Summary Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca2+] which control aerobic metabolism, cell death and survival pathways. We have investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function and trophism, by overexpressing or silencing the Mitochondrial Calcium Uniporter (MCU). The results demonstrate that both in developing and in adult muscles MCU-dependent mitochondrial Ca2+ uptake has a marked trophic effect that does not depend on aerobic control, but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-AKT/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca2+-dependent organelle-to-nucleus signaling route, which links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss.

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

The type of neuronal activity required for circuit development is a matter of significant debate. We addressed this issue by analyzing the topographic organization of the olfactory bulb in transgenic mice engineered to have very little afferent spontaneous activity due to the overexpression of the inwardly rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). In these conditions, the topography of the olfactory bulb was unrefined. Odor-evoked responses were readily recorded in glomeruli with reduced spontaneous afferent activity, although the functional maps were coarser than in controls and contributed to altered olfactory discrimination behavior. In addition, overexpression of Kir2.1 in adults induced a regression of the already refined connectivity to an immature (i.e., coarser) status. Our data suggest that spontaneous activity plays a critical role not only in the development but also in the maintenance of the topography of the olfactory bulb and in sensory information processing.

BackgroundThe mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.ResultsBy taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.ConclusionsOur study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.

A distinctive feature in the topographic organization of the olfactory system in mammals is the dual function of the odorant receptor (OR): it detects odors in the nasal epithelium and plays an instructive role in the axonal convergence of olfactory sensory neurons (OSN) into the olfactory bulb (OB). The latter function is supported by genetic experiments and by the expression of the OR not only on the cilia, but also on the axon termini of the OSN. The signaling pathway coupled to the OR on the cilia is well known and is recognized to involve cAMP and Ca 2؉ , whereas, until now, nothing was known on the functional characteristics of the OR on the axon termini-growth cone. Here, by analyzing the spatiotemporal dynamics of cAMP and Ca 2؉ in living OSN in vitro and in situ, we found that the OR at the growth cone is capable of binding odors and is coupled to cAMP synthesis and Ca 2؉ influx through cyclic nucleotide gated (CNG) channels. Furthermore we found that selective odor activation of the OR on the growth cone is followed by nuclear translocation of protein kinase A catalytic subunit. These results define the functional properties of the OR on the growth cone and suggest a potential role of OR activation in axonal convergence and sensory map formation.olfactory sensory neurons ͉ real-time imaging ͉ second messengers I n sensory systems, neurons in the peripheral sheet send their axons in precise loci of the CNS to create an internal representation of the external world. The spatial segregation of afferent inputs from primary sensory neurons provides a topographic map that defines the quality and the location of the stimulus. In the olfactory system, each OSN expresses only one OR gene out of a repertoire of approximately 1000 (1). OSNs expressing different ORs are randomly dispersed in the nasal epithelium. However, spatial order is achieved in the olfactory bulb (OB), where OSNs expressing the same odorant receptor converge with exquisite precision to form glomeruli both on the lateral and the medial side of each OB. Each odor is thus encoded by a specific spatial pattern of activated glomeruli. The OR, a G-protein-coupled receptor, upon binding of odorant ligands at the cilia, activates a specific G protein, Golf, that stimulates adenylyl cyclase, AC, to synthesize cAMP. The cAMP then directly activates cyclic nucleotide gated (CNG) channels, leading to action potential generation (2, 3).A unique feature in the topographic organization of the OB is the ''dual role'' of the OR. Although it is well established that the OR is involved in the transduction of chemical signals (odors) at the cilia level, a number of evidence suggests that this receptor plays also an instructive role in glomerular convergence of OSN axons to the bulb (4-6). The latter property is supported by genetic observations demonstrating that alterations of OR sequence perturb the sensory map (7-9) and by the demonstration that the OR is expressed not only at the cilia but also on the OSN axon termini (10, 11). The OR is not the only determ...

It is becoming increasingly evident that the freely diffusible second messenger cAMP can transduce specific responses by localized signalling. The machinery that underpins compartmentalized cAMP signalling is only now becoming appreciated. Adenylate cyclases, the enzymes that synthesize cAMP, are localized at discrete parts of the plasma membrane, and phosphodiesterases, the enzymes that degrade cAMP, can be targeted to selected subcellular compartments. A-kinase-anchoring proteins then serve to anchor PKA (protein kinase A) close to specific targets, resulting in selective activation. The specific activation of such individual subsets of PKA requires that cAMP is made available in discrete compartments. In this presentation, the molecular and structural mechanisms responsible for compartmentalized PKA signalling and restricted diffusion of cAMP will be discussed.

ThemechanismofcGMPproductioninolfactorysensoryneurons(OSNs)ispoorlyunderstood,althoughthismessengertakespartinseveralkey processes such as adaptation, neuronal development, and long-term cellular responses to odorant stimulation. Many aspects of the regulation of cGMP in OSNs are still unknown or highly controversial, such as its subcellular heterogeneity, mechanism of coupling to odorant receptors and downstream targets. Here, we have investigated the dynamics and the intracellular distribution of cGMP in living rat OSNs in culture transfected with a genetically encoded sensor for cGMP. We demonstrate that OSNs treated with pharmacological stimuli able to activate membrane or soluble guanylyl cyclase (sGC) presented an increase in cGMP in the entire neuron, from cilia-dendrite to the axon terminus-growth cone. Upon odorant stimulation, a rise in cGMP was again found in the entire neuron, including the axon terminus, where it is locally synthesized. The odorant-dependent rise in cGMP is due to sGC activation by nitric oxide (NO) and requires an increase of cAMP. The link between cAMP and NO synthase appears to be the rise in cytosolic Ca 2ϩ concentration elicited by either plasma membrane Ca 2ϩ channel activation or Ca 2ϩ mobilization from stores via the guanine nucleotide exchange factor Epac. Finally, we show that a cGMP rise can elicit both in vitro and in vivo the phosphorylation of nuclear CREB, suggesting that this signaling pathway may be relevant for both local events (pathfinding, neurotransmitter release) and more distal processes involving gene expression regulation.

Highlights d Axonal odorant receptors respond to cues elaborated in the olfactory bulb d PEBP1, expressed in the olfactory bulb, is a putative ligand of axonal receptors d Genetic ablation of PEBP1 results in disrupted olfactory map in vivo d Axonal odorant receptors modulate axon targeting in the sensory map formation