Dynamics of Intracellular Signaling and Therapeutic Targets

Intracellular signals determine the properties of a cell within an organism, and participates in the intregration of extracellular cues leading to an appropriate cellular response. Our approach focuses on the dynamic aspects of this integration process, in particular with the use of real-time biosensor imaging. We use this experimental framework to monitor adaptations in physiopathological conditions and analyze the effects of pharmacological compounds. This will ultimately lead to novel therapeutic strategies.

Trans-differentiation of vascular smooth muscle cells in atherosclerosis

A short splice variant of adenylyl cyclase perturbs cAMP signaling.

Vascular smooth muscle cells submitted to chronic inflammation - such as that involved in athrosclerotic plaque formation or post-angiopalsty restenosis - show a prominent reduction in their cAMP production, leading to increased cell motility and proliferation. We showed that this transdifferentiation depends on the de-novo expression of a short splice variants of adenylyl cyclase 8, which lacks the first 5 transmembrane segments. These short forms of AC8 exert a dominant-negative effect by dimerizing with the other full-length functional adenylyl cyclases, retaining them in the endoplasmic reticulum and thus reducing global cAMP production within the cell.

We are now exploring how to inhibit this deleterious process in the prospect of preventing vascular diseases.

More on the short AC8 isoforms

Biosensor imaging to analyze the dysfunctions in cAMP signaling

Live-imaging of cAMP in primary cultures of control vascular smooth muscle cells (top) and the same cell type after chronic interleukine treatment (bottom). Forskolin was used to increase cAMP production. In control cells, this leads to a maximal response, whereas transdifferentiated cells show a limited cAMP production.

Référence: Vallin et al, 2018.

Dopamine and other neuromodulators in the striatum

A number of neuromodulators, a complex integrating scheme

Our objective is to analyze the dynamics of the complex signaling events, looking at the signaling events in real time in living neurons using genetically-encoded fluorescent biosensors. We also analyze how these events are altered in physiopathological conditions, such as the chronic loss of dopamine in Parkinson's disease.

The striatum, a brain structure with complex neuromodulatory processes

95 % of striatal neurons are medium spiny neurons, of two distinct types: neurons of the anatomical "direct pathway" express D1 dopamine and M4 muscarinic receptors; neurons of the "indirect pathway" express D2 dopamine and A2A adenosine receptors. D1 and A2A receptors trigger an increase in cAMP production, whereas D2 and M4 inhibit cAMP production. Many other neuromodulators, including nitric oxide, are also part of the game.

Dopamine action in the striatum

Dopamine signals are integrated in a non-linear way

It is commonly accepted that a low level of dopamine preferentially activate D2 dopamine receptors, while D1 receptors would respond selectively to a high level of dopamine such as that associated with a reward. This assumption is challenged by simultaneously imaging cAMP in D1 and D2 neurons: both neurons respond to dopamine with almost the same sensitivity. Integration down the cascade make things more complicated, with D2 neurons appear ideally geared (with DARPP-32) to detect a pause in the release of dopamine.

DARPP-32 promotes all-or-none responsiveness

Nucleus story.

Cellular mechanisms involved in the dopamine / acetylcholine balance

Acetylcholine gates the action of dopamine

M4 muscarinic are negatively coupled to cAMP production, and co-expressed with D1 dopamine receptors by medium spiny neurons of the direct pathway. Activation of these receptors could in theory oppose the positive action of dopamine, and a pause in the firing of cholinergic neurons is required to allow for a positive response to dopamine. This was studied by numeric simulation. Experiments revealed that M4 receptors were highly sensitive to very low doses of acetylcholine, and these receptors therefore exert a powerful negative control on cAMP.

We are now studying the control of acetylcholine release by cholinergic interneurons.

Phosphodiesterases

Phosphodiesterases determine the dynamics of cyclic nucleotide signals

The main phosphodiesterases expressed in the striatum are PDE1B, PDE2A and PDE10A, degrading both cAMP and cGMP. PDE4 is marginally expressed and degrades specifically cAMP. We use biosensor imaging to analyze their functional role and determine their potential as therapeutic targets.

PDE10A is responsible for controlling baseline cAMP levels in the striatum (Polito, 2015).

PDE2A in the striatum can be efficiently activated by cGMP, and controls the higher levels of concentration of both cAMP and cGMP (Polito, 2013). In the mouse model of Fragile X Syndrome, PDE2A is expressed to a higher level than in control mice, leading to abnormal cAMP signaling (Maurin, 2018).

We are currently exploring the functional role played by PDE1A.

cAMP signaling during neuronal migration

Newly formed neurons

This project developed through a collaboration with Isabelle Caillé aims at analyzing the transient cAMP signals that occurr in the cytosol of neurons during their migration from the sub-ventricular zone toward the olfactory bulb.

Alterations in cAMP signaling in Alzheimer's disease

Neurons and vessels undergo a coicident degeneration during Alzheimer's disease.