Abstract
Alzheimer’s Disease is the most common form of dementia worldwide and has become a major public health problem. This pathology is characterized by the presence of two main features in the brain: neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau and amyloid plaques, dense aggregates of hydrophobic β-amyloid peptide (Aβ). Aβ peptide results from the amyloidogenic degradation of the Amyloid Precursor Protein (APP) by β- and ɣ-secretases. On the other hand, the non-amyloidogenic proteolysis of APP, within the Aβ sequence by an α-secretase (mainly ADAM10), releases the extracellular fragment of APP (sAPPα), which is neurotrophic and neuroprotective. The 5-HT4 receptor (5-HT4R) is a G protein coupled receptor (GPCR) which interacts with APP and ADAM10, modulates their trafficking and maturation, and which pharmacological activation promotes the non-amyloidogenic cleavage of APP. The 5-HT6 receptor (5-HT6R) is another serotoninergic receptor which pharmacological modulation induces pro-cognitive effects. Thus, 5-HT4R and 5 HT6R constitute interesting therapeutic targets in the context of Alzheimer’s Disease. Nevertheless, the molecular and cellular mechanisms underlying these effects linked to 5-HT4R expression and activation remain elusive. This thesis aimed, in a first part, to develop a screening technique to isolate nanobodies (single domain antibody fragment from camelids) directed against 5-HT4R and 5 HT6R, by using TR FRET and flow cytometry approaches. In a second part, the use of a proteomic approach combining affinity purification of 5-HT4R partner proteins followed by their systematic identification by mass spectrometry (AP-MS) lead to the identification of potential partner proteins associated with 5-HT4R in HEK-293N cells. Amongst proteins recruited by 5 HT4Rs, we identified IRS4 (Insulin Receptor Substrate 4) protein. IRS4 protein is a transducer of the insulin signaling pathway. Given that a dysregulation of the insulin signaling pathway is one of the major events involved in Alzheimer’s Disease development, we decided to focus on this partner. We validated the interaction by Western blotting and showed a colocalization of endogenous IRS4 with 5 HT4R in HEK 293 cells. Then, functional studies showed that IRS4 downregulation induced 5 HT4R overexpression in HEK-293 cells, associated with an increase in the canonical signaling of the receptor through its Gs protein pathway.These results suggest a modulation of 5-HT4R trafficking and of its associated complex including APP and ADAM10 by IRS4 protein. This thesis work reveals for the first time a functional link between 5-HT4R signaling and insulin signaling pathway via IRS4 protein which could be of interest in the context of Alzheimer’s disease. It also establishes the preliminary basis for a fast identification of nanobodies targeting 5-HT4 and 5-HT6 receptors for which immunohistological tools are lacking to explore their underlying mechanisms.