The 5-HT4 receptor networks: bodyguards of ADAM10 and APP trafficking – ADAMGUARD

The characterization of the protein networks associated with the serotonin type 4 receptor and ADAM10 is carried out by mass spectrometry. This technology enables to accurately determine the protein composition of a biologic sample. This unbiased approach provides an exhaustive list of network partners, within which we focus our attention on the proteins likely having a role in intracellular trafficking. We over-express or suppress these relevant proteins in the cells to analyse the impact on amyloid peptide production. Then, we try to decipher the action mechanisms of the more efficient candidates and to propose innovative strategies that may have an application in vivo. Finally, our hypotheses are validated with the help of transgenic animal models of Alzheimer's disease.
Moreover, in cellular models and in vivo, we study the effect of the serotonin receptor activation by chemicals, drug candidates.

The first proteomic screens have driven our attention on two proteins of the network associated with the serotonin receptor and ADAM10 that are of particular interest in the context of Alzheimer's disease. Mutations of the first candidate protein have already been reported in neurodegenerative pathologies. The second protein has not been clearly involved in physiopathological mechanisms of Alzheimer's disease, despite its link with the amyloid precursor protein. The two proteins have a function related to intracellular trafficking, a fact that support our hypothesis. We are characterizing the role of these molecular actors.
In the in vivo part of this project, we have demonstrated that chronic and early administration of molecules activating the serotonin receptor is able to slow down the pathology course of a mouse model of Alzheimer’s disease. We also conducted a longitudinal analysis of amyloid-dependant damages to the cerebrovasculature and the integrity of the blood-brain barrier.
In collaboration with two teams from the Caen University, we designed of a innovative multi-target directed molecule that is really promising for the treatment of Alzheimer’s disease. The originality of donecopride lies in the fact that, on one side, it may have symptomatic effects similar to the donepezil, which is the actual medicine used for Alzheimer’s disease and on the other side it may slow down the disease’s course by its abilities to activate the serotonin receptor.

This project should provide, for the fist time, the description of the entire protein network associated with a serotonin receptor, in a physiological context. This characterization should open new therapeutical strategies in order to slow down Alzheimer's disease.

A publication in ACS Chemical Neuroscience described the existence of the complex between serotonin receptor, the amyloid protein precursor and the alpha-secretase ADAM10.
A publication in Frontiers in Aging Neuroscience demonstrated the positive effects of chronic treatments with molecules that activate the serotonin receptor.
An article in Cell Death & Disease precised the modulation mechanisms of ADAM10 trafficking.
Two publications in PNAS and The Journal of Medical Chemistry related the conception and characterisation of donécopride, an original and promising molecule for the treatment of Alzheimer’s disease by its simultaneous action on two distinct targets.
A viewpoint in ACS Chemical Neuroscience outlined the interest of multiple intervention strategy within the serotonin system to fight against Alzheimer's disease.
A publication in Neurobiology of Disease presented our longitudinal analysis of amyloid-dependant damages to the cerebrovasculature and the integrity of the blood-brain barrier.

Alzheimer's disease (AD) is the most common form of dementia affecting 35 million individuals worldwide and over 800 000 in France. It is characterized by the presence in the brain of neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau and of "amyloid" plaques mainly composed of aggregates of hydrophobic ß-amyloid peptide (Aß). Aß peptide formation results from the "amyloidogenic" degradation of a transmembrane precursor, the APP (Amyloid Precursor Protein) by ß- and gamma-secretases mainly in early/sorting and late endosomes. The "non-amyloidogenic" proteolysis of APP within the Aß sequence by alpha-secretase releases the extracellular fragment of APP (sAPPalpha), which is neurotrophic and increases long term potentiation. Non-amyloidogenic APP processing mainly occurs at the plasma membrane level. The most active alpha-secretase in brain is ADAM10. Several G protein-coupled receptors (GPCRs) stimulate the amyloidogenic pathway by increasing the endocytosis of ß- and gamma-secretases. These GPCRs include GPR3 which acts via a physical association with these enzymes. Our preliminary experiments show that the 5-HT4 receptor (5-HT4R) physically associates directly or indirectly with the alpha-secretase ADAM10 and APP. Moreover 5-HT4 receptor enhances the trafficking of ADAM10 from the endoplasmic reticulum, where it is retained by a specific sequence (RRR), to the plasma membrane. This effect is "constitutive" and agonist-independent. Using a preliminary proteomic approach in HEK-293 cells, we have also shown that 5-HT4 receptors interact with numerous GIPs (GPCR interacting proteins). Some of them are implicated in the trafficking of membrane proteins such as APP. We hypothesize that the receptor itself or the receptor/GIP complex might mask the endoplasmic reticulum (ER) retention site in ADAM10 C-terminus and thereby promote its ER sorting and trafficking to the plasma membrane. The objective of this proposal is to get, in a neuronal context and for the first time, a global picture of protein networks underlying the regulation of the trafficking of ADAM10 and APP to the plasma membrane and processing of APP by a GPCR, the 5-HT4 receptor. For this objective we will: 1) characterize the protein networks associated with 5-HT4 receptors, APP and ADAM10 in neurons by means of proteomic approaches; 2) characterize the role of selected proteins within these networks in the trafficking of the 5-HT4R/ADAM10/APP complex to the plasma membrane where ADAM10 can release sAPPalpha from APP; 3) characterize if the presence or absence of 5-HT4 receptor impacts APP processing and the formation of amyloid plaques, in vivo, crossing the 5-HT4 receptor -/- mice with some AD mice models (APPswe/PS1deltaE9 and 5XFAD). Conjointly, we will study the effect of a chronic 5-HT4R agonist-treatment on APP processing and plaque formation in the 5XFAD model. Indeed, in addition to the "constitutive" regulation of ADAM10 by 5-HT4 receptors, which constitutes the main and entirely original focus of this study, activation of 5-HT4 receptors, by agonists, also exerts a known "regulated" activation of alpha-secretases.