Regulation of GABA(A) and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc)) of the natural cellular prion protein (PrP(c)) encoded by the Prnp gene. Although several roles have been attributed to PrP(c), its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c) studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Principal Findings: Here we explore the role of PrP(c) expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c)-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A) and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrP(c) is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA(A) and AMPA-Kainate neurotransmission. New PrP(c) functions have recently been described, which point to PrP(c) as a target for putative therapies in Alzheimer's disease. However, our results indicate that a "gain of function" strategy in Alzheimer's disease, or a "loss of function" in prionopathies, may impair PrP(c) function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgito-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(AWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-NWASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP¿dependent manner.

Calmodulin Regulates Intracellular Trafficking of Epidermal Growth Factor Receptor and the MAPK Signaling Pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13¿mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the alpha1B-adrenergic receptor (alpha1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the alpha1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.

Ubiquitination of the Epstein-Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1(-)) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wild-type LMP1 with respect to membrane localization. However, CTAR1(-) does not bind TRAF3. We demonstrate that ubiquitination of CTAR1(-) is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1(-) is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.

Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission.

Endogenous and infectious mouse mammary tumor viruses (MMTVs) encode in their 3' long terminal repeat a protein that exerts superantigen activity; that is, it is able to interact with T cells via the variable domain of the T cell receptor (TCR) beta chain. We show here that transmission of an infectious MMTV is prevented when superantigen-reactive cells are absent through either clonal deletion due to the expression of an endogenous MTV with identical superantigen specificity or exclusion due to expression of a transgenic TCR beta chain that does not interact with the viral superantigen. A strict requirement for superantigen-reactive T cells is also seen for a local immune response following MMTV infection. This immune response locally amplifies the number of MMTV-infected B cells, most likely owing to their clonal expansion. Collectively, our data indicate that a superantigen-induced immune response is critical for the MMTV life cycle.

Penicillin-binding protein gene alterations in Streptococcus uberis isolates presenting decreased susceptibility to penicillin.

Streptococcus uberis is an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis (MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberis can readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberis in that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q(554)E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberis might follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.

Identification of CT46/HORMAD1, an immunogenic cancer/testis antigen encoding a putative meiosis-related protein.

Transcripts with ESTs derived exclusively or predominantly from testis, and not from other normal tissues, are likely to be products of genes with testis-restricted expression, and are thus potential cancer/testis (CT) antigen genes. A list of 371 genes with such characteristics was compiled by analyzing publicly available EST databases. RT-PCR analysis of normal and tumor tissues was performed to validate an initial selection of 20 of these genes. Several new CT and CT-like genes were identified. One of these, CT46/HORMAD1, is expressed strongly in testis and weakly in placenta; the highest level of expression in other tissues is <1% of testicular expression. The CT46/HORMAD1 gene was expressed in 31% (34/109) of the carcinomas examined, with 11% (12/109) showing expression levels >10% of the testicular level of expression. CT46/HORMAD1 is a single-copy gene on chromosome 1q21.3, encoding a putative protein of 394 aa. Conserved protein domain analysis identified a HORMA domain involved in chromatin binding. The CT46/HORMAD1 protein was found to be homologous to the prototype HORMA domain-containing protein, Hop1, a yeast meiosis-specific protein, as well as to asy1, a meiotic synaptic mutant protein in Arabidopsis thaliana.

The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription.

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT2A receptor.

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT(2A) receptor distribution. MAP1A co-localized mainly with 5-HT(2A) receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.