HTT is a repressor of ABL activity required for APP induced axonal growth

ABL tyrosine kinase activity controls several aspects of development including axon patterning. Amyloid precursor protein (APP) is linked to Alzheimer's disease and previous work established that ABL is a downstream effector in an Appl, the Drosophila App ortholog, signaling pathway which modulates axon outgrowth in the mushroom bodies (MBs), the fly memory center. Here we show that Abl is required for the MB neuron axonal growth. Importantly, both Abl overexpression and lack of expression produce a similar phenotype in the MBs indicating the necessity of tightly regulating ABL activity. We find that the fly huntingtin protein (HTT), the homolog of the protein involved in Huntington's disease, behaves genetically as a repressor of ABL activity. Supporting this, FRET-based measurements of in vivo ABL activity in the MBs reveal a clear increase in its activity when HTT levels are reduced. Thus, in addition to its many other reported roles, HTT acts as a negative regulator of ABL activity, at least in the MBs, to maintain its appropriate physiological levels necessary for axon growth.

Rôle de la protéine APPL dans la croissance axonale des corps pédonculés chez Drosophila melanogaster

In the drosophila brain, mushroom bodies are involved in olfactory memory and learning. This structure is composed of different types of / neurons. These neurons form an orthogonal structure, with the branch projecting dorsally and the branch projecting medially. The aim of this study is to understand mechanisms and pathways involved during the development of these neurons.The drosophila APPL protein (Amyloïd Precursor Protein-Like) is the homologue of the human APP, known to be involved in Alzheimer’s disease. This pathology is characterized by neuronal degeneration inducing cognitive and memory defects. In spite of the numerous studies focused on the pathological function of APP during the last decades, few things are known on the physiological functions of this protein and more particularly during the development. This is from this perspective that we studied the APPL function and its interaction with proteins during the mushroom bodies development.The APPL protein was identified as a co-receptor of the PCP pathway (Planar Cell Polarity), involved in the axonal growth regulation. During the development, APPL allows the recruitment and the activation of the ABL protein (Abelson Tyrosine Kinase), which phosphorylates DSH (dishevelled) and so activates the axonal growth pathway.The first part investigates the regulation of ABL activity during the / neuron development. If it’s already established that APPL regulates positively the kinase activity of ABL, I show here that the HTT protein (Huntingtin) allows a negative regulation of ABL activity. In human, HTT is involved in the Huntington’s disease, another neurodegenerative disorder. This thesis work shows that HTT regulates the phosphorylation level of ABL, and therefore its activity.The second part investigates the interaction between APPL and ARM (armadillo), the homologue of the human -catenin, during the development of the / neurons. I show that this interaction is independent of the APPL function in the PCP pathway. Moreover, this interaction between APPL and ARM involves the actin cytoskeleton dynamic function of ARM, and not its Wnt pathway function.The third and last part presents new mutant alleles of APPL obtained with the CRISPR-CAS9 technique. The creation and analysis of these new alleles lead us to propose that vnd (ventral nervous system defective), neighbor gene of Appl, is also involved in / neurons development, and can interact genetically with Appl.Le cerveau de drosophile est constitué entre autres des mushroom body, siège de la mémoire et de l’apprentissage. Cette structure est composée de différents types de neurones, parmi lesquels les neurones /. Ces neurones se présentent sous une forme orthogonale, avec l’axone qui se divise en une branche dorsale : la branche et une branche médiale : la branche . Le but de cette étude est de comprendre les mécanismes et voies de signalisation mis en jeu lors du développement de ces neurones.Chez la drosophile, la protéine APPL (Amyloïd Precursor Protein-Like) est l’homologue de la protéine APP humaine, connue pour son implication dans la maladie d’Alzheimer chez l’homme. Cette pathologie est caractérisée par une dégénérescence neuronale entraînant des défauts cognitifs et mnésiques. Malgré les nombreuses études focalisées sur la fonction pathologique d’APP durant les dernières décennies, peu de choses sont actuellement connues sur les fonctions physiologiques de cette protéine et notamment pendant le développement. C’est dans cette optique que nous avons étudié la fonction d’APPL et son interaction avec différentes protéines lors du développement des mushroom body. La protéine APPL a été identifiée comme étant un co-récepteur de la voie PCP (Planar Cell Polarity), permettant la régulation de la croissance axonale. Lors du développement, APPL permet le recrutement et l’activation de la protéine ABL (Abelson Tyrosine Kinase), qui phosphoryle DSH (dishevelled) et ainsi active la voie de signalisation permettant la croissance axonale.Le premier volet de cette thèse porte sur la régulation de l’activité ABL lors du développement des neurones /. S’il est établi qu’APPL permet une régulation positive de l’activité kinase d’ABL, je montre ici que la protéine HTT (huntingtine) permet de réguler négativement cette activité. Cette protéine HTT est impliquée dans la maladie de Huntington chez l’homme, une autre pathologie neurodégénérative. Ces travaux démontrent qu’HTT régule le niveau de phosphorylation d’ABL et par conséquent son activité. Le deuxième volet de cette thèse porte sur l’interaction d’APPL avec la protéine ARM (armadillo), homologue de la -caténine, lors du développement des neurones /. Je démontre que cette interaction est indépendante du rôle d’APPL dans la voie PCP. Je démontre aussi que cette interaction entre APPL et ARM est dépendante uniquement de la fonction d’ARM dans la dynamique du cytosquelette d’actine.Enfin le troisième volet de cette thèse porte sur la création de nouveaux allèles mutants pour Appl grâce à la technique du CRISPR-CAS9. La production de ces allèles permet d’avancer d’une part un possible rôle du gène voisin vnd (ventral nervous system defective) dans le développement des mushroom body, et d’autre part une interaction génétique entre Appl et vnd

Function of APPL during axonal growth in the Drosophila brain

Le cerveau de drosophile est constitué entre autres des mushroom body, siège de la mémoire et de l’apprentissage. Cette structure est composée de différents types de neurones, parmi lesquels les neurones /. Ces neurones se présentent sous une forme orthogonale, avec l’axone qui se divise en une branche dorsale : la branche et une branche médiale : la branche . Le but de cette étude est de comprendre les mécanismes et voies de signalisation mis en jeu lors du développement de ces neurones.Chez la drosophile, la protéine APPL (Amyloïd Precursor Protein-Like) est l’homologue de la protéine APP humaine, connue pour son implication dans la maladie d’Alzheimer chez l’homme. Cette pathologie est caractérisée par une dégénérescence neuronale entraînant des défauts cognitifs et mnésiques. Malgré les nombreuses études focalisées sur la fonction pathologique d’APP durant les dernières décennies, peu de choses sont actuellement connues sur les fonctions physiologiques de cette protéine et notamment pendant le développement. C’est dans cette optique que nous avons étudié la fonction d’APPL et son interaction avec différentes protéines lors du développement des mushroom body. La protéine APPL a été identifiée comme étant un co-récepteur de la voie PCP (Planar Cell Polarity), permettant la régulation de la croissance axonale. Lors du développement, APPL permet le recrutement et l’activation de la protéine ABL (Abelson Tyrosine Kinase), qui phosphoryle DSH (dishevelled) et ainsi active la voie de signalisation permettant la croissance axonale.Le premier volet de cette thèse porte sur la régulation de l’activité ABL lors du développement des neurones /. S’il est établi qu’APPL permet une régulation positive de l’activité kinase d’ABL, je montre ici que la protéine HTT (huntingtine) permet de réguler négativement cette activité. Cette protéine HTT est impliquée dans la maladie de Huntington chez l’homme, une autre pathologie neurodégénérative. Ces travaux démontrent qu’HTT régule le niveau de phosphorylation d’ABL et par conséquent son activité. Le deuxième volet de cette thèse porte sur l’interaction d’APPL avec la protéine ARM (armadillo), homologue de la -caténine, lors du développement des neurones /. Je démontre que cette interaction est indépendante du rôle d’APPL dans la voie PCP. Je démontre aussi que cette interaction entre APPL et ARM est dépendante uniquement de la fonction d’ARM dans la dynamique du cytosquelette d’actine.Enfin le troisième volet de cette thèse porte sur la création de nouveaux allèles mutants pour Appl grâce à la technique du CRISPR-CAS9. La production de ces allèles permet d’avancer d’une part un possible rôle du gène voisin vnd (ventral nervous system defective) dans le développement des mushroom body, et d’autre part une interaction génétique entre Appl et vnd.In the drosophila brain, mushroom bodies are involved in olfactory memory and learning. This structure is composed of different types of / neurons. These neurons form an orthogonal structure, with the branch projecting dorsally and the branch projecting medially. The aim of this study is to understand mechanisms and pathways involved during the development of these neurons.The drosophila APPL protein (Amyloïd Precursor Protein-Like) is the homologue of the human APP, known to be involved in Alzheimer’s disease. This pathology is characterized by neuronal degeneration inducing cognitive and memory defects. In spite of the numerous studies focused on the pathological function of APP during the last decades, few things are known on the physiological functions of this protein and more particularly during the development. This is from this perspective that we studied the APPL function and its interaction with proteins during the mushroom bodies development.The APPL protein was identified as a co-receptor of the PCP pathway (Planar Cell Polarity), involved in the axonal growth regulation. During the development, APPL allows the recruitment and the activation of the ABL protein (Abelson Tyrosine Kinase), which phosphorylates DSH (dishevelled) and so activates the axonal growth pathway.The first part investigates the regulation of ABL activity during the / neuron development. If it’s already established that APPL regulates positively the kinase activity of ABL, I show here that the HTT protein (Huntingtin) allows a negative regulation of ABL activity. In human, HTT is involved in the Huntington’s disease, another neurodegenerative disorder. This thesis work shows that HTT regulates the phosphorylation level of ABL, and therefore its activity.The second part investigates the interaction between APPL and ARM (armadillo), the homologue of the human -catenin, during the development of the / neurons. I show that this interaction is independent of the APPL function in the PCP pathway. Moreover, this interaction between APPL and ARM involves the actin cytoskeleton dynamic function of ARM, and not its Wnt pathway function.The third and last part presents new mutant alleles of APPL obtained with the CRISPR-CAS9 technique. The creation and analysis of these new alleles lead us to propose that vnd (ventral nervous system defective), neighbor gene of Appl, is also involved in / neurons development, and can interact genetically with Appl

HTT is a repressor of ABL activity required for APP induced axonal growth

ABL tyrosine kinase activity controls several aspects of development including axon patterning. Amyloid precursor protein (APP) is linked to Alzheimer's disease and previous work established that ABL is a downstream effector in an Appl, the Drosophila App ortholog, signaling pathway which modulates axon outgrowth in the mushroom bodies (MBs), the fly memory center. Here we show that Abl is required for the MB neuron axonal growth. Importantly, both Abl overexpression and lack of expression produce a similar phenotype in the MBs indicating the necessity of tightly regulating ABL activity. We find that the fly huntingtin protein (HTT), the homolog of the protein involved in Huntington's disease, behaves genetically as a repressor of ABL activity. Supporting this, FRET-based measurements of in vivo ABL activity in the MBs reveal a clear increase in its activity when HTT levels are reduced. Thus, in addition to its many other reported roles, HTT acts as a negative regulator of ABL activity, at least in the MBs, to maintain its appropriate physiological levels necessary for axon growth.

Htt is a repressor of Abl activity required for APP-induced axonal growth.

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αβ neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αβ neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth

Guidance of Drosophila Mushroom Body Axons Depends upon DRL-Wnt Receptor Cleavage in the Brain Dorsomedial Lineage Precursors

In vivo axon pathfinding mechanisms in the neuron-dense brain remain relatively poorly characterized. We study the Drosophila mushroom body (MB) axons, whose α and β branches connect to different brain areas. We show that the Ryk family WNT5 receptor, DRL (derailed), which is expressed in the dorsomedial lineages, brain structure precursors adjacent to the MBs, is required for MB α branch axon guidance. DRL acts to capture and present WNT5 to MB axons rather than transduce a WNT5 signal. DRL’s ectodomain must be cleaved and shed to guide α axons. DRL-2, another Ryk, is expressed within MB axons and functions as a repulsive WNT5 signaling receptor. Finally, our biochemical data support the existence of a ternary complex composed of the cleaved DRL ectodomain, WNT5, and DRL-2. Thus, the interaction of MB-extrinsic and -intrinsic Ryks via their common ligand acts to guide MB α axons

Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by GIRK4 inactivation

The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific I-f silencing caused altered [Ca2+](i) release and Ca2+ handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of I-f silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in I-f-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases