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.

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.

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.

Loss of ULK1 Attenuates Cholesterogenic Gene Expression in Mammalian Hepatic Cells.

The hepatic mevalonate (MVA) pathway, responsible for cholesterol biosynthesis, is a therapeutically important metabolic pathway in clinical medicine. Using an unbiased transcriptomics approach, we uncover a novel role of Unc-51 like autophagy activating kinase 1 (ULK1) in regulating the expression of the hepatic de novo cholesterol biosynthesis/MVA pathway genes. Genetic silencing of ULK1 in non-starved mouse (AML-12) and human (HepG2) hepatic cells as well as in mouse liver followed by transcriptome and pathway analysis revealed that the loss of ULK1 expression led to significant down-regulation of genes involved in the MVA/cholesterol biosynthesis pathway. At a mechanistic level, loss of ULK1 led to decreased expression of SREBF2/SREBP2 (sterol regulatory element binding factor 2) via its effects on AKT-FOXO3a signaling and repression of SREBF2 target genes in the MVA pathway. Our findings, therefore, discover ULK1 as a novel regulator of cholesterol biosynthesis and a possible druggable target for controlling cholesterol-associated pathologies.

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.

Both 3,5-Diiodo-L-Thyronine and 3,5,3'-Triiodo-L-Thyronine Prevent Short-term Hepatic Lipid Accumulation via Distinct Mechanisms in Rats Being Fed a High-Fat Diet.

3,3',5-triiodo-L-thyronine (T3) improves hepatic lipid accumulation by increasing lipid catabolism but it also increases lipogenesis, which at first glance appears contradictory. Recent studies have shown that 3,5-diiodothyronine (T2), a natural thyroid hormone derivative, also has the capacity to stimulate hepatic lipid catabolism, however, little is known about its possible effects on lipogenic gene expression. Because genes classically involved in hepatic lipogenesis such as SPOT14, acetyl-CoA-carboxylase (ACC), and fatty acid synthase (FAS) contain thyroid hormone response elements (TREs), we studied their transcriptional regulation, focusing on TRE-mediated effects of T3 compared to T2 in rats receiving high-fat diet (HFD) for 1 week. HFD rats showed a marked lipid accumulation in the liver, which was significantly reduced upon simultaneous administration of either T3 or T2 with the diet. When administered to HFD rats, T2, in contrast with T3, markedly downregulated the expression of the above-mentioned genes. T2 downregulated expression of the transcription factors carbohydrate-response element-binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) involved in activation of transcription of these genes, which explains the suppressed expression of their target genes involved in lipogenesis. T3, however, did not repress expression of the TRE-containing ChREBP gene but repressed SREBP-1c expression. Despite suppression of SREBP-1c expression by T3 (which can be explained by the presence of nTRE in its promoter), the target genes were not suppressed, but normalized to HFD reference levels or even upregulated (ACC), partly due to the presence of TREs on the promoters of these genes and partly to the lack of suppression of ChREBP. Thus, T2 and T3 probably act by different molecular mechanisms to achieve inhibition of hepatic lipid accumulation.

Metabolomic analysis shows differential hepatic effects of T2 and T3 in rats after short-term feeding with high fat diet.

Nonalcoholic fatty liver disease (NAFLD) is a major health problem worldwide, and is often associated with lipotoxic injury, defective mitochondrial function, and insulin resistance. Thyroid hormones (THs) are important regulators of hepatic lipid metabolism. Among the THs, diiodothyronine (T2) and triiodothyronine (T3) have shown promising results in lowering hepatic fat content in various models of NAFLD. In this study, we used a targeted metabolomics approach to investigate the differential effects of T2 and T3 on the early metabolic adaptation in the livers of rats fed high fat diet (HFD), a period when hepatosteatosis is reversible. Our results showed that both T2 and T3 strongly induced autophagy and intra-hepatic acylcarnitine flux but prevented the generation of sphingolipid/ceramides in animals fed HFD. Interestingly, although both T2 and T3 decreased hepatic fat content, only T2 was able to rescue the impairment in AKT and MAPK/ERK pathways caused by HFD. In summary, we have identified and characterized the effects of T2 and T3 on hepatic metabolism during short-term exposure to HFD. These findings illuminate the common and divergent metabolic pathways by T2 and T3 that also may be important in the prevention and treatment of NAFLD.

Short chain fatty acids induce UCP2-mediated autophagy in hepatic cells.

Short-chain fatty acids (SCFAs) are gut microbial fermentation products derived from dietary fiber sources. Although depletion of gut microflora has been linked to the development of liver disease, the direct effects of SCFAs on intracellular hepatic processes are not well understood. In this study, we demonstrated that the SCFAs, propionate and butyrate, regulated autophagic flux in hepatic cells in a cell-autonomous manner. Induction of autophagy by SCFAs required PPARγ stimulation of Uncoupling Protein 2 (UCP2) expression that was associated with reduced intracellular ATP levels and activation of PRKAA1/AMPK (protein kinase, AMP-activated, alpha 1 catalytic subunit). In addition, elimination of gut flora by chronic antibiotic treatment diminished basal hepatic autophagy in mice suggesting that gut microbiota can regulate hepatic autophagy. These findings provide novel insights into the interplay between diet, gut microbiota, short chain fatty acids, and hepatic autophagic signaling.