Contrôle transcriptionnel associé au domaine intracellulaire du précurseur du peptide amyloïd

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by neurofibrillary tangles and deposition of β-amyloïd peptide (Aβ) derived from β-amyloïd precursor protein (APP). APP belongs to a gene family including APP-like proteins (APLPs), which could display overlapping functions. Although the mechanisms of Aβ production have been intensely studied, the biological functions of APP and of its proteolytic fragments are poorly understood. APP is cleaved by alpha-, beta- and gamma-secretase activities leading to the production of soluble alpha APP (sαAPP), soluble beta APP (sβAPP), Aβ and an intracellular fragment named APP intracellular domain (AICD). Biochemical and genetic interaction screens have also led to the identification of multiple intracellular binding partners including the adaptor protein Fe65. Several studies suggested that this protein is able to modulate the APP processing, signaling and Aβ production. AICD is thought to regulate gene transcription but the identity of the target genes as well as the mechanisms involved remain highly controversial. The aim of this work was to clarify the role of Fe65 in APP processing, Aβ production and APP signaling. We also wanted to identify new genes regulated by APP. Using a human APPGal4 fusion protein in a luciferase-based reporter assay, we showed that AICD triggers the transcription of reporter genes. Coexpression of Fe65 and human APP in CHO cells had no effect on sαAPP, sβAPP and endogenous AICD production but increased luciferase activity and decreased Aβ production, indicating that Fe65 is able to enhance transcription independently of AICD. We profiled gene expression using Gene-chip Micro array in mouse embryonic cells (MEFs) expressing or not APP and presenilins (presenilin 1 and 2), the catalytic core of the gamma-secretase. We found Aquaporin1 (AQP1) as new APP target gene and confirmed our results by qRT-PCR, Western blotting and rescue experiments using retroviral and lentiviral expression of APP and presenilins. In addition, we found, in vivo, an up-regulation of AQP1 in astrocytes located near senile plaques of AD and lower levels of AQP1 in APP knock-out mice. Our results indicate that a PS2-mediated cleavage of APP releases an AICD fragment that interacts with histone deacetylase activities and controls indirectly AQP1 expression. This interaction confers to APP a key role in histone acetylation and chromatin remodelling, a new mechanism possibly involved in the etiology of Alzheimer's diseaseLa maladie d'Alzheimer est une démence neurodégénérative caractérisée par la présence de dégénérescences neurofibrillaires et de plaques séniles dans le cerveau. Celles-ci sont constituées d'un noyau de peptide amyloïde (Aβ) provenant de la protéolyse d'une protéine appelée Précurseur du Peptide Amyloïde (APP). Bien que les voies métaboliques de l'APP aient largement été étudiées, la fonction physiologique de l'APP demeure, à ce jour, peu connue. L'APP est clivé par différentes sécrétases ce qui aboutit à la libération d'APP soluble alpha (APPsα), beta (APPsβ), d’Aβ et du domaine intracellulaire de l'APP ou AICD. Ce dernier fragment interagit avec de nombreuses protéines intracellulaires parmi lesquelles on compte la protéine adaptatrice Fe65. Ces dernières années, différents travaux montrent, d'une part, que Fe65 peut moduler le métabolisme de l'APP, la signalisation dépendante de l'AICD ainsi que la production d'Aβ. D'autre part, il est apparu que l'AICD pouvait contrôler la transcription de certains gènes bien que ce point fasse l'objet d'une vive controverse. L'objectif de ce travail a été, dans un premier temps, de tenter de clarifier le rôle de Fe65 dans le métabolisme de l'APP, la production d'Aβ et la signalisation dépendante de l'APP. Nous avons ensuite voulu identifier de nouveaux gènes cibles de l'APP. Nous avons montré, à l'aide de protéines de fusion APPGal4, que l'AICD est capable d'induire la transcription de gènes rapporteurs. Par ailleurs, la co-expression de Fe65 et de l'APP n'a pas d'effet sur la production APPsα, APPsβ et d'AICD endogène. Nos résultats indiquent également que l'expression de Fe65 augmente l'activité transcriptionnelle tout en diminuant la production de peptide amyloïde, ce qui suggère un rôle propre de Fe65 sur la transcription, du moins indépendant de l'AICD. En analysant le profil d'expression génique par Micro array sur des cellules embryonnaires de souris (MEFs) exprimant ou non l'APP et les présénilines (préséniline 1 et 2), nous avons identifié un nouveau gène régulé par l'expression de l'APP : l'aquaporine 1. Nous avons confirmé les résultats obtenus par qRT-PCR et Western blotting. De plus, la réexpression de l'APP ou de la préséniline 2 dans des cellules APPKO ou PSKO restaure l'expression d'AQP1. In vivo, nous avons pu observer une augmentation de l'expression d'AQP1 dans les astrocytes localisés près des plaques séniles des patients Alzheimer ainsi qu'une diminution d'AQP1 dans des souris APP knock-out. Dans leur ensemble, nos résultats indiquent que le clivage PS2-dépendant de l'APP libère un AICD capable d'interagir avec des histones déacétylases et de contrôler indirectement l'expression de l'AQP1. Cette interaction confère à l'APP un rôle clé dans l'acétylation des histones et dans le remodelage de la chromatine, un nouveau mécanisme potentiellement impliqué dans l'étiologie de la maladie d'AlzheimerThèse de doctorat en sciences biomédicales et pharmaceutiques (SBIM 3) --UCL, 201

Fe65 does not stabilize AICD during activation of transcription in a luciferase assay.

The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment

Unravelling the roles of lysine acetyl-transferase activity of Elongator complex during inner ear development

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P15. To find new potential targets for Elp3, transcriptomes from wild-type, heterozygous and Elp3 cKO mice were analysed by RNA-Seq at E14.5 and E18.5. Surprisingly, we observed that hair cell markers were upregulated in the Elp3 cKO at E14.5, suggesting a premature differentiation in these mice that was confirmed by in situ hybridisation. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3

Rôles du complexe Elongator dans le développement embryonnaire de l'oreille interne

The inner ear is composed of the vestibular system that controls balance, and the cochlea, which is dedicated to hearing. In both parts of the inner ear, sensory epithelia comprise supporting cells surrounding the sensory hair cells. These cells bear at their apical surface a staircase-structured bundle, consisting of multiple rows of actin-based stereocilia and a single tubulin-based kinocilium. This hair bundle allows the transduction from mechanical stimuli, initiated by sound or gravitational changes, to electrical signals that will then be transmitted by neurons from the spiral ganglion (innervating hair cells of the cochlea) or the vestibular ganglion. The inner ear organogenesis requires a tightly regulated transcriptional program that can be affected by post-transcriptional and post-translational modifications among which lysine acetylation. Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first determine the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we have generated conditional knock-out mice (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, compared to wild-type mice, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not found any structural abnormalities nor cell patterning impairments that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P0 and P15. In conclusion, our results clearly showed a role of Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins acetylated by Elp3

UNRAVELLING THE ROLES OF LYSINE ACETYLATION BY ELP3 DURING INNER EAR DEVELOPMENT

Given the importance of acetylation homeostasis in controlling developmental processes [1-3], we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis [4]. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of neurons and fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P15. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding hair cells innervation (misorientation of fibers). In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3

Unravelling the roles of lysine acetylation by Elp3 during inner ear development

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons P15. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3