RESUMO
For Alzheimer's disease (AD), aging is the main risk factor, but whether cognitive impairments due to aging resemble early AD deficits is not yet defined. When working with mouse models of AD, the situation is just as complicated, because only a few studies track the progression of the disease at different ages, and most ignore how the aging process affects control mice. In this work, we addressed this problem by comparing the aging process of PS2APP (AD) and wild-type (WT) mice at the level of spontaneous brain electrical activity under anesthesia. Using local field potential recordings, obtained with a linear probe that traverses the posterior parietal cortex and the entire hippocampus, we analyzed how multiple electrical parameters are modified by aging in AD and WT mice. With this approach, we highlighted AD specific features that appear in young AD mice prior to plaque deposition or that are delayed at 12 and 16 months of age. Furthermore, we identified aging characteristics present in WT mice but also occurring prematurely in young AD mice. In short, we found that reduction in the relative power of slow oscillations (SO) and Low/High power imbalance are linked to an AD phenotype at its onset. The loss of SO connectivity and cortico-hippocampal coupling between SO and higher frequencies as well as the increase in UP-state and burst durations are found in young AD and old WT mice. We show evidence that the aging process is accelerated by the mutant PS2 itself and discuss such changes in relation to amyloidosis and gliosis.
Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Potenciais de Ação/fisiologia , Doença de Alzheimer/complicações , Doença de Alzheimer/fisiopatologia , Amiloidose/complicações , Amiloidose/patologia , Amiloidose/fisiopatologia , Animais , Ritmo Delta/fisiologia , Progressão da Doença , Gliose/complicações , Gliose/patologia , Gliose/fisiopatologia , Hipocampo/patologia , Camundongos Endogâmicos C57BL , Rede Nervosa/fisiopatologia , Placa Amiloide/complicações , Placa Amiloide/patologia , Placa Amiloide/fisiopatologiaRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Animais , Autofagia , Camundongos , Fosforilação , TransfecçãoRESUMO
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder in which learning, memory and cognitive functions decline progressively. Familial forms of AD (FAD) are caused by mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes. Presenilin 1 (PS1) and its homologue, presenilin 2 (PS2), represent, alternatively, the catalytic core of the γ-secretase complex that, by cleaving APP, produces neurotoxic amyloid beta (Aß) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently, PSEN1 FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to PSEN2 FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca2+ signaling and mitochondrial function. By exploiting neurons derived from a PSEN2 knock-out (PS2-/-) mouse model, we found that, upon IP3-generating stimulation, cytosolic Ca2+ handling is not altered, compared to wild-type cells, while mitochondrial Ca2+ uptake is strongly compromised. Accordingly, PS2-/- neurons show a marked reduction in endoplasmic reticulum-mitochondria apposition and a slight alteration in mitochondrial respiration, whereas mitochondrial membrane potential, and organelle morphology and number appear unchanged. Thus, although some alterations in mitochondrial function appear to be shared between PS2-/- and FAD-PS2-expressing neurons, the mechanisms leading to these defects are quite distinct between the two models. Taken together, our data appear to be difficult to reconcile with the proposal that FAD-PS2 mutants are loss-of-function, whereas the concept that PS2 plays a key role in sustaining mitochondrial function is here confirmed.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sinalização do Cálcio , Mitocôndrias/metabolismo , Presenilina-2/deficiência , Trifosfato de Adenosina/biossíntese , Animais , Respiração Celular , Ciclo do Ácido Cítrico , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicólise , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fosforilação Oxidativa , Fenótipo , Presenilina-2/metabolismoRESUMO
Mitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).
Assuntos
Mitocôndrias , Organelas , Camundongos , Animais , Camundongos Transgênicos , Mitocôndrias/genética , Proteínas de Fluorescência Verde/genética , Organelas/metabolismo , Sinalização do Cálcio , Cálcio da Dieta/metabolismoRESUMO
Cytosolic cAMP signalling in live cells has been extensively investigated in the past, while only in the last decade the existence of an intramitochondrial autonomous cAMP homeostatic system began to emerge. Thanks to the development of novel tools to investigate cAMP dynamics and cAMP/PKA-dependent phosphorylation within the matrix and in other mitochondrial compartments, it is now possible to address directly and in intact living cells a series of questions that until now could be addressed only by indirect approaches, in isolated organelles or through subcellular fractionation studies. In this contribution we discuss the mechanisms that regulate cAMP dynamics at the surface and inside mitochondria, and its crosstalk with organelle Ca2+ handling. We then address a series of still unsolved questions, such as the intramitochondrial localization of key elements of the cAMP signaling toolkit, e.g., adenylate cyclases, phosphodiesterases, protein kinase A (PKA) and Epac. Finally, we discuss the evidence for and against the existence of an intramitochondrial PKA pool and the functional role of cAMP increases within the organelle matrix.
Assuntos
AMP Cíclico/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Animais , Sinalização do Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , FosforilaçãoRESUMO
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.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Linhagem Celular , AMP Cíclico/metabolismo , Espaço Extracelular/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Miócitos Cardíacos/citologiaRESUMO
Herein, we report on the synthesis of a small set of linear precursors of an inosine analogue of cyclic ADP-ribose (cADPR), a second messenger involved in Ca2+ mobilization from ryanodine receptor stores firstly isolated from sea urchin eggs extracts. The synthesized compounds were obtained starting from inosine and are characterized by an N1-alkyl chain replacing the "northern" ribose and a phosphate group attached at the end of the N1-alkyl chain and/or 5'-sugar positions. Preliminary Ca2+ mobilization assays, performed on differentiated C2C12 cells, are reported as well.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Ouriços-do-Mar/química , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ovos , Relação Estrutura-AtividadeRESUMO
Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described.
RESUMO
An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Organelas/metabolismo , Presenilina-1/genética , Presenilina-2/genética , Cálcio/análise , Humanos , Mutação , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Microdomínios da Membrana/enzimologia , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microdomínios da Membrana/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Ratos , Ratos Sprague-DawleyRESUMO
A typical characteristic of eukaryotic cells compared to prokaryotes is represented by the spatial heterogeneity of the different structural and functional components: for example, most of the genetic material is surrounded by a highly specific membrane structure (the nuclear membrane), continuous with, yet largely different from, the endoplasmic reticulum (ER); oxidative phosphorylation is carried out by organelles enclosed by a double membrane, the mitochondria; in addition, distinct domains, enriched in specific proteins, are present in the plasma membrane (PM) of most cells. Less obvious, but now generally accepted, is the notion that even the concentration of small molecules such as second messengers (Ca2+ and cAMP in particular) can be highly heterogeneous within cells. In the case of most organelles, the differences in the luminal levels of second messengers depend either on the existence on their membrane of proteins that allow the accumulation/release of the second messenger (e.g., in the case of Ca2+, pumps, exchangers or channels), or on the synthesis and degradation of the specific molecule within the lumen (the autonomous intramitochondrial cAMP system). It needs stressing that the existence of a surrounding membrane does not necessarily imply the existence of a gradient between the cytosol and the organelle lumen. For example, the nuclear membrane is highly permeable to both Ca2+ and cAMP (nuclear pores are permeable to solutes up to 50 kDa) and differences in [Ca2+] or [cAMP] between cytoplasm and nucleoplasm are not seen in steady state and only very transiently during cell activation. A similar situation has been observed, as far as Ca2+ is concerned, in peroxisomes.
Assuntos
Sinalização do Cálcio/fisiologia , AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Membrana Nuclear/metabolismo , Animais , AMP Cíclico/genética , Retículo Endoplasmático/genética , Humanos , Membrana Nuclear/genéticaRESUMO
Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 µmâ s-1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Modelos Biológicos , Animais , Animais Recém-Nascidos , CamundongosRESUMO
The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic ß2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.
Assuntos
Saúde , Homeostase , Doenças do Sistema Nervoso/patologia , Junção Neuromuscular/patologia , Sistema Nervoso Simpático/patologia , Transporte Ativo do Núcleo Celular , Animais , Técnicas Biossensoriais , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Músculo Esquelético/inervação , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fenótipo , Transdução de Sinais , Simpatectomia , Sistema Nervoso Simpático/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277-302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272-481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions.
Assuntos
Diferenciação Celular , Metabolismo Energético , Fatores de Transcrição Forkhead/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Linhagem Celular , Fatores de Transcrição Forkhead/genética , Proteínas de Fluorescência Verde/genética , Potencial da Membrana Mitocondrial , Camundongos , Proteínas do Tecido Nervoso/genéticaRESUMO
Glomerulosa cells secrete aldosterone in response to agonists coupled to Ca(2+) increases such as angiotensin II and corticotrophin, coupled to a cAMP dependent pathway. A recently recognized interaction between Ca(2+) and cAMP is the Ca(2+)-induced cAMP formation in the mitochondrial matrix. Here we describe that soluble adenylyl cyclase (sAC) is expressed in H295R adrenocortical cells. Mitochondrial cAMP formation, monitored with a mitochondria-targeted fluorescent sensor (4mtH30), is enhanced by HCO3(-) and the Ca(2+) mobilizing agonist angiotensin II. The effect of angiotensin II is inhibited by 2-OHE, an inhibitor of sAC, and by RNA interference of sAC, but enhanced by an inhibitor of phosphodiesterase PDE2A. Heterologous expression of the Ca(2+) binding protein S100G within the mitochondrial matrix attenuates angiotensin II-induced mitochondrial cAMP formation. Inhibition and knockdown of sAC significantly reduce angiotensin II-induced aldosterone production. These data provide the first evidence for a cell-specific functional role of mitochondrial cAMP.
Assuntos
Aldosterona/metabolismo , AMP Cíclico/biossíntese , Mitocôndrias/metabolismo , Adenilil Ciclases/metabolismo , Aldosterona/biossíntese , Angiotensina II/fisiologia , Sinalização do Cálcio , Linhagem Celular , HumanosRESUMO
While mitochondrial Ca(2+) homeostasis has been studied for several decades and many of the functional roles of Ca(2+) accumulation within the matrix have been at least partially clarified, the possibility of modulation of the organelle functions by cAMP remains largely unknown. In this contribution we briefly summarize the key aspects of Ca(2+) and cAMP signalling pathways in mitochondria. In particular, we focus on recent findings concerning the discovery of an autonomous cAMP toolkit within the mitochondrial matrix, its crossroad with mitochondrial Ca(2+) signalling and its role in controlling ATP synthesis. The discovery of a cAMP signalling, and the demonstration of a cross-talk between cAMP and Ca(2+) inside mitochondria, open the way to a re-evaluation of these organelles as integrators of multiple second messengers. A description of the main methods presently available to measure Ca(2+) and cAMP in mitochondria of living cells with genetically encoded probes is also presented.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , AMP Cíclico/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Humanos , Microscopia de Fluorescência/métodosRESUMO
The Golgi apparatus (GA) is a dynamic intracellular Ca(2+) store endowed with complex Ca(2+) homeostatic mechanisms in part distinct from those of the endoplasmic reticulum (ER). We describe the generation of a novel fluorescent Ca(2+) probe selectively targeted to the medial-Golgi. We demonstrate that in the medial-Golgi: (i) Ca(2+) accumulation takes advantage of two distinct pumps, the sarco/endoplasmic reticulum Ca(2+) ATPase and the secretory pathway Ca(2+) ATPase1; (ii) activation of IP3 or ryanodine receptors causes Ca(2+) release, while no functional two-pore channel was found; (iii) luminal Ca(2+) concentration appears higher than that of the trans-Golgi, but lower than that of the ER, suggesting the existence of a cis- to trans-Golgi Ca(2+) concentration gradient. Thus, the GA represents a Ca(2+) store of high complexity where, despite the continuous flow of membranes and luminal contents, each sub-compartment maintains its Ca(2+) identity with specific Ca(2+) homeostatic characteristics. The functional role of such micro-heterogeneity in GA Ca(2+) handling is discussed.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Complexo de Golgi/metabolismo , Animais , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/fisiologia , Linhagem Celular , Cricetinae , Retículo Endoplasmático/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Homeostase , HumanosRESUMO
While the role of mitochondrial Ca²âº homeostasis in cell pathophysiology is widely accepted, the possibility that cAMP regulates mitochondrial functions has only recently received experimental support. The site of cAMP production, its targets, and its functions in the organelles remain uncertain. Using a variety of genetic/pharmacological tools, we here demonstrate that the mitochondrial inner membrane is impermeable to cytosolic cAMP, while an autonomous cAMP signaling toolkit is expressed in the matrix. We demonstrate that rises in matrix Ca²âº powerfully stimulate cAMP increases within mitochondria and that matrix cAMP levels regulate their ATP synthesizing efficiency. In cardiomyocyte cultures, mitochondrial cAMP can be increased by treatments that augment the frequency and amplitude of Ca²âº oscillations within the cytosol and organelles, revealing that mitochondria can integrate an oscillatory Ca²âº signal to increase cAMP in their matrix. The present data reveal the existence, within mitochondria, of a hitherto unknown crosstalk between Ca²âº and cAMP.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , AMP Cíclico/biossíntese , Homeostase , Humanos , Miócitos Cardíacos/metabolismoRESUMO
Mitochondrial ability of shaping Ca(2+) signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca(2+) uniporter (MCU) and of unique targeted Ca(2+) probes to directly address this issue. We demonstrate that, during spontaneous Ca(2+) pacing, Ca(2+) peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca(2+) hot spots generated on the OMM surface. Cytoplasmic Ca(2+) peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca(2+) rises.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Citoplasma/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Membranas Mitocondriais/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Ratos WistarRESUMO
UNLABELLED: Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2-defective mice, cyclic 3',5'-adenosine monophosphate/ protein kinase A (cAMP/PKA)-dependent activation of extracellular signal-regulated kinase/ mammalian target of rapamycin (ERK-mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca(2+) and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO (knockout) mice (Pkd2(flox/-) :pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild-type mice (WT). Our results showed that, compared to WT cells, in PC2-defective cholangiocytes (Pkd2KO), cytoplasmic and ER-Ca(2+) (measured with Fura-2 and Mag-Fluo4) levels are decreased and store-operated Ca(2+) entry (SOCE) is inhibited, whereas the expression of Ca(2+) -sensor stromal interaction molecule 1 (STIM1) and store-operated Ca(2+) channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca(2+) depletion increases cAMP and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6). CONCLUSION: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca(2+) depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store-operated cAMP production.