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

Abstract

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 doseresponse 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 (B142%), membranes (B23%) and nucleus (B54%). 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 effectFie 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.

Chronic MDMA induces neurochemical changes in the hippocampus of adolescent and young adult rats: Down-regulation of apoptotic markers

“…122 Total homogenate of rat HC was prepared for immunoblotting 123 standard procedures (García-Fuster et al, 2009) and brain proteins 124 (40 mg protein) were resolved by electrophoresis on 10-15% SDS-…”

Decreased Proliferation of Adult Hippocampal Stem Cells During Cocaine Withdrawal: Possible Role of the Cell Fate Regulator FADD

“…Hippocampal proteins (40 mg) were separated under non-reducing conditions on 10% SDS-PAGE minigels (Bio-Rad Laboratories, Hercules, CA), followed by standard immunoblotting procedures (García-Fuster et al, 2003). The primary antibodies used (overnight incubation at 4 1C) were selected based on previous experiments and characterization in rat brain (see García-Fuster et al, 2009): anti-FADD (dilution 1 : 2500; sc-5559; Santa Cruz); and anti-b-actin (dilution 1 : 10 000; clone AC-15; Sigma). The secondary antibody (anti-rabbit or -mouse) was incubated for 1 h at room temperature at a 1 : 5000 dilution in blocking solution.…”

“…Briefly, before probe hybridization, slide-mounted tissue sections (approximately 30 mm) from the left-half brain were fixed for 1 h in 4% PFA at room temperature, rinsed with salt buffers, and dehydrated with graded alcohols. After air-drying, the sections were hybridized with a 35 S-labeled cRNA probe previously cloned from cDNA fragments with specific primers using standard in vitro transcription methodology (FADD: 542 nucleotide fragment directed against the rat FADD mRNA coding region, nucleotides 355-898; see García-Fuster et al, 2009). The probes were labeled by incorporation of 35 S-UTP and 35 S-CTP and hybridized to tissue overnight at 551C.…”

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

“…9C) Álvaro-Bartolomé et al, 2011). It is worth noting that there was a positive correlation between FADD protein and the levels of FADD mRNA in rat brain cortex (r=0.43; n=29; p<0.05, see García-Fuster et al, 2009).…”

“…norcocaine) can induce various forms of neurotoxicity (Büttner, 2011), including apoptotic effects in both cultured cells (Xiao et al, 2000;Cunha-Oliveira et al, 2008) and the developing brain (Novikova et al, 2005). However, the aberrant activation of several cell death mechanisms by cocaine, including those mediated by the Fas/FADD complex, in the adult rat brain remains inconclusive (Dietrich et al, 2005;García-Fuster et al, 2009). Nevertheless, self-exposure to cocaine in humans was recently shown to enhance the degradation of a DNA-repairing enzyme in the PFC/BA9 of long-term addicts, which is compatible with the induction of aberrant cell death by the psychostimulant (Álvaro-Bartolomé et al, 2011).…”