Effect of cocaine on Fas-associated protein with death domain in the rat brain: individual differences in a model of differential vulnerability to drug abuse.

This study was designed to (1) assess the effects of cocaine on Fas-associated protein with death domain (FADD) system and its role in the activation of apoptotic vs nonapoptotic events and (2) ascertain whether animals selectively bred for their differential propensity to drug-seeking show differences in FADD levels or response to cocaine. Acute cocaine, through D(2) dopamine receptors, induced a dose-response increase in FADD protein in the cortex, with opposite effects over pFADD (Ser191/194), and no induction of apoptotic cell death (poly-(ADP-ribose) polymerase cleavage). FADD was increased by cocaine in cytosol (approximately 142%), membranes (approximately 23%) and nucleus (approximately 54%). The modulation of the FADD system showed tolerance of the acute effect over time, as well as a compensatory response on withdrawal that mirrored the acute effect--ie a transient FADD decrease on day 3 of withdrawal, both at mRNA and protein levels. In a second experiment, possible FADD differences were investigated in rats selectively bred for differential responsiveness to novelty, propensity for drug-seeking and cocaine sensitization. High-responders (HR), who were more prone to drug abuse, exhibited higher FADD and lower pFADD levels than low-responder (LR) rats. However, HR and LR rats showed similar rates of cocaine-induced apoptosis, and exhibited a parallel impact of cocaine over FADD within each phenotype. Thus, FADD is a signaling protein modulated by cocaine, regulating apoptosis/proliferative mechanisms in relation to its FADD/pFADD content. Interestingly, animals selectively bred for differential propensity to substance abuse show basal differences in the expression of this protein, suggesting FADD may also be a molecular correlate for the HR/LR phenotype.

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.

This study was designed to assess the effects of opiate treatment on the expression of Fas-associated protein with death domain (FADD) in the rat brain. FADD is involved in the transmission of Fas-death signals that have been suggested to contribute to the development of opiate tolerance and addiction. Acute treatments with high doses of sufentanil and morphine (mu-agonists), SNC-80 (delta-agonist), and U50488H (kappa-agonist) induced significant decreases (30-60%) in FADD immunodensity in the cerebral cortex, through specific opioid receptor mechanisms (effects antagonized by naloxone, naltrindole, or nor-binaltorphimine). The cannabinoid CB1 receptor agonist WIN 55,212-2 did not alter FADD content in the brain. Chronic (5 days) morphine (10-100 mg/kg), SNC-80 (10 mg/kg), or U50488H (10 mg/kg) was associated with the induction of tachyphylaxis to the acute effects. In morphine- and SNC-80-tolerant rats, antagonist-precipitated (2 h) or spontaneous withdrawal (24-48 h) induced a new and sustained inhibition of FADD (13-50%). None of these treatments altered the densities of caspases 8/3 (including the active cleaved forms) in the brain. Pretreatment of rats with SL 327 (a selective MEK1/2 inhibitor that blocks ERK activation) fully prevented the reduction of FADD content induced by SNC-80 in the cerebral cortex (43%) and corpus striatum (29%), demonstrating the direct involvement of ERK1/2 signaling in the regulation of FADD by the opiate agonist. The results indicate that mu- and delta-opioid receptors have a prominent role in the modulation of FADD (opposite to that of Fas) shortly after initiating treatment. Opiate drugs (and specifically the delta-agonists) could promote survival signals in the brain through inhibition of FADD, which in turn is dependent on the activation of the antiapoptotic ERK1/2 signaling pathway.

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.