Mitochondrial communication in the context of aging.

Mitochondria constantly contribute to the cell homeostasis and this, during the lifespan of a cell, takes its toll. Indeed, the functional decline of mitochondria appears correlated to the aging of the cell. The initial idea was that excessive production of reactive oxygen species (ROS) by functionally compromised mitochondria was the causal link between the decline of the organelle functions and cellular aging. However, in recent years accumulating evidence suggests that the contribution of mitochondria to cellular aging goes beyond ROS production. In this short review, we discuss how intracellular signalling, specifically the cAMP-signalling cascade, is involved in the regulation of mitochondrial functions and potentially in the processes that link mitochondrial status to cellular aging.

Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane.

Evidence supporting the heterogeneity in cAMP and PKA signaling is rapidly accumulating and has been largely attributed to the localization or activity of adenylate cyclases, phosphodiesterases, and A-kinase-anchoring proteins in different cellular subcompartments. However, little attention has been paid to the possibility that, despite homogeneous cAMP levels, a major heterogeneity in cAMP/PKA signaling could be generated by the spatial distribution of the final terminators of this cascade, i.e., the phosphatases. Using FRET-based sensors to monitor cAMP and PKA-dependent phosphorylation in the cytosol and outer mitochondrial membrane (OMM) of primary rat cardiomyocytes, we demonstrate that comparable cAMP increases in these two compartments evoke higher levels of PKA-dependent phosphorylation in the OMM. This difference is most evident for small, physiological increases of cAMP levels and with both OMM-located probes and endogenous OMM proteins. We demonstrate that this disparity depends on differences in the rates of phosphatase-dependent dephosphorylation of PKA targets in the two compartments. Furthermore, we show that the activity of soluble phosphatases attenuates PKA-driven activation of the cAMP response element-binding protein while concurrently enhancing PKA-dependent mitochondrial elongation. We conclude that phosphatases can sculpt functionally distinct cAMP/PKA domains even in the absence of gradients or microdomains of this messenger. We present a model that accounts for these unexpected results in which the degree of PKA-dependent phosphorylation is dictated by both the subcellular distribution of the phosphatases and the different accessibility of membrane-bound and soluble phosphorylated substrates to the cytosolic enzymes.

Cyclic AMP induces reversible EPAC1 condensates that regulate histone transcription.

The second messenger cyclic AMP regulates many nuclear processes including transcription, pre-mRNA splicing and mitosis. While most functions are attributed to protein kinase A, accumulating evidence suggests that not all nuclear cyclic AMP-dependent effects are mediated by this kinase, implying that other effectors may be involved. Here we explore the nuclear roles of Exchange Protein Activated by cyclic AMP 1. We find that it enters the nucleus where forms reversible biomolecular condensates in response to cyclic AMP. This phenomenon depends on intrinsically disordered regions present at its amino-terminus and is independent of protein kinase A. Finally, we demonstrate that nuclear Exchange Protein Activated by cyclic AMP 1 condensates assemble at genomic loci on chromosome 6 in the proximity of Histone Locus Bodies and promote the transcription of a histone gene cluster. Collectively, our data reveal an unexpected mechanism through which cyclic AMP contributes to nuclear spatial compartmentalization and promotes the transcription of specific genes.

Ca2+ oscillation frequency decoding in cardiac cell hypertrophy: role of calcineurin/NFAT as Ca2+ signal integrators.

The role of Ca(2+) signaling in triggering hypertrophy was investigated in neonatal rat cardiomyocytes in vitro. We show that an increase in cell size and sarcomere reorganization were elicited by receptor agonists such as Angiotensin II, aldosterone, and norepinephrine and by a small rise in medium KCl concentration, a treatment devoid of direct effects on receptor functions. All these treatments increased the frequency of spontaneous [Ca(2+)] transients, caused nuclear translocation of transfected NFAT(GFP), and increased the expression of a NFAT-sensitive reporter gene. There was no increase in Ca(2+) spark frequency in the whole cell or in the perinuclear region under these conditions. Hypertrophy and NFAT translocation but not the increased frequency of [Ca(2+)] transients were inhibited by the calcineurin inhibitor cyclosporine A. Hypertrophy by the different stimuli was insensitive to inhibition of myofilament contraction. We concluded that calcineurin-NFAT can act as integrators of the contractile Ca(2+) signal, and that they can decode alterations in the frequency even of rapid Ca(2+) oscillations.

PKA compartmentalization links cAMP signaling and autophagy.

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.

Compartmentalized Signaling in Aging and Neurodegeneration.

The cyclic AMP (cAMP) signalling cascade is necessary for cell homeostasis and plays important roles in many processes. This is particularly relevant during ageing and age-related diseases, where drastic changes, generally decreases, in cAMP levels have been associated with the progressive decline in overall cell function and, eventually, the loss of cellular integrity. The functional relevance of reduced cAMP is clearly supported by the finding that increases in cAMP levels can reverse some of the effects of ageing. Nevertheless, despite these observations, the molecular mechanisms underlying the dysregulation of cAMP signalling in ageing are not well understood. Compartmentalization is widely accepted as the modality through which cAMP achieves its functional specificity; therefore, it is important to understand whether and how this mechanism is affected during ageing and to define which is its contribution to this process. Several animal models demonstrate the importance of specific cAMP signalling components in ageing, however, how age-related changes in each of these elements affect the compartmentalization of the cAMP pathway is largely unknown. In this review, we explore the connection of single components of the cAMP signalling cascade to ageing and age-related diseases whilst elaborating the literature in the context of cAMP signalling compartmentalization.

Cholesterol Activates Cyclic AMP Signaling in Metaplastic Acinar Cells.

Cholesterol is a non-essential metabolite that exerts both structural and signaling functions. However, cholesterol biosynthesis is elevated, and actively supports, pancreatic carcinogenesis. Our previous work showed that statins block the reprogramming of mutant KRAS-expressing acinar cells, that spontaneously undergo a metaplastic event termed acinar-to-ductal metaplasia (ADM) to initiate carcinogenesis. Here we tested the impact of cholesterol supplementation on isolated primary wild-type acinar cells and observed enhanced ductal transdifferentiation, associated with generation of the second messenger cyclic adenosine monophosphate (cAMP) and the induction of downstream protein kinase A (PKA). Inhibition of PKA suppresses cholesterol-induced ADM ex vivo. Live imaging using fluorescent biosensors dissected the temporal and spatial dynamics of PKA activation upon cholesterol addition and showed uneven activation both in the cytosol and on the outer mitochondrial membrane of primary pancreatic acinar cells. The ability of cholesterol to activate cAMP signaling is lost in tumor cells. Qualitative examination of multiple normal and transformed cell lines supports the notion that the cAMP/PKA axis plays different roles during multi-step pancreatic carcinogenesis. Collectively, our findings describe the impact of cholesterol availability on the cyclic AMP/PKA axis and plasticity of pancreatic acinar cells.

A Novel Loss of Function Melanocortin-4-Receptor Mutation (MC4R-F313Sfs*29) in Morbid Obesity.

CONTEXT: Melanocortin receptor-4 (MC4R) gene mutations are associated with early-onset severe obesity, and the identification of potential pathological variants is crucial for the clinical management of patients with obesity. OBJECTIVE: To explore whether and how a novel heterozygous MC4R variant (MC4R-F313Sfs*29), identified in a young boy (body mass index [BMI] 38.8 kg/m2) during a mutation analysis conducted in a cohort of patients with obesity, plays a determinant pathophysiological role in the obesity development. DESIGN SETTING AND PATIENTS: The genetic screening was carried out in a total of 209 unrelated patients with obesity (BMI ≥ 35 kg/m2). Structural and functional characterization of the F313Sfs*29-mutated MC4R was performed using computational approaches and in vitro, using HEK293 cells transfected with genetically encoded biosensors for cAMP and Ca2+. RESULTS: The F313Sfs*29 was the only variant identified. In vitro experiments showed that HEK293 cells transfected with the mutated form of MC4R did not increase intracellular cAMP or Ca2+ levels after stimulation with a specific agonist in comparison with HEK293 cells transfected with the wild type form of MC4R (∆R/R0 = -90% ± 8%; P < 0.001). In silico modeling showed that the F313Sfs*29 mutation causes a major reorganization in the cytosolic domain of MC4R, thus reducing the affinity of the putative GalphaS binding site. CONCLUSIONS: The newly discovered F313Sfs*29 variant of MC4R may be involved in the impairment of α-MSH-induced cAMP and Ca2+ signaling, blunting intracellular G protein-mediated signal transduction. This alteration might have led to the dysregulation of satiety signaling, resulting in hyperphagia and early onset of obesity.

Studying ß1 and ß2 adrenergic receptor signals in cardiac cells using FRET-based sensors.

Cyclic 3'-5' adenosine monophosphate (cAMP) is a key modulator of cardiac function. Thanks to the sophisticated organization of its pathway in distinct functional units called microdomains, cAMP is involved in the regulation of both inotropy and chronotropy as well as transcription and cardiac death. While visualization of cAMP microdomains can be achieved thanks to cAMP-sensitive FRET-based sensors, the molecular mechanisms through which cAMP-generating stimuli are coupled to distinct functional outcomes are not well understood. One possibility is that each stimulus activates multiple microdomains in order to generate a spatiotemporal code that translates into function. To test this hypothesis here we propose a series of experimental protocols that allow to simultaneously follow cAMP or Protein Kinase A (PKA)-dependent phosphorylation in different subcellular compartments of living cells. We investigate the responses of ß Adrenergic receptors (ß1AR and ß2AR) challenged with selective drugs that enabled us to measure the actions of each receptor independently. At the whole cell level, we used a combination of co-culture with selective ßAR stimulation and were able to molecularly separate cardiac fibroblasts from neonatal rat ventricular myocytes based on their cAMP responses. On the other hand, at the subcellular level, these experimental protocols allowed us to dissect the relative weight of ß1 and ß2 adrenergic receptors on cAMP signalling at the cytosol and outer mitochondrial membrane of NRVMs. We propose that experimental procedures that allow the collection of multiparametric data are necessary in order to understand the molecular mechanisms underlying the coupling between extracellular signals and cellular responses.