María Álvaro-Bartolomé

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.

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).

The neuroplastic index p-FADD/FADD and phosphoprotein PEA-15, interacting at GABAA receptor, are upregulated in brain cortex during midazolam-induced hypnosis in mice

Fas-associated death domain (FADD) adaptor is involved in the signaling of metabotropic G protein-coupled receptors, whose agonists stimulate its phosphoryaltion (p) increasing p-FADD/FADD ratio in brain. Whether FADD might also participate in the activation of dissimilar receptors such as the ligand-gated ion channels is not known. This study investigated the role of FADD and phosphoprotein-enriched in astrocytes of 15 kDa (PEA-15, a FADD partner) in the activation of γ-aminobutyric acid-A (GABAA) receptor, which mediates the hypnotic effect of midazolam. The main findings revealed that during the time course of midazolam (60 mg/kg)-induced hypnosis in mice (about 2 h) p-FADD (and p-FADD/FADD ratio) as well as p-PEA (and its phosphorylating Akt1 kinase) were markedly increased (36-80%) in brain cortex, and these effects were partially (only p-FADD) or fully prevented by flumazenil (a neutral allosteric ligand) and FG 7142 (a partial negative allosteric ligand) acting at GABAA receptors. The upregulation of cortical p-FADD/FADD was exclusively observed in the nucleus (up to 2.8-fold), where the transciption factor NF-κB was also increased (up to 46%), and that of p-PEA/p-Akt1 only in the cytosol (up to 53%), suggesting that p-FADD/p-PEA/p-Akt1 are involved in sleep-induced neuroplasticity. Repeated treatment with midazolam (60 mg/kg, 4 days) induced behavioral (prolonged sleep latency and reduced sleeping time) and neurochemical (reduced p-FADD/p-PEA contents) tolerance. These findings indicated that p-FADD/p-PEA are novel molecules in GABAA receptor signaling and that cortical p-PEA and p-FADD, working in tandem, are involved in the complex molecular processes leading to the hypnotic effect of midazolam in mice.

The Fas Receptor/Fas-Associated Protein and Cocaine

Abstract This chapter summarizes the current data studying the effects of cocaine on altering the balance between the induction of aberrant apoptotic cell death (i.e., neurotoxicity) and nonapoptotic events (i.e., neuroplasticity) mediated through the activation of Fas receptor and Fas-associated protein (FADD) in the rat brain and the human postmortem brain. The findings presented here reveal important differences between humans and rats concerning the long-term regulation of the extrinsic apoptotic pathway by cocaine in the brain. The Fas/FADD complex is downregulated in the prefrontal cortex of human cocaine addicts, while it shows tolerance to an acute upregulation in the brain of cocaine-treated rats. The modulation of Fas receptor and more interestingly that of FADD adaptor by cocaine is a new and relevant molecular process in the complex neurobiology of cocaine addiction, suggesting long-term contraregulatory adaptations or nonapoptotic actions.

Regulation of cannabinoid CB2 receptor constitutive activity in vivo: repeated treatments with inverse agonists reverse the acute activation of JNK and associated apoptotic signaling in mouse brain

RationaleCB2 receptors express constitutive activity and inverse agonists regulate receptor basal activity, which might be involved in death mechanisms. This study assessed the effects of a selective CB2 agonist (JWH133) and different CB2 inverse agonists (AM630, JTE907, raloxifene) on death pathways in brain.ObjectivesThe acute (JWH13) and the acute/chronic effects (AM630, JTE907, raloxifene) of CB2 ligands regulating pro-apoptotic c-Jun NH2-terminal kinase (p-JNK/JNK ratio) and associated signaling of extrinsic (Fas receptor, Fas-Associated death domain protein, FADD) and intrinsic (Bax, cytochrome c) death pathways (nuclear poly (ADP-ribose) polymerase PARP) were investigated in mouse brain.MethodsMice were treated with CB2 drugs and target protein contents were assessed by western blot analysis.ResultsJWH133 reduced cortical JNK (−27–45%) whereas AM630 acutely increased JNK in cortex (+61–148%), cerebellum (+34–40%), and striatum (+33–42%). JTE907 and raloxifene also increased cortical JNK (+31%–57%). Acute AM630, but not JWH133, increased cortical FADD, Bax, cytochrome c, and PARP cleavage. Repeated treatments with the three CB2 inverse agonists were associated with a reversal of the acute effects resulting in decreases in cortical JNK (AM630: −36%; JTE907: −25%; raloxifene: −11%). Chronic treatments also induced a reversal with down-regulation (AM630) or only tolerance (JTE907 and raloxifene) on other apoptotic markers (FADD, Bax, cytochrome c, PARP).ConclusionsAM630 and JTE907 are CB2 protean ligands. Thus, chronic inverse agonists abolished CB2 constitutive activity and then the ligands behaved as agonists reducing (like JWH133) JNK activity. Acute and chronic treatments with CB2 inverse agonists regulate in opposite directions brain death markers.

Disruption of brain MEK-ERK sequential phosphorylation and activation during midazolam-induced hypnosis in mice: Roles of GABAA receptor, MEK1 inactivation, and phosphatase MKP-3

ABSTRACT Midazolam is a positive allosteric modulator at GABAA receptor that induces a short hypnosis and neuroplasticity, in which the sequential phosphorylation of MEK1/2 and ERK1/2 was shown to play a role. This study investigated the parallel activation of p‐MEK and p‐ERK and regulatory mechanisms induced by midazolam through the stimulation of GABAA receptors in the mouse brain. During the time course of midazolam (60 mg/kg)‐induced sleep in mice (lasting for about 2 h) p‐Ser217/221 MEK1/2 was increased (+ 146% to + 258%) whereas, unexpectedly, p‐Tyr204/Thr202 ERK1/2 was found decreased (− 16% to − 38%), revealing uncoupling of MEK to ERK signals in various brain regions. Midazolam‐induced p‐MEK1/2 upregulation was prevented by pretreatment (30 min) with flumazenil (10 mg/kg), indicating the involvement of GABAA receptors. Also unexpectedly, midazolam‐induced p‐ERK1/2 downregulation was not prevented by flumazenil (10 or 30 mg/kg). Notably, during midazolam‐induced sleep the content of inactivated p‐Thr286 MEK1, which can dampen ERK1/2 activation, was increased (+ 33% to + 149%) through a mechanism sensitive to flumazenil (10 mg/kg). Midazolam also increased MKP‐3 (+ 13% to + 73%) content and this upregulation was prevented by flumazenil (10 mg/kg); an effect suggesting ERK inactivation because MKP‐3 is the phosphatase selective for ERK1/2 dephosphorylation. The results indicate that during midazolam‐induced sleep in mice there is an uncoupling of p‐MEK (increased) to p‐ERK (decreased) signals. p‐ERK1/2 downregulation (not involving GABAA receptors) is the result of increased inactivated MEK1 and phosphatase MKP‐3 (both effects involving GABAA receptors). These findings are relevant for the neurobiology and clinical use of benzodiazepines. HIGHLIGHTSDisruption of MEK‐ERK activation during midazolam‐induced sleep in miceUpregulation of p‐MEK prevented by flumazenil (GABAA receptor‐mediated)Downregulation of p‐ERK not prevented by flumazenil

P.1.g.040 Effects of CB2 receptor inverse agonists on JNK activation and pro-apoptotic Bax in mouse brain

TNFa, BDNF, NR2B) and astroglial connexin markers (Cx30, Cx43). Pressure threshold values to elicit nocifensive responses (paw withdrawal and vocalization) increased progressively during amitriptyline treatment, confirming the anti-hyperalgesic action of this antidepressant in CCI rats. Although the connexin inhibitor THN01 was inactive on its own, it significantly enhanced amitriptyline-induced reduction of mechanical hyperalgesia. Thus, at the end of co-treatment, pressure threshold values in CCI rats were similar to those in naive healthy rats. On the other hand, mechanical hyperalgesia was associated with significantly upregulated levels of the microglia marker Ox42, the injured neuron marker ATF3 and the proinflammatory cytokine IL-6 in dorsal root ganglia and/or the dorsal spinal cord ipsilateral to sciatic nerve CCI, but neither amitriptyline or THN01 alone, nor the combined treatment, significantly reduced such CCI-induced effects. These results show that the connexin inhibition by THN01 enhanced the anti-hyperalgesic effect of amitriptyline in the rat CCI model. The unchanged overexpression of neuroinflammatory markers suggests that the anti-hyperalgesic effect of combined amitriptyline+THN01 treatment involved mechanisms downstream of these processes. Therefore, inhibition of connexinbased channel functions in astroglia could represent a promising and novel approach toward improving current neuropathic pain therapy.

Ketamine-induced hypnosis and neuroplasticity in mice is associated with disrupted p-MEK/p-ERK sequential activation and sustained upregulation of survival p-FADD in brain cortex: Involvement of GABAA receptor

Abstract Ketamine (KET) is an antidepressant and hypnotic drug acting as an antagonist at excitatory NMDA glutamate receptors. The working hypothesis postulated that KET‐induced sleep in mice results in dysregulation of mitogen‐activated protein kinases (MAPK) MEK‐ERK sequential phosphorylation and upregulation of survival p‐FADD and other neuroplastic markers in brain. Low (5–15 mg/kg) and high (150 mg/kg) doses of KET on target proteins were assessed by Western immunoblot in mouse brain cortex. During the time course of KET (150 mg/kg)‐induced sleep (up to 50 min) p‐MEK was increased (up to +79%) and p‐ERK decreased (up to −46%) indicating disruption of MEK to ERK signal. Subhypnotic KET (5–15 mg/kg) also revealed uncoupling of p‐MEK (+13–81%) to p‐ERK (unchanged content). KET did not alter contraregulatory MAPK mechanisms such as inactivated p‐MEK1 (ERK dampening) and phosphatases MKP1/2/3 (ERK dephosphorylation). As other relevant findings, KET (5, 15 and 150 mg/kg) upregulated p‐FADD in a dose‐dependent manner, and for the hypnotic dose the effect paralleled the time course of sleep which resulted in increased p‐FADD/FADD ratios. KET (150 mg/kg) also increased NF‐&kgr;B and PSD‐95 neuroplastic markers. Flumazenil (a neutral allosteric antagonist at GABAA receptor) prolonged KET sleep and blocked p‐MEK upregulation, indicating the involvement of this receptor as a negative modulator. SL‐327 (a MEK inhibitor) augmented KET sleep, further indicating the relevance of reduced p‐ERK1/2 in KET‐induced hypnosis. These findings suggest that hypnotic and subhypnotic doses of KET inducing uncoupling of p‐MEK to p‐ERK signal and regulation of p‐ERK (downregulation) and p‐FADD (upregulation) may participate in the expression of some of its adverse effects (e.g. amnesia, dissociative effects). HighlightsDisruption of MEK‐ERK activation during ketamine‐induced sleep in miceUpregulation of neuroplastic/survival p‐FADD by ketamine during sleepUpregulation of neuroplastic NF‐&kgr;B and PSD‐9 by ketamineEffects of flumazenil (GABAA antagonist) and SL‐327 (MEK inhibitor)

A Biomarker to Differentiate between Primary and Cocaine-Induced Major Depression in Cocaine Use Disorder: The Role of Platelet IRAS/Nischarin (I1-Imidazoline Receptor)

The association of cocaine use disorder (CUD) and comorbid major depressive disorder (MDD; CUD/MDD) is characterized by high prevalence and poor treatment outcomes. CUD/MDD may be primary (primary MDD) or cocaine-induced (CUD-induced MDD). Specific biomarkers are needed to improve diagnoses and therapeutic approaches in this dual pathology. Platelet biomarkers [5-HT2A receptor and imidazoline receptor antisera selected (IRAS)/nischarin] were assessed by Western blot in subjects with CUD and primary MDD (n = 16) or CUD-induced MDD (n = 9; antidepressant free, AD−; antidepressant treated, AD+) and controls (n = 10) at basal level and/or after acute tryptophan depletion (ATD). Basal platelet 5-HT2A receptor (monomer) was reduced in comorbid CUD/MDD subjects (all patients: 43%) compared to healthy controls, and this down-regulation was independent of AD medication (decreases in AD−: 47%, and in AD+: 40%). No basal differences were found for IRAS/nischarin contents in AD+ and AD− comorbid CUD/MDD subjects. The comparison of IRAS/nischarin in the different subject groups during/after ATD showed opposite modulations (i.e., increases and decreases) in response to low plasma tryptophan levels with significant differences discriminating between the subgroups of CUD with primary MDD and CUD-induced MDD. These specific alterations suggested that platelet IRAS/nischarin might be useful as a biomarker to discriminate between primary and CUD-induced MDD in this dual pathology.

Chronic Effects of Cannabinoid Drugs on Monoaminergic Systems and the Role of Endocannabinoids and Cannabinoid Receptors in Human Brain Disorders

The endocannabinoid system and cannabinoid (CB) receptors participate in the regulation of a variety of psychiatric and neurological disorders through a functional coupling with the monoaminergic systems in the brain. Norepinephrine, serotonin (5-HT) and dopamine systems are modulated via inhibitory CB1 receptors by direct or indirect effects. The repeated stimulation of CB1 receptors (and receptor desensitization) can lead to the induction of tolerance on the activity of monoaminergic systems. The chronic administration of CB drugs can also alter the function of presynaptic inhibitory monoamine autoreceptors and heteroreceptors and thus modulate the final effects on these systems. The functional interactions between endocannabinoids, CB receptors, and monoaminergic systems suggest a potential role for CB receptor signaling in the pathophysiology and treatment of various psychiatric and neurological disorders, including drug addiction, which are discussed on evidence from postmortem and living human brain studies.