Caractérisation immunohistologique de la pathologie amyloïde et des symptômes associés dans un modèle murin dela maladie d'Alzheimer. (Conférencière invitée)

International audienc

Primary culture of mouse dopaminergic neurons

International audienceDopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson's disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment

Serotonin type 4 receptor dimers

Chapitre 7International audienceNumerous class A G protein-coupled receptors and especially biogenic amine receptors have been reported to form homodimers. Indeed, the dimerization process might occur for all the metabotropic serotonergic receptors. Moreover, dimerization appears to be essential for the function of serotonin type 2C (5-HT2C) and type 4 (5-HT4) receptors and required to obtain full receptor activity. Several techniques have been developed to analyze dimer formation and properties. Due to our involvement in deciphering 5-HT4R transduction mechanisms, we improved and set up new procedures to study 5-HT4R dimers, by classical methods or modern tools. This chapter presents detailed protocols to detect 5-HT4R dimers by western blotting and co-immunoprecipitation, including the optimizations that we routinely carry out. We developed an innovative method to achieve functional visualization of 5-HT4R dimers by immunofluorescence, taking advantage of the 5-HT4-RASSL (Receptor Activated Solely by Synthetic Ligand) mutant that was engineered in the laboratory. Finally, we adapted the powerful Time-resolved FRET technology to assess a relative quantification of dimer formation and affinit

Gαolf Mutation Allows Parsing the Role of cAMP-Dependent and Extracellular Signal-Regulated Kinase-Dependent Signaling in L-3,4-Dihydroxyphenylalanine-Induced Dyskinesia.

International audienceAlthough l-3,4-dihydroxyphenylalanine (l-DOPA) remains the reference treatment of Parkinson's disease, its long-term beneficial effects are hindered by l-DOPA-induced dyskinesia (LID). In the dopamine (DA)-denervated striatum, l-DOPA activates DA D(1) receptor (D(1)R) signaling, including cAMP-dependent protein kinase A (PKA) and extracellular signal-regulated kinase (ERK), two responses associated with LID. However, the cause of PKA and ERK activation, their respective contribution to LID, and their relationship are not known. In striatal neurons, D(1)R activates adenylyl-cyclase through Gα(olf), a protein upregulated after lesion of DA neurons in rats and in patients. We report here that increased Gα(olf) levels in hemiparkinsonian mice are correlated with LID after chronic l-DOPA treatment. To determine the role of this upregulation, we performed unilateral lesion in mice lacking one allele of the Gnal gene coding for Gα(olf) (Gnal(+/-)). Despite an increase in the lesioned striatum, Gα(olf) levels remained below those of unlesioned wild-type mice. In Gnal(+/-) mice, the lesion-induced l-DOPA stimulation of cAMP/PKA-mediated phosphorylation of GluA1 Ser845 and DARPP-32 (32 kDa DA- and cAMP-regulated phosphoprotein) Thr34 was dramatically reduced, whereas ERK activation was preserved. LID occurrence was similar in Gnal(+/+) and Gnal(+/-) mice after a 10-d l-DOPA (20 mg/kg) treatment. Thus, in lesioned animals, Gα(olf) upregulation is critical for the activation by l-DOPA of D(1)R-stimulated cAMP/PKA but not ERK signaling. Although the cAMP/PKA pathway appears to be required for LID development, our results indicate that its activation is unlikely to be the main source of LID. In contrast, the persistence of l-DOPA-induced ERK activation in Gnal(+/-) mice supports its causal role in LID development

Prodromal stimulation of 5-HT4 receptors reduces amyloid pathology in 5XFAD mice.

International audienc

5-HT4 agonists: novel promising agents for AD prevention. (Conférencière invitée)

International audienc

Activation chronique du récepteur de la sérotonine de type 4 : Effet préventif dans un model murin de la maladied’Alzheimer. (Conférencière invitée)

International audienc

5 HT4 agonists: novel promising agents for AD prevention.

International audienc

Activation chronique du récepteur de la sérotonine de type 4 : Effet préventif dans un model murin de la maladied’Alzheimer. (Conférencière invitée)

International audienc