Cellular integration of neuromodulatory processes

Neuromodulatory signals are transduced inside the cell through a variety of signaling cascades, among which the cyclic AMP (cAMP)/PKA cascade is an archetype. The striatum is a brain region where neuromodulators like dopamine play critical physiological functions in motor control, motivation or reward-mediated learning, and severe neuropsychiatric diseases (Parkinson, schizophrenia, addiction…) are associated with alterations in the neuronal response to dopamine

The team uses state of the art biosensor imaging methods to monitor the cAMP/PKA signaling cascade in real time and with sub-cellular spatial resolution in mature living neurons. This approach gives a mechanistic understanding of the molecular processes underlying signal integration, and we are currently studying the mechanism of action of novel phosphodiesterase inhibitors which are currently tested in clinics as antipsychotic treatment.

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Neuromodulators such as monoamines (dopamine, noradrenaline, serotonine...) or neuropeptides (enkephalines…) play a critical role in the regulation of neuronal activities and dysfunction in neuromodulatory systems are involved in a number of neuropsychiatric diseases, including Parkinson's disease or schizophrenia. Neuromodulators activate membrane receptors which transduce the signal through intracellular signaling cascades like cAMP and protein kinase A (PKA). These highly dynamic processes regulate a number of cellular events such as neurotransmission, excitability, gene expression…

Roger Y. Tsien was awarded the Nobel Prize of Chemistry in 2008 for his seminal work on fluorescent proteins and the concept of genetically-encoded fluorescent biosensors. We use such biosensors to measure in real time in living cells changes in intracellular cAMP concentration or activation of PKA. Because biosensors are genetically encoded, they can be expressed in virtually any cell type, from primary cultures to tissue preparations, and we routinely monitor cAMP/PKA signals in anatomically intact neurons in brain slice preparations. We focus on the functional role played by phosphodiesterases, the enzymes which degrade cAMP and thereby determine the temporal and spatial extent of the cAMP signal. We study how these processes are altered in pathological situations and how they might be restored by pharmacological treatment.

Highlights

By combining electophysiology and biosensor imaging in cortical neurons, we analyzed the PKA signal as it slows down and dampens during its propagation from the membrane through the cytosol and into the nucleus (Gervasi, 2007). Two-photon microscopy revealed a spatial compartmentation of this cAMP/PKA signal: in the somatic cytosol, phosphodiesterases maintain a low (sub-micromolar) cAMP level, whereas in the dendrites, cAMP increases faster and reaches much higher values (Castro, 2010). Type 4 phosphodiesterase appears critical to maintain this spatial compartmentation.

Dopamine activates the cAMP/PKA signaling cascade in both striatum and cortex. Biosensor imaging however revealed that the striatum was atypical in that the cAMP/PKA signal was particularly fast and powerful: a single sub-second dopaminergic stimulation was sufficient to trigger a large cytosolic signal and trigger c-Fos expression in the nucleus. We showed that this amazing striatal responsiveness stems from a high adenylyl cyclase activity, low phosphodiesterase activity and the presence of DARPP-32 (Castro, 2013). We suggest that the high sensitivity to brief dopamine transients is an essential property of striatum neurons which is critical for incentive learning.

Type 2 phosphodiesterase (PDE2) degrades both cAMP and cGMP, and this enzyme is activated by cGMP. We showed that, in the striatum, PDE2 indeed controls cGMP level. In addition, PDE2 controls the amplitude of transient cAMP responses, and its activity is under the control of cGMP (Polito, 2013). PDE2 thus mediates a cross-talk between two neuromodulators, dopamine and nitric oxide.

Future directions

  • Striatal neurons specifically express high levels of type 10 phosphodiesterase (PDE10), which makes this enzyme an interesting pharmaceutical target. PDE10 blockers show promising antipsychotic effects, but their mechanism of action remains uncertain. We are currently analyzing the functional effects of PDE10 inhibition on the dynamics of the cAMP/PKA signaling cascade.
  • Signaling cascades are profoundly affected by chronic conditions such as lack of dopaminergic input (Parkinson's disease), drug intake or chronic treatment with antipsychotic drugs. These adaptative changes are reported by genomics or proteomics analysis, but the actual consequence on the dynamics of intracellular signaling remain unknown. In adult animals with a chronic condition, we will analyze the changes undergone by striatal neurons during the adaptative process, and analyze the mechanism of action of drugs aiming at curing such disorders.

Collaborations

  • Isabelle Limon, team "phenotypic control of vascular smooth muscle cells", UMR8256, Paris.
  • Jean-Antoine Girault and Denis Hervé, Neurotransmission and Signal transduction, UMR-S839, Institut du Fer a Moulin, Paris
  • Fabienne Merola, Laboratoire de Chimie Physique, Université Paris-Sud, Orsay.
  • Kees Jalink, Cellular Biophysics team, The Netherlands Cancer Institute, Amsterdam.
  • Franck Riquet, Inflammation Research Center, VIB-UGent, Ghent, Belgium