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Yes / The activity-regulated cytoskeleton-associated (Arc) protein control synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-excitatory postsynaptic currents (mEPSC). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, effect that is restored by re-introducing µ2. The Arc/AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. This data provides a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. / This work was supported by the BBSRC_FAPPA BB/J02127X/1 and BBSRC-BB/H018344/1 to SALC and by the FAPESP_RCUK_FAPPA 2012/50147-5 and FAPESP_Young Investigator’s grant 2009/50650-6 to LLdS. SCW was a PhD Student supported be the BBSRC/GSK PhD-CASE Studentship, LPdA is a postdoc fellow supported by FAPESP, YCJ was supported by a FAPESP scientific initiation scholarship.

Le cancer du sein est le cancer le plus fréquent chez la femme (Boyle and Ferlay, 2005; Ferlay et al., 2007). Même si les traitements sont de plus en plus efficaces, il est responsable denviron 130 000 décès en Europe. Environ 20-30% des cancers du sein surexprime le gène ERBB2. Cette surexpression confère à la cellule un profil tumoral très agressif, et résistant aux chimiothérapies conventionnelles. Létude des facteurs responsable de la surexpression du gène ERBB2 dans les cancers du sein est le thème principal de recherche du laboratoire doncologie moléculaire. Notre laboratoire, a étudié les mécanismes moléculaires responsables de la surexpression du gène ERBB2 dans les cancers du sein. Nous avons montré que la surexpression est la conséquence dune stimulation de la transcription et non de la stabilisation de lARN (Pasleau et al., 1993). Pour étudier la régulation de la transcription un fragment de 6 kb du promoteur du gène ERBB2 a été cloné et séquencé. Différentes régions régulatrices ont été identifiées (Grooteclaes et al., 1994). Plusieurs sites de liaison pour des facteurs AP-2 ont été identifiés (Delacroix et al., 2005; Grooteclaes et al., 1999; Vernimmen et al., 2003a). La fixation des facteurs au promoteur a été vérifiée par Chromatin Immuniprecipitation (ChIP assay) (Begon et al., 2005; Delacroix et al., 2005). Afin de mieux comprendre le fonctionnement des facteurs de transcription AP-2, nous avons cherché les protéines interagissant avec ce facteur et contribuant à la surexpression dERBB2 dans des lignées de cancer du sein. Précédemment, Dominique Begon a montré linteraction de la protéine YY1 avec le facteur de transcription AP-2α et leur implication sur le promoteur du gène ERBB2 (Begon et al., 2005). La première partie de mon travail a été de mettre en corrélation par immunohistochimie lexpression des protéines YY1 et AP-2α avec la surexpression dERBB2 dans des tumeurs primaires. Nous avons également voulu étudier leffet dune diminution des protéines YY1 et AP-2 sur lexpression dERBB2, à laide de siRNAs (Voir article 1 en annexe, (Allouche A and Nolens G. et al., 2008). La deuxième partie de ce travail a porté sur lidentification dautres protéines pouvant interagir avec les protéines AP-2. Après purification, les protéines Ku70 et Ku80 furent identifiées. Nous avons voulu étudier leffet de cette interaction sur lactivité des protéines AP-2 (α et γ) et sur lexpression dERBB2 (voir article 2 en annexe).

Previous studies from our lab have shown that the Activating Protein- 2 (AP-2) transcription factors, AP-2α and AP-2β, are important in retinal development. It was discovered that these are co-expressed in developing horizontal cells and postmitotic amacrine cells. To understand their role in retinogenesis, and the impact of their deletion on the adult retina, a double mutant mouse model was created, AP-2αKI/flox/AP-2β-/flox. The neural retina of the AP-2αKI/flox/AP-2β-/flox mice was examined in the current study using histological, immunofluorescent and electron microscopy (EM) techniques at embryonic, post-natal and adult stages. These double mutants displayed a variety of abnormalities in the inner retina. Loss of AP-2α and AP-2β at E10.5 led to a complete absence of developing and mature horizontal cells. This loss was associated with changes in the outer plexiform layer, which diminished from two to four months of age. There were also defects with photoreceptor ribbons in which triad synapses failed to form, and instead led to rudimentary, spherical-shaped ribbons. There was also significant retraction of photoreceptor axons. Furthermore, this study was able to infer a role of AP-2α and AP-2β as acting upstream of the Onecut-1 protein, which targets Lim1 and Prox1 to direct horizontal cell genesis. Examining amacrine cells of the double mutants shows evidence that AP-2α and AP-2β are involved in the mosaic arrangement pattern of amacrine cell bodies and axons. Previous work on embryonic double mutants displayed clustering of amacrine cells. This study observed abnormalities in the dendrites of the inner plexiform layer, which consists of amacrine cell processes. Taken together, the work presented in this thesis implicates the redundant requirement of both AP-2α and AP-2β in development of horizontal cells and patterning of amacrine cells in the neural retina. / Thesis / Master of Health Sciences (MSc)

Introduction AP2α is a 52 kDa retinoic acid inducible and developmentally regulated activator of transcription, which binds to the DNA in a sequence-specific manner. Transcription factor AP-2α was isolated from HeLa cells by affinity chromatography using specific binding sites with in SV40 and human metallothionein promoters. Further screening of HeLa cDNA library with oligonucleotide probes predicted partial peptide sequence which led to the isolation of AP-2α cDNA and subsequently it was mapped to chromosome 6 near HLA locus. A differentially spliced version of AP-2α, which lacks most of the C-terminus, encodes a dominant negative protein (AP-2B). Subsequent studies led to the identification of four more isoforms: AP-2β, AP-2γ, AP-2δ and AP-2ε. AP-2 family members can form homo or hetero dimers among themselves through the unique C-terminal helix span helix motif and bind DNA through basic domain lies N-terminus of DNA binding domain. Several evidences suggest that AP-2α can act as a tumor suppressor gene. It has been shown that AP-2α can activate growth suppressor genes like p21WAF1/CIP1. Transforming viral oncogenes like adenovirus E1A and SV40 large T antigen have been shown to alter AP-2α function. In addition, reduced expression of AP-2α has been reported in human breast, ovary, colon, skin, brain and prostate cancers. Further, supporting evidences suggest that more invasiveness and tumorogenicity was observed when dominant negative mutant of AP-2α was expressed in melanoma cells. In this work, we have carried out a systematic study to find the various signal transduction pathways which regulate AP-2 activity as well as we attempted to demonstrate the importance of DNA binding domain in the growth inhibitory functions of AP-2α. HDAC inhibitors (HDIs) activate AP-2 activity through spleen tyrosine kinase (Syk) In the literature, ample evidences are available that genotoxic drugs such as adriamycin, induce tumor suppressors like p53 and p73. In this study, we have screened pharmacological drugs which damage DNA and specific inhibitors of various signal transduction pathways for their ability to activate AP-2 activity. AP-2 specific reporter, 3Χ-AP2-CAT was used in this study to measure the AP-2 activity. Of all the compounds studied, we found that Histone Deacetylase Inhibitors (HDIs) efficiently activated AP-2 activity and was found to be specific as they failed to activate 3X-AP2 mut CAT, which contains mutated AP-2 binding sites as well as pGL tk Luc, which contains thymidine kinase minimal promoter and no AP-2 binding sites. To understand the mechanism of HDI-mediated of AP-2 activation, AP-2 isoforms and its coactivators transcript and protein levels were analyzed. We found significant change in transcript levels of the some of the molecules tested. While the endogenous protein levels of various AP-2 isoforms were undetectable, we found stabilization of AP-2α protein expressed from exogenous source in cells treated with HDIs. HDI stabilized AP-2α was found to be functionally active as it showed increased sequence-specific DNA-binding as well as increased apoptosis. While HDIs known for their ability to modulate the gene activities by chromatin remodeling, it is also known that they alter various signal transduction pathways. In an effort to find pathway(s) by which HDIs activate AP-2 activity, we found that HDIs failed to activate AP-2 reporter in the presence of staurosporine suggesting the involvement a staurosporine sensitive pathway(s) in this process. Stauosporine is a non-specific kinase inhibitor of different signaling pathways. Further studies using different pathway specific inhibitors identified that spleen tyrosine kinase (Syk) is essential for HDIs mediated activation of AP-2 activity. Syk is a non receptor tyrosine kinase which is known to be activated in stress conditions. Syk is considered to be a tumor suppressor since Syk over expression leads to growth suppression of breast cancer cells and is also inactivated in a subset of breast cancers. These results suggest that HDI mediated activation of AP-2 involves AP-2α stabilization through Syk pathway. Regulation of AP-2 by MAP kinase pathway Cell growth, differentiation, and apoptosis are mediated by the activation of mitogenactivated protein kinase (MAPK) pathways. These kinases constitute MAP kinase cascades mainly regulated through phosphorylation status. In mammalian cells, at least four MAPKs, namely, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), p38 and ERK5/big MAP kinase have been identified. The ERKs are usually activated by mitogenic stimuli which in turn increase the proliferation and survival. Over expression of any activator of this signaling cascade lead to the unregulated proliferation of cells. In many cancers, ERK pathways are known to be up regulated. In this study, we found that MEK (MEK is the immediate upstream regulator of ERK) inhibitors - PD98059 and U0126 activate 3X-AP2-CAT suggesting that AP-2 activity is repressed by activated MAP kinase pathway. MEK inhibitor mediated activation was found to be specific because they failed to activate transcription from pGL tk Luc which contains thymidine kinase minimal promoter and no AP-2 binding sites. To understand the mechanism of MEK inhibitor-mediated of AP-2 activation, AP-2 isoforms and its coactivators transcript and protein levels were analyzed. We found significant change in transcript levels of the some of the molecules tested. The endogenous protein levels of various AP-2 isoforms were undetectable. When AP-2α was exogenously expressed, while no change in protein levels and DNA-binding ability was seen, we found evidence for appearance of post-ranslationally modified AP-2α protein in U0126 treated cells. We also found CITED2 (CBP/p300-interacting transactivator 2, co-activator of AP-2α) transcript levels were up regulated in UO126 treated cells. Post translational modifications of AP-2α and increased and increased CITED2 levels may be responsible for MEK inhibitor mediated AP-2 activation. Thus we conclude that ERK pathway, which is an oncogenic MAP kinase pathway, inhibits AP-2 activity thereby suggesting the importance of down regulation of AP-2 activity during transformation. Essential role of DNA-binding domain of AP-2α for its growth inhibitory functions Transcription factor AP-2α has three distinct domains, N-terminal transactivation domain (52-108 aa), C-terminal DNA binding domain (204-408 aa) and dimerization domain (277-395 aa) which lies within the DNA binding domain. AP-2α exerts its effects through binding to specific DNA sequence in the promoter of its target genes leading to either repression or activation. Recent evidences suggest that AP-2α represses many genes through its competitive binding to overlapping AP-2 and other transcription factor binding sites. This suggests an important role exclusively for the DNA binding domain in AP-2α mediated functions. To address the importance of DNA binding domain for AP-2α mediated apoptosis, we have tested the ability different deletion/point mutants of AP-2α with varying DNA binding and transactivation capability to perform growth suppressor function and ability to induce apoptosis. Replication-deficient recombinant adenoviruses expressing different mutants were used in this study. We found that an intact DNA-binding domain alone even in the absence of activation domain is sufficient for AP-2α to inhibit colony formation and to induce significant levels of apoptosis. These results suggest an important role for DNA binding domain growth inhibitory functions of AP-2α and thereby implying the importance of transcriptional repression in AP-2α functions.

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<p>Eukaryotic gene transcription plays a regulatory role in mammalian developmental processes. It has been shown that transcriptional control is an important mechanism for specification of neurotransmitter phenotypes. In the mammalian central nervous system, the transcription factor AP-2 family is one of the critical regulatory factors for neural gene expression and neuronal development. It has been shown that several genes in the monoaminergic systems have AP-2 binding sites in regulatory regions, suggesting a regulatory role of AP-2 also in the adult brain. Brainstem monoamines are implicated in the expression of personality traits and imbalances in these systems may give rise to psychiatric disorders. </p><p>The gene encoding AP-2β includes a polymorphic region consisting of a tetranucleotide repeat of [CAAA]_4-5 in intron 2. Studies on AP-2β genotype in relation to personality and platelet MAO activity, a trait-dependant marker for personality, are presented in this thesis. Furthermore, correlations between brainstem levels of AP-2α and AP-2β and monoamine turnover in projection areas in rat forebrain are reported. These results strengthen the notion that the AP-2 family is important regulators of the monoaminergic systems in the adult brain. Furthermore, two studies are presented in this thesis with analyses indicating a role for AP-2 in the molecular mechanism of antidepressant drugs.</p><p>Altogether, this thesis presents data supporting our notion that the transcription factor AP-2 family is involved in the regulation of the monoaminergic systems both pre- and postnatally, and, therefore, might be involved in the pathophysiology of neuropsychiatric disorders.</p>

<p>The CNS monoaminergic systems are considered as the head engine regulating neuropsychiatric functions and personality. Transcription factor AP-2 is known to be essential for the development of the brainstem including the monoaminergic nuclei, and has the ability to regulate many genes in the monoaminergic systems. The ability of transcription factors to regulate specific gene expression, has lately made them hot candidates as drug targets. In this thesis, results indicating a role of AP-2 in the molecular effects of the antidepressant drugs citalopram and phenelzine, are presented. </p><p>A polymorphism in the second intron of the gene encoding AP-2ß has previously been associated with anxiety-related personality traits as estimated by the Karolinska Scales of Personality (KSP). In this thesis, results confirming this association, gained by using a larger material and several different personality scales, are presented. Furthermore, data is presented showing an association between the activity of platelet monoamine oxidase, a trait-dependent marker for personality, and the genotype of the AP-2ß intron 2 polymorphism. </p><p>The functional importance of the AP-2ß intron 2 polymorphism has not yet been elucidated. Included in this thesis are results showing that the AP-2ß intron 2 polymorphism is not in linkage disequilibrium with the only other described polymorphism in the AP-2ß gene, i.e. in the AP-2ß promoter (-67 G/A). Introns have in several studies been shown to include binding sites for regulatory proteins, and thus, to be important in transcriptional regulation. Results are presented demonstrating that one human brain nuclear protein binds only to the long variant of the AP-2ß intron 2 polymorphism. If this protein is involved in the regulation of the AP-2ß gene, it would affect the expression levels of the AP-2ß protein. </p><p>In general, this thesis further establishes the role of transcription factor AP-2 as a regulatory factor of importance for personality and monoaminergic functions.</p>

Eukaryotic gene transcription plays a regulatory role in mammalian developmental processes. It has been shown that transcriptional control is an important mechanism for specification of neurotransmitter phenotypes. In the mammalian central nervous system, the transcription factor AP-2 family is one of the critical regulatory factors for neural gene expression and neuronal development. It has been shown that several genes in the monoaminergic systems have AP-2 binding sites in regulatory regions, suggesting a regulatory role of AP-2 also in the adult brain. Brainstem monoamines are implicated in the expression of personality traits and imbalances in these systems may give rise to psychiatric disorders. The gene encoding AP-2β includes a polymorphic region consisting of a tetranucleotide repeat of [CAAA]4-5 in intron 2. Studies on AP-2β genotype in relation to personality and platelet MAO activity, a trait-dependant marker for personality, are presented in this thesis. Furthermore, correlations between brainstem levels of AP-2α and AP-2β and monoamine turnover in projection areas in rat forebrain are reported. These results strengthen the notion that the AP-2 family is important regulators of the monoaminergic systems in the adult brain. Furthermore, two studies are presented in this thesis with analyses indicating a role for AP-2 in the molecular mechanism of antidepressant drugs. Altogether, this thesis presents data supporting our notion that the transcription factor AP-2 family is involved in the regulation of the monoaminergic systems both pre- and postnatally, and, therefore, might be involved in the pathophysiology of neuropsychiatric disorders.