Crosstalk between Rap1 and Rac regulates secretion of sAPPalpha.

Cyclic AMP (cAMP) is produced by activation of Gs protein-coupled receptors and regulates many physiological processes through activation of protein kinase A (PKA). However, a large body of evidence indicates that cAMP also regulates specific cellular functions through PKA-independent pathways. Here, we show that a small GTPase of the Rho family, Rac, is regulated by cAMP in a PKA-independent manner. We also show that Rac activation results from activation of Rap1 through the cAMP guanine nucleotide-exchange factor (GEF) Epac1. Activation of the Gs-coupled serotonin 5-HT(4) receptor initiates this signalling cascade in various cell types. Furthermore, we demonstrate that crosstalk between the Ras and Rho GTPase families is involved in cAMP-dependent processing of amyloid precursor protein (APP), a key protein in Alzheimer's disease. Indeed, Epac1 regulates secretion of the non-amyloidogenic soluble form of APP (sAPPalpha) through Rap1 and Rac. Our data identify an unsuspected connection between two families of small GTPases and imply that Rac can function downstream of cAMP/Epac1/Rap1 in a novel signal transduction secretory pathway.

Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells.

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-ß. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis.

Cytokines in neuroinflammation and Alzheimer's disease.

Inflammation has been reported in numerous neurodegenerative disorders such as Parkinson's disease, stroke and Alzheimer's disease (AD). In AD, the inflammatory response is mainly located to the vicinity of amyloid plaques. Cytokines, such as Interleukin-1 (IL-1), Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-alpha) and Transforminng Growth Factor beta (TGF-beta) have been clearly involved in this inflammatory process. Although their expression is induced by the presence of amyloid-beta peptide, these cytokines are also able to promote the accumulation of amyloid-beta peptide. Altogether, IL-1, IL-6, TNF-alpha and TGF-beta should be considered as key players of a vicious circle leading to the progression of the disease.

Ageing and amyloid-beta peptide deposition contribute to an impaired brain tissue plasminogen activator activity by different mechanisms.

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder in the ageing population. It is characterized by the cerebral accumulation of toxic amyloid-beta peptide assemblies (Abeta). The serine protease plasmin, which is generated from the inactive zymogen plasminogen through its proteolytic cleavage by tissue- (tPA) or urokinase-type plasminogen activator, has been implicated in the catabolism of Abeta peptides. In this report, we studied the regulation of tPA activity in vivo during ageing in normal mice and in a mouse model of AD characterized by an exacerbated endogenous Abeta accumulation. We observed that cerebral tPA activity was decreased during ageing in normal mice and that this effect was worsened in mice overproducing Abeta peptides. These phenomena result, respectively, from a decrease in tPA expression and from an increase in the production of one of the tPA inhibitors, the plasminogen activator inhibitor type 1 (PAI-1). A similar study in sporadic AD and age-matched control brain tissues revealed that the tPA proteolytic activity was negatively correlated to Abeta peptides levels supporting the data observed in mice. Altogether, our data support a model in which amyloid deposition induces a decrease in tPA activity through the overproduction of PAI-1 by activated glial cells.

Neurotrophin-3-induced PI-3 kinase/Akt signaling rescues cortical neurons from apoptosis.

A number of cytokines including neurotrophins have been tested for their neuroprotective activity against different paradigms of neuronal death. However, as for neurotrophin-3 (NT-3), their mechanisms of action have not been fully identified. By using cultures of mouse cortical neurons, we have investigated the molecular mechanisms by which neurotrophin-3 could protect cortical neurons against apoptosis. In a model of caspase-dependent apoptosis leading to the recruitment of active initiators caspase-8 and -9 and of executioner caspase-3, we have evidenced that NT-3 displayed an anti-apoptotic effect in a dose-dependent manner. First, we showed that, in cultured cortical neurons, NT-3 could promote extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol-3' (PI-3) kinase/Akt phosphorylation. Second, we showed that although the blockade of the Akt pathway prevented the anti-apoptotic effect of NT-3, blockade of the ERK pathway did not. Altogether, our data demonstrate that NT-3 displayed an anti-apoptotic effect on cultured cortical neurons through a mechanism involving the recruitment of the PI-3 kinase/Akt signaling pathway.

Is tissue-type plasminogen activator a neuromodulator?

In the last few years, it has been evidenced that serine proteases play key roles in the mammalian brain, both in physiological and pathological conditions. It has been well established that among these serine proteases, the tissue-type plasminogen activator (t-PA) is critically involved in development, plasticity, and pathology of the nervous system. However, its mechanism of action remains to be further investigated. By using pharmacological and immunological approaches, we have evidenced in the present work that t-PA should be considered as a neuromodulator. Indeed, we have observed that: (i). neuronal depolarization induces a release of t-PA; (ii). this release of t-PA is sensitive to exocytosis inhibition and calcium chelation; (iii). released t-PA modulates NMDA receptor signaling and (iv). astrocytes are able to recapture extracellular t-PA through a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent mechanism.

Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling.

Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-D-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59-64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function.