjects, the immunodensities of cytosolic GRK2 and beta-arrestin-2 correlated, respectively, with those of membrane-associated GRK2 (r=0.67, P=0.049, n=9) and membrane-associated beta-arrestin-2 (r=0.77, P=0.01, n=9). The results of this study emphasize the importance of examining relevant variables (PMD, age) and potential artifacts (individual variation, freeze-thawing effect) when designing signal transduction studies in neuropsychiatric disorders using the postmortem human brain.