Alfredo Ramos-Miguel

Opioid receptor agonists enhance the phosphorylation state of Fas-associated death domain (FADD) protein in the rat brain: Functional interactions with casein kinase Iα, Gαi proteins, and ERK1/2 signaling

Opioid drugs have been proposed to promote anti-apoptotic signals in brain through inhibition of FADD protein [García-Fuster et al., 2007. Effects of opiate drugs on Fas-associated protein with death domain (FADD) and effector caspases in the rat brain: Regulation by the ERK1/2 MAP kinase pathway. Neuropsychopharmacology 32, 399-411]. FADD phosphorylation by casein kinase Ialpha (CKIalpha) appears to regulate its non-apoptotic activity. This study investigated the effects of opioids on p-FADD in rat brain, as well as various mechanisms that could link opioid receptors with p-FADD, including the modulation of CKIalpha, Galpha(i) proteins and ERK1/2 signaling. In rat, mouse and human brains, various anti-p-FADD antibodies immunodetected the monomeric and oligomeric forms of this protein, irrespective of the antibody origin and specific Ser191 or Ser194 phosphorylation site. Acute mu- and delta-agonists increased, through specific opioid receptor mechanisms, the content of oligomeric and monomeric p-FADD forms in rat cortical homogenates (25-61%) and subcellular compartments, with most relevant effects for sufentanil in membrane (239%) and nucleus (136%). p-FADD induction vanished with repeated (5days) morphine but not SNC-80, and opioid withdrawal induced a new (morphine) or sustained (SNC-80) stimulatory effect (32-33%). The kappa-agonist (-)-U-50488H failed to stimulate p-FADD. Sufentanil reduced CKI protein and kinase activity in the cytosol (30-37%). Morphine, but not SNC-80, augmented CKIalpha in cytosol, membrane and nucleus (36-104%). In contrast to FADD, the ability of SNC-80 to stimulate p-FADD was not sensitive to ERK1/2 blockade. Pertussis toxin did not prevent the opposite effects of SNC-80 on p-FADD and FADD because the toxin by itself markedly altered their basal contents, indicating that FADD could be a novel toxin target. The upregulation of p-FADD induced by mu/delta-agonists could play a relevant role in the anti-apoptotic and/or neuroplastic effects of opioids.

The time course of unconditioned morphine-induced psychomotor sensitization mirrors the phosphorylation of FADD and MEK/ERK in rat striatum: role of PEA-15 as a FADD-ERK binding partner in striatal plasticity.

Drugs of abuse induce behavioral neuroadaptations whose molecular mechanisms, partly known, are crucial to understanding drug addictions. The multifunctional adaptor Fas-associated protein with death domain (FADD) was recently associated with the induction of neuroplasticity. This study investigated the modulation of FADD and MAP kinase signaling, as well as their interactions with PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) and Akt1 pathways, during the expression of unconditioned morphine-induced psychomotor sensitization. In morphine-pretreated rats (10mg/kg during 5 days), a challenge dose of the opiate induced a robust psychomotor sensitization at early withdrawal (3 days, SW 3), but not after a prolonged abstinence period (14 days), which was coincident with an accelerated dopamine turnover in the striatum. Marked concomitant increases in the content of p-FADD (48%) and the activation of MEK-ERK (46-79%) were quantified during the short-term expression of morphine sensitization (SW 3, in the absence of morphine challenge). At SW 3, p-PEA-15, a FADD-ERK binding partner, was also upregulated (51%) as well as the activation of its phosphorylating Akt1 kinase (49%). Notably, the MEK inhibitor SL 327 attenuated (58%) the expression of morphine-induced psychomotor sensitization (SW 3) and fully prevented the upregulation of p-FADD, p-PEA-15 and p-Akt1 at SW 3. The results indicate that the activation of MEK/ERK, the upregulation of p-FADD and that of the linking partners PEA-15/Akt1 have a major role in mediating the short-lasting expression of unconditioned psychomotor sensitization induced by morphine in rats.

Reduced platelet G protein-coupled receptor kinase 2 in major depressive disorder: Antidepressant treatment-induced upregulation of GRK2 protein discriminates between responder and non-responder patients

The homologous regulation of neurotransmitter receptors by G protein-coupled receptor kinases (GRKs) is important in the pathogenesis and treatment of major depressive disorder (MDD). Previous studies have reported that the basal status of GRK2 is different in brains (upregulation) and platelets (downregulation) of subjects with MDD. The principal aim of this study was to re-examine the status of platelet membrane GRK2 protein in patients with MDD, along with GRK3 (a close kinase homolog) and GRK5 (a kinase with different properties), before and after treatment with serotonin-selective reuptake inhibitor (SSRI) or serotonin noradrenaline reuptake inhibitor (SNRI) antidepressants. The main findings indicated that platelet GRK2 and p-Ser670 GRK2 were reduced (36-41%) in unmedicated MDD subjects, and that GRK2 content correlated inversely with the severity of depression (r=-0.51). Effective antidepressant treatments normalized platelet GRK2, and, notably, GRK2 upregulation discriminated between responder and non-responder patients. Other findings revealed a modest reduction of platelet GRK3 (23%) and no alteration of platelet GRK5 content. In untreated subjects with MDD, lymphocyte GRK2 and GRK5 mRNAs were unaltered but antidepressant treatment-induced upregulation of GRK2 mRNA expression. The reduced content of platelet GRK2 protein is a relevant target in MDD. Although this peripheral GRK2 defect does not mirror the canonical regulation of brain GRK2 in depressed suicides, it could well represent a disease state marker as well as a surrogate of response to effective antidepressant treatment.

Regulation of munc18-1 and syntaxin-1A interactive partners in schizophrenia prefrontal cortex: down-regulation of munc18-1a isoform and 75 kDa SNARE complex after antipsychotic treatment

Munc18-1 and syntaxin-1 are crucial interacting molecules for synaptic membrane fusion and neurotransmitter release. Contrasting abnormalities of several proteins of the exocytotic machinery, including the formation of SNARE (synaptobrevin, SNAP-25 and syntaxin-1) complexes, have been reported in schizophrenia. This study quantified in the dorsolateral prefrontal cortex (PFC, Brodmann area 9) the immunocontent of munc18-1a/b isoforms, syntaxin-1A, other presynaptic proteins (synaptotagmin, synaptophysin), and SNARE complexes, as well as the effects of psychoactive drug exposure, in schizophrenia (SZ, n=24), non-schizophrenia suicide (SD, n=13) and major depression (MD, n=15) subjects compared to matched controls (n=39). SZ was associated with normal expression of munc18-1a/b and increased syntaxin-1A (+44%). The presence of antipsychotic drugs reduced the basal content of munc18-1a isoform (-23%) and synaptobrevin (-32%), and modestly reduced that of up-regulated syntaxin-1A (-16%). Munc18-1a and syntaxin-1A protein expression correlated positively in controls but showed a markedly opposite pattern in SZ, regardless of antipsychotic treatment. Thus, the ratio of syntaxin-1A to munc18-1a showed a net increase in SZ (+53/114%). The SNARE complex (75 kDa) was found unaltered in antipsychotic-free and reduced (-28%) in antipsychotic-treated SZ subjects. None of these abnormalities were observed in SD and MD subjects, unexposed or exposed to psychoactive drugs. The results reveal some exocytotic dysfunctions in SZ that are probably related to an imbalance of the interaction between munc18-1a and SNARE (mainly syntaxin-1A) complex. Moreover, antipsychotic drug treatment is associated with lower content of key proteins of the exocytotic machinery, which could result in a destabilization/impairment of neurosecretion.

Substituting a long-acting dopamine uptake inhibitor for cocaine prevents relapse to cocaine seeking

The treatment of cocaine addiction remains a challenge. The dopamine replacement approach in cocaine addiction involves the use of a competing dopaminergic agonist that might suppress withdrawal and drug craving in abstinent individuals. Although it has long been postulated that such an approach may be therapeutically successful, preclinical or clinical evidence showing its effectiveness to prevent relapse is scant. We used in rats a procedure that involved substitution of the N‐substituted benztropine analog 3α‐[bis(4′‐fluorophenyl)methoxy]‐tropane (AHN‐1055), a long‐acting dopamine uptake inhibitor (DUI), for cocaine. Maintenance treatment was self‐administered. After extinction, reinstatement of drug seeking was induced by cocaine priming. We measured the contents of brain‐derived neurotrophic factor (BDNF), c‐Fos and Fas‐associated death domain (FADD) proteins in the medial prefrontal cortex (mPFC) following reinstatement. DUI, but not amphetamine, substitution led to extinction of active lever presses, as did saline substitution. DUI substitution significantly reduced cocaine‐induced reinstatement of drug‐seeking behavior, which was strongly elicited after saline substitution. Rats passively yoked to DUI also showed reduced cocaine‐primed reinstatement. Reductions in drug seeking during reinstatement were matched by downward shifts in the contents of BDNF, c‐Fos and FADD proteins in the mPFC, which were elevated in relapsing rats. These data indicate that DUI substitution not only leads to extinction of self‐administration behavior but also prevents reinstatement of drug seeking induced by cocaine re‐exposure. Thus, DUI substitution therapy using compounds with low abuse potential, even if received passively in the context previously paired with drug taking, may provide an effective treatment for stimulant addiction.

Correlation of rat cortical Fas-associated death domain (FADD) protein phosphorylation with the severity of spontaneous morphine abstinence syndrome: role of α2-adrenoceptors and extracellular signal-regulated kinases

Fas-associated death domain (FADD) phosphorylation was recently implicated in opiate-induced neuroplasticity. To further explore the role of FADD in the mechanisms of morphine-induced physical dependence, the regulation of cortical p-FADD (and their interactions with α2-adrenoceptors and other signalling pathways) was assessed during spontaneous opiate withdrawal (SW) in morphine-dependent rats (10–100 mg/kg for 6 days). The main results indicated that oligomeric p-FADD in the cerebral cortex mirrored the time course of morphine SW (12–96 h), which resulted in a striking correlation between p-FADD and the intensity (behavioural scores) of morphine abstinence (Spearman correlation coefficient: 0.59, n = 39, p < 0.0001). The inactivation of brain α2-adrenoceptors (EEDQ at SW 12 h) further enhanced morphine abstinence intensity and cortical p-FADD content at SW 24 h. The disruption of ERK1/2 signalling (SL 327 at SW 4 h and SW 8 h) did not alter morphine abstinence at SW 12 h, but it attenuated the behavioural syndrome at SW 24 h. This inhibition of ERK1/2, however, did not prevent the up-regulation of oligomeric p-FADD at SW 12 h and 24 h. These data indicate that cortical oligomeric p-FADD, mainly through an interaction with inhibitory α2-adrenoceptors, plays a functional role in the behavioural expression of morphine abstinence in rats.

Role of Multifunctional FADD (Fas-Associated Death Domain) Adaptor in Drug Addiction

Human drug addictions are chronic medical disorders characterized by tolerance and dependence to the abused substance, incentive sensitization, loss of control over drug use that becomes compulsive, relapse (Belin & Everitt, 2010), and in some cases high mortality. A large body of research has established that the majority of drugs leading to addiction stimulate dopamine release through the meso-cortico-limbic circuit in laboratory animals and humans (e.g. see Badiani et al., 2011). Brain neuroadaptations along the reward system are a focus of current research, especially those induced in the prefrontal cortex of human addicts (Goldstein & Volkow, 2011). These persistent neuroplastic events appear to be major causes for compulsive drug-seeking behavior despite the negative effects (e.g., neurotoxicity) induced by drugs of abuse in humans (Nutt et al., 2007).

Cyclin-dependent kinase-5 and p35/p25 activators in schizophrenia and major depression prefrontal cortex: basal contents and effects of psychotropic medications.

Cyclin-dependent kinase-5 (CDK5) and p35/p25 activators, interacting with the exocytotic machinery (e.g. munc18-1 and syntaxin-1A), play critical roles in neurosecretion. The basal status of CDK5/p35/p25 and the effect of psychotropic drugs (detected in blood/urine samples) were investigated in post-mortem prefrontal cortex (PFC)/Brodmanns area 9 of schizophrenia (SZ) and major depression (MD) subjects. In SZ (all subjects, n = 24), CDK5 and p35, but not p25, were reduced (-28 to -58%) compared to controls. In SZ antipsychotic-free (n = 12), activator p35 was decreased (-52%). In SZ antipsychotic-treated (n = 12), marked reductions of CDK5 (-47%), p35 (-76%) and p25 (-36%) were quantified. In MD (n = 13), including antidepressant-free/treated subgroups, CDK5, p35 and p25 were unaltered. In SZ (n = 24), CDK5, p35 or p25 correlated with munc18-1a, but not with syntaxin-1A. The results demonstrate reduced p35 basal content and down-regulation of CDK5/p35/p25 by antipsychotics in SZ. The suggested CDK5/munc18-1a functional interaction may lead to dysregulated neurosecretion in SZ PFC.

P.6.d.014 The severity of morphine abstinence correlates with brain FADD phosphorylation: interaction with α2-adrenoceptors

INTRODUCTION The phosphorylation status of Fas-associated death domain (FADD) protein has recently been implicated in various forms of neuroplasticity in brains of chronic morphine-treated rats [1,2] and postmortem brains of long-term opiate abusers [3]. To further explore the role of multifunctional FADD in the molecular mechanisms of morphine-induced physical dependence, the regulation of cortical phosphorylated (p-) FADD and its interaction with inhibitory α2-adrenoceptors were assessed during the induction of spontaneous opiate withdrawal (SW) in morphine-dependent rats. METHODS Groups of rats (adult male Sprague-Dawley) were chronically treated with escalating doses of morphine (10-100 mg/kg, i.p.), three times daily, for 6 days. Then, some rats (groups of n = 4-12) were left undisturbed for 2, 12, 24, 48, 72 and/or 96 h (SW), and others (n = 8) treated with the α2-adrenoceptor alkylating agent EEDQ (low dose of 1.6 mg/kg, i.p.) at SW 12 h, and target proteins were assessed at SW 24 h (see figure 1). Control rats received saline or vehicle solutions in parallel (n= 13-8). In chronic morphine-withdrawn rats (SW 2-96 h) some classical behavioral signs were checked (hostility, lacrimation, diarrhea) or counted (wet-dog shakes, teeth chattering, exploring behaviors) for a 10-min period, to evaluate the intensity of morphine SW. After the behavioral assays, the rats were killed by decapitation and the brains immediately removed to dissect the cerebral cortices. The contents of oligomeric and monomeric p-Ser194 FADD in the cortical samples were assessed by Western blot analyses with specific antibodies. Results are expressed as means ± SEM of n rats per group. Statistical analyses of data from the behavioral (Kruskal-Wallis) and neurochemical (one-way ANOVA) assays were followed by Dunn’s or Bonferroni’s post hoc test respectively. In all figures, *p < 0.05; **p < 0.01; ***p < 0.001 when compared with the control group.