Fas-associated Protein With Death Domain Research Articles

Deciphering DED assembly mechanisms in FADD-procaspase-8-cFLIP complexes regulating apoptosis

Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.

Characterization and expression profiling of fadd gene in response to exogenous Aeromonas hydrophila or Edwardsiella tarda challenge in the hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂)

In mammals, fas-associated protein with death domain (FADD) is involved in the process of cell apoptosis and plays a key role in innate immune signaling. Nevertheless, its detailed molecular mechanisms underlying apoptosis and immune responses to exogenous bacterial infections in teleosts remain largely unknown. In this study, a group of 60 hybrid yellow catfish (with the body weight of 25 ± 0.5 g) were used in subsequent experiments, we examined the expression profiling of fadd gene through comparative genomics and comparative immunological methods. Our results showed that fadd in the hybrid yellow catfish (hycfadd) exhibited similar gene and spatial structures to those in other vertebrates, and formed an independent clade in phylogeny. An expression pattern analysis revealed that hycfadd widely transcribed in various tissues, with the highest transcription level in the liver. Furthermore, expression profiling of hycfadd when intraperitoneally infected with 50 μL of exogenous Aeromonas hydrophila (2.0 × 107 CFU/mL) or Edwardsiella tarda (2.0 × 107 CFU/mL) within 48 h were significantly up-regulated in the kidney, spleen, liver and intestine. Important genes in the toll like receptor (tlr) 1-tlr2- myeloid differentiation primary response 88 (MyD88)-fadd-caspase (casp) 8 cascades of TLR signaling pathway in liver were significantly up-regulated after the A. hydrophila stimulation, suggesting that apoptosis through the TLR signaling pathway may have been triggered and activated, which were further verified in the liver, kidney, spleen, intestine and gill by a TUNEL assay. Overall, this study provides solid evidence for the bacterial induction of fadd-related apoptosis in teleosts.

The metabolite alpha-ketoglutarate: a novel target of gasdermin C-dependent pyroptosis.

The metabolite alpha-ketoglutarate: a novel target of gasdermin C-dependent pyroptosis.

Erianin as a Promising Novel Agent in the Treatment of Neuroblastoma: The Anticancer Effects and Underlying Molecular Mechanisms.

Erianin is an active dibenzyl compound isolated from Dendrobium officinale and Dendrobium chrysotoxum and there are very few studies on molecular mechanisms and drug targets of erianin. In addition, there is no study investigating the anti-cancer effect of erianin on neuroblastoma cells. The aim of the study is to investigate the anticancer effect of erianin and the underlying mechanism of this effect on SH-SY5Y cells. The effects of erianin on cell viability, invasion and migration were determined by XTT, matrigel chamber and wound healing evaluation, respectively. Expression changes of miRNAs (microRNA) and apoptosis-related genes were evaluated by RT-PCR, and the apoptosis rate was supported by Annexin V evaluation. Erianin significantly decreased cell proliferation, invasion and migration. Erianin administration caused apoptosis by significantly increasing caspase-7, FADD (Fas-associated protein with death domain), BID (BH3 Interacting Domain Death Agonist) and DR5 (Death receptor 5) gene expressions. While the rate of total apoptotic cells was 45.35 ± 6.80% in SH-SY5Y cells treated with erianin, it was 0.133 ± 0.05% in the control group (p = 0.000). In addition, erianin administration significantly decreased the expressions of hsa-miR-155-5p (p = 0.014) and hsa-miR-223-3p (p = 0.004). Also, our study demonstrated for the first time the relationship between erianin and mi-RNAs in a cancer cell. Our study suggests that erianin may be a natural, safe and easily accessible drug candidate that can be used in the treatment of neuroblastoma.

Regulation of Cortico-Thalamic JNK1/2 and ERK1/2 MAPKs and Apoptosis-Related Signaling Pathways in PDYN Gene-Deficient Mice Following Acute and Chronic Mild Stress.

The crosstalk between the opioidergic system and mitogen-activated protein kinases (MAPKs) has a critical role in mediating stress-induced behaviors related to the pathophysiology of anxiety. The present study evaluated the basal status and stress-induced alterations of cortico-thalamic MAPKs and other cell fate-related signaling pathways potentially underlying the anxiogenic endophenotype of PDYN gene-deficient mice. Compared to littermates, PDYN knockout (KO) mice had lower cortical and or thalamic amounts of the phospho-activated MAPKs c-Jun N-terminal kinase (JNK1/2) and extracellular signal-regulated kinase (ERK1/2). Similarly, PDYN-KO animals displayed reduced cortico-thalamic densities of total and phosphorylated (at Ser191) species of the cell fate regulator Fas-associated protein with death domain (FADD) without alterations in the Fas receptor. Exposure to acute restraint and chronic mild stress stimuli induced the robust stimulation of JNK1/2 and ERK1/2 MAPKs, FADD, and Akt-mTOR pathways, without apparent increases in apoptotic rates. Interestingly, PDYN deficiency prevented stress-induced JNK1/2 and FADD but not ERK1/2 or Akt-mTOR hyperactivations. These findings suggest that cortico-thalamic MAPK- and FADD-dependent neuroplasticity might be altered in PDYN-KO mice. In addition, the results also indicate that the PDYN gene (and hence dynorphin release) may be required to stimulate JNK1/2 and FADD (but not ERK1/2 or Akt/mTOR) pathways under environmental stress conditions.

Meloxicam Inhibits Apoptosis in Neurons by Deactivating Tumor Necrosis Factor Receptor Superfamily Member 25, Leading to the Decreased Cleavage of DNA Fragmentation Factor Subunit α in Alzheimer's Disease.

Neuronal apoptosis is considered to be a critical cause of Alzheimer's disease (AD). Recently, meloxicam has shown neuroprotective effects; however, the inherent mechanisms are highly overlooked. Using APP/PS1 transgenic (Tg) mice as in vivo animal models, we found that meloxicam inhibits apoptosis in neurons by deactivating tumor necrosis factor receptor superfamily member 25 (TNFRSF25), leading to the suppression of the expression of fas-associated protein with death domain (FADD) and the cleavage of DNA fragmentation factor subunit α (DFFA) and cysteine aspartic acid protease-3 (caspase 3) via β-amyloid protein (Aβ)-depressing mechanisms. Moreover, the meloxicam treatment blocked the effects of β-amyloid protein oligomers (Aβo) on stimulating the synthesis of tumor necrosis factor α (TNF-α) and TNF-like ligand 1A (TL1A) in neuroblastoma (N) 2a cells. TNF-α and TL1A induce apoptosis in neurons via TNFR- and TNFRSF25-dependent caspase 3-activating mechanisms, respectively. Knocking down the expression of TNFRSF25 blocked the effects of TL1A on inducing apoptosis in neurons by deactivating the signaling cascades of FADD, caspase 3, and DFFA. Consistently, TNFRSF25 shRNA blocked the effects of Aβo on inducing neuronal apoptosis, which was corroborated by the efficacy of meloxicam in inhibiting Aβo-induced neuronal apoptosis. By ameliorating neuronal apoptosis, meloxicam improved memory loss in APP/PS1 Tg mice.

Identification of a novel necroptosis-related classifier to predict prognosis and guide immunotherapy in breast invasive carcinoma.

BackgroundNecroptosis plays a crucial function in the progression of breast invasive carcinoma (BRCA). It may be triggered in cancer therapy to enhance anti-tumor immunity. However, the functions of necroptosis in tumors and its relationship with the tumor microenvironment (TME) remain largely unclear.MethodsNecroptosis-related genes (NRGs) were collated from high-quality literature reviews. A robust risk model was constructed to systematically evaluate the clinical value, functional status, effects exerted by the risk model on the TME, and the genomic variations based on the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) meta-cohorts.ResultsA risk model was constructed which comprised of six NRGs, including TNF receptor-associated factor 5 (TRAF5), Toll-like receptor 3 (TLR3), a riboflavin kinase (RFK), Fas ligand (FASLG), Fas-associated protein with death domain (FADD), and baculoviral IAP repeat-containing 3 (BIRC3). The stability and accuracy of the risk model were demonstrated for both the training and validation cohorts and its utility as an independent prognostic model for BRCA was verified. Patients in the low-risk group exhibited “hot” tumors having active immune and cell killing functions, while those in the high-risk group showed “cold” tumors having active tumor proliferation and immunosuppression. Moreover, patients in the high-risk group had a greater number of CNV events in their genome, while the somatic mutations were fewer. Furthermore, patients in the low-risk group showed high sensitivity toward immunotherapy and chemotherapy.ConclusionA reliable risk model based on NRGs to assess patient prognoses and guide clinical decision-making was constructed and validated. Our findings may contribute to the understanding of necroptosis and aid clinical management, along with precision treatment in BRCA.

The dephosphorylation of FADD at S191 induces an excessive expansion of TCRαβ+ IELs in the intestinal mucosa.

Intestinal intraepithelial lymphocytes (IELs) play a crucial role in host defence against pathogens in the intestinal mucosa. The development of intestinal IELs is distinct from peripheral T lymphocytes and remains elusive. Fas-associated protein with death domain (FADD) is important for T cell development in the thymus. Here we describe a novel function of FADD in the IEL development. FADD (S191A), a mouse FADD mutant at Ser191 to Ala mimicking constitutively unphosphorylated FADD, promoted a rapid expansion of TCRαβ+ IELs, not TCRγδ+ IELs. Mechanism investigation indicated that the dephosphorylation of FADD was required for cell activation mainly in TCRαβ+ CD8+ T cells. Consistently, FADD (S191A) as dephosphorylated FADD led to a high NF-κB activation in the TCR-dependent cell expansion. In addition, The FADD (S191A)-induced abnormal IEL populations resulted in the increased incidence and severity of colitis in mice. In summary, FADD signalling is involved in the intestinal IEL development and might be a regulator for intestinal mucosal homeostasis.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

A second FADD mediates coelomocyte apoptosis response to Vibrio splendidus infection in sea cucumber Apostichopus japonicus

Fas-associated protein with death domain (FADD) is a pivotal adaptor protein that functions in mediating cell death, cell cycle regulation, and particular in innate immunity by the main death receptors. In this study, a second FADD gene in sea cucumber Apostichopus japonicus (termed AjFADD-2) was cloned and its potential function in the innate responses was analyzed. The full-length cDNA of AjFADD-2 consists of 2405 bp and contains a 47 bp 5′-untranslated region (UTR), a 1629 bp 3′-UTR, and a 729 bp ORF encoding 242 amino acids. AjFADD-2 possesses two conserved domains of intracellular N-terminal death effector domain and an extracellular C-terminal death domain, which is different from the first cloned FADD gene in A. japonicus that only possesses the death domain. AjFADD-2 was examined in all sampled six tissues and was significantly induced in V. splendidus-challenged sea cucumbers and LPS-exposed coelomocytes. Subcellular localization detection showed that AjFADD-2 was primarily observed in the coelomocyte cytoplasm, and transferred to the nucleus post V. splendidus challenge. Consistently, AjFADD-2 knockdown significantly inhibited apoptosis in V. splendidus-challenged sea cucumbers and LPS-exposed coelomocytes. Taken together, our results provided evidence that AjFADD functioned as a positive regulator of coelomocytes apoptosis in response to pathogen V. splendidus challenge.

Targeting Fas-associated protein with death domain gene with chitosan-lipid nanoparticles inhibits coronary heart disease progression in mice

Cell apoptosis or programmed cell death is a physiological phenomenon during the development of the body; however, under certain pathological conditions, insufficient, or excessive apoptosis can cause tissue damage and physiological dysfunction. This study investigated the inhibition of oxidized low-density lipoprotein (oxLDL)-induced macrophage apoptosis in mice with coronary heart disease by regulating the target Fasassociated protein with death domain (FADD) gene. We used Western blotting to measure the effect of oxLDL on mouse macrophages and observe the effects of adding simvastatin, 3-methyladenine (3-MA), self-blocking, and other drugs. The results indicated that the target FADD gene played an important role in inhibiting lactate dehydrogenase release by 13%, in the process of oxLDL-induced lipid aggregation in macrophages. The EC50 observed by dual acridine orange/ethidium bromide fluorescence-staining accounts for the proportion of FADD being reduced by 32%, indicating that the target FADD gene exhibited a significant inhibitory effect on oxLDL-induced macrophage apoptosis in coronary heart disease. This study used a novel chitosan-lipid nanoparticle as a gene carrier to significantly improve efficacy. Therefore, targeting the FADD gene using novel chitosan-lipid nanoparticles to deliver siRNA could be a potential clinical treatment in coronary heart disease.

FADD regulates antibacterial immune responses via the immune deficiency signaling pathway in the Chinese mitten crab

In invertebrates, innate immune responses are the only defense against invading pathogens. The immune deficiency (IMD) signaling pathway protects invertebrates from bacterial infection by secreting antimicrobial peptides (AMPs). Fas-associated protein with death domain (FADD) activates AMPs and triggers apoptosis. However, FADD's function in crustaceans is unclear. Herein, the full-length FADD cDNA (EsFADD) was cloned from the Chinese mitten crab, Eriocheir sinensis. Vibrio parahaemolyticus infection upregulated EsFADD expression markedly. Knockdown of EsFADD in hemocytes suppressed the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation, which in turn reduced the expression of several AMPs. In vivo, silencing of EsFADD rendered crabs susceptible to bacterial infection and impaired their bacterial clearance. The results suggest that EsFADD is indispensable in IMD signal transduction in E. sinensis. In contrast to Drosophila, EsFADD barely promoted apoptosis. Our findings revealed the evolutionary conservation of FADD in crustaceans and provided insights into IMD signaling in invertebrates.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) loss in canine mammary carcinoma.

Escaping apoptosis is a hallmark of cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), a central molecule that regulates the extrinsic apoptotic pathway, has been widely investigated in human oncology; however, investigations focusing on the endogenous expression of TRAIL in canine tumours are lacking. Therefore, we aimed to examine the expression of endogenous TRAIL in canine mammary tumours and analysed its correlation with downstream molecules Fas-associated protein with death domain (FADD) and caspase-3, and to the apoptotic index. A total of 147 samples, classified as normal mammary gland (n= 9), mammary adenoma (n= 30), low-grade carcinoma (n= 42) and high-grade carcinoma (n= 66), were included in the immunohistochemical analyses, and 43 samples with sufficient levels of RNA were analysed via RNA in situ hybridization and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. In immunohistochemistry, TRAIL protein expression was significantly decreased in high-grade carcinoma compared to those in normal mammary gland and adenoma, with similar downregulation of TRAIL mRNA expression. Also, FADD and caspase-3 expression positively correlated with TRAIL expression. However, the apoptotic index was paradoxically elevated in high-grade tumours. Overall, these results suggest that the loss of TRAIL accompanied by dysregulation of TRAIL-induced extrinsic apoptotic pathway molecules could affect malignant progression of canine mammary tumours.

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Add necroptosis to your asthma action plan.

Add necroptosis to your asthma action plan.

Molecular cloning and characterization of FADD from the grass carp (Ctenopharyngodon idellus) in response to bacterial infection

Fas-associated protein with Death Domain (FADD) is closely associated with cell death and plays a key role in hematopoiesis, cell cycle regulation, embryogenesis, and particularly, innate immune signaling. However, the mechanisms underlying the apoptotic and immune responses against bacterial infection associated with FADD in teleost fish have yet to be elucidated. In this study, we attempted to identify these mechanisms by cloning and characterizing the FADD gene in Ctenopharyngodon idellus (grass carp) (GcFADD). The ORF (open reading frame) of GcFADD was 579 bp and encoded a 22.169 kDa putative protein consisting of 192 amino acids. Phylogenetically, the identity of the deduced amino acid sequence was similar to that of other teleost and contained both DEATH and DED domains. In healthy grass carp, the mRNA transcripts of GcFADD were detectable in a range of tissues, in the following order of expression (high to low): liver > middle kidney > head-kidney > heart > gills > spleen > intestines > muscle > brain. In addition, the mRNA transcription and protein expression of GcFADD following infection by Aeromonas hydrophila and Staphylococcus aureus were predominantly up-regulated in the head-kidney, spleen, and liver tissues. Moreover, when a C. idellus kidney (CIK) cell line was incubated with LPS and LTA, the expression of GcFADD transcripts was also considerably up-regulated. In addition, the overexpression of GcFADD in CIK cells activated the significant expression of apoptosis-related genes, including p53, CytoC, caspase-3, caspase-8, and caspase-9. Herein, we demonstrated that GcFADD plays a significant role in innate immunity. Our findings indicate the potential framework by which teleost acquire antibacterial immunity.

Effects of apple extract on the mechanism of apoptosis during acetic acid-induced colitis in Wistar rats

The aim of this study was to evaluate the immunoreactivity of Fas-L, Fas, FADD (Fas-associated protein with death domain), Smac/DIABLO, apoptosis-inducing factor (AIF), cytochrome c, and cleaved caspase 3 in the colon of rats with colitis and treated with apple extract. For this purpose, Wistar rats were submitted to the following experimental design: Group 1 (Sh) was kept as normal and received 1.0 mL of saline solution by intra-colonic injection. Group 2 (AE) only received the concentrated apple extract (CAE) treatment. Groups 3 (AA), 4 (CT), and 5 (PT) were subjected to the induction of experimental colitis by intra-colonic injection of 1 mL acetic acid (7%). Group 3 (AA) simulated as AA-colitis control. Group 4 (CT) was treated with CAE after AA-colitis induction for 7 days, and group 5 (PT) was treated 7 days before colitis induction and followed CAE treatment after colitis induction. The AE, CT, and PT groups received the CAE (1 mL) orally by gavage. All rats were euthanized on day 8 following the experiment. There were no significant statistical differences in the immunoreactivity of Fas-L, Fas, Smac/DIABLO, AIF, and cytochrome c. The expression of FADD in the PT group was similar to that of the AE group and three times greater than in the CT group. For this preventive group (PT), the expression of FADD was restored by apple extract. Our results demonstrated that concentrated apple extract was able to interfere with either intrinsic or extrinsic apoptotic pathways in the colon of rats with colitis.

Differentially expressed immune response genes in COVID-19 patients based on disease severity.

Background: Dysregulated immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are thought to underlie the progression of coronavirus disease 2019 (COVID-19). We sought to further characterize host antiviral and cytokine gene expression in COVID-19 patients based on illness severity.Methods: In this case-control study, we retrospectively analyzed 46 recovered COVID-19 patients and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We assessed changes in antiviral gene expression between healthy controls and patients with mild/moderate (MM) and severe/critical (SC) disease.Results: We found that type I interferon signaling (IFNA2, TLR8, IFNA1, IFNAR1, TLR9, IRF7, ISG15, APOBEC3G, and MX1) and genes encoding proinflammatory cytokines (IL12B, IL15, IL6, IL12A and IL1B) and chemokines (CXCL9, CXCL11 and CXCL10) were upregulated in patients with MM and SC disease. Moreover, we found that IFNA1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), and Fas-associated protein with death domain (FADD) were significantly downregulated (P < 0.05) in the SC group compared to the MM group. We also observed that microRNA (miR)-155 and miR-130a levels were markedly higher in the MM group compared to the SC group.Conclusion: COVID-19 is associated with the activation of host antiviral genes. Induction of the IFN system appears to be particularly important in controlling SARS-CoV-2 infection, as decreased expression of IFNA1, APOBEC3G and FADD genes in SC patients, relative to MM patients, may be associated with disease progression.

Loss of FADD and Caspases Affects the Response of T-Cell Leukemia Jurkat Cells to Anti-Cancer Drugs

Programmed cell death (PCD) pathways play a crucial role in the response of cancer cells to treatment. Their dysregulation is one of the cancer hallmarks and one of the reasons of drug resistance. Here, we studied the significance of the individual members of PCD signaling pathways in response to treatment with common anti-cancer drugs using the T-cell leukemia Jurkat cells with single or double knockouts of necroptosis and/or apoptosis genes. We identified apoptosis as the primary cell death pathway upon anti-cancer drugs treatment. The cells with knocked out either Fas-associated protein with death domain (FADD) or all executioner caspases were resistant. This resistance could be partially overcome by induction of RIP1-dependent necroptosis through TNFR1 activation using combined treatment with TNF-α and smac mimetic (LCL161). RIP1 was essential for cellular response to TNF-α and smac mimetic, but dispensable for the response to anti-cancer drugs. Here, we demonstrated the significance of FADD and executioner caspases in carrying out programmed cell death upon anti-cancer drug treatments and the ability of combined treatment with TNF-α and smac mimetic to partially overcome drug resistance of FADD and/or CASP3/7/6-deficient cells via RIP1-dependent necroptosis. Thus, a combination of TNF-α and smac mimetic could be a suitable strategy for overcoming resistance to therapy in cells unable to trigger apoptosis.

Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex.

Alpha2A-adrenergic (α2A-AR) and cannabinoid CB1 (CB1-R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α2A-AR- and CB1-R in vivo are unclear. This study investigated the roles of Gαi2 and Gαz proteins in mediating α2A-AR- and CB1-R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. Gαi2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3nmol/day, 5days) prior to UK-14,304 (UK or brimonidine; 1mg/kg) or WIN55212-2 (WIN; 8mg/kg) acute treatments. Inactivated (p-T286) MEK1, activated (p-S217/221) MEK1/2, activated (p-T202/Y204) ERK1/2, p-S191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. Increased (+ 88%) p-T286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi2- and Gαz-independent upregulations of p-T286 MEK1 (+ 63%), p-S217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (- 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi2 or Gαz protein integrity. The results indicate that α2A-AR (UK), but not CB1-R (WIN), agonists use Gαi2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.