RESUMEN
Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.
Asunto(s)
Enfermedad de Alzheimer , Anciano , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Homeostasis , Humanos , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMEN
Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.
Asunto(s)
Calcio/metabolismo , Núcleo Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Fenómenos Biofísicos , Señalización del Calcio , Citosol/metabolismo , Células HeLa , Humanos , CinéticaRESUMEN
Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca2+ signaling comprises complex pathways that impact on virtually every aspect of cellular physiology. Within such a complex field of study, Drosophila offers the advantages of consolidated molecular and genetic techniques, lack of genetic redundancy and a completely annotated genome since 2000. These and other characteristics provided the basis for the identification of many genes encoding Ca2+ signaling molecules and the disclosure of conserved Ca2+ signaling pathways. In this review, we will analyze the applications of Ca2+ imaging in the fruit fly model, highlighting in particular their impact on the study of normal brain function and pathogenesis of neurodegenerative diseases.
Asunto(s)
Calcio , Drosophila melanogaster , Animales , Encéfalo/fisiología , Encéfalo/fisiopatología , Calcio/metabolismo , Drosophila melanogaster/fisiología , Humanos , Modelos AnimalesRESUMEN
Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔµH+) across the inner membrane. Although several drugs can modulate ΔµH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic ß-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.
Asunto(s)
Señalización del Calcio/fisiología , Channelrhodopsins/metabolismo , Células Secretoras de Insulina/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Optogenética , Animales , Células HEK293 , Células HeLa , Humanos , Células Secretoras de Insulina/citología , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5â µm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.
Asunto(s)
Calcio/metabolismo , Colorantes Fluorescentes/uso terapéutico , Mitocondrias/metabolismo , Colorantes Fluorescentes/metabolismo , HumanosRESUMEN
Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.
Asunto(s)
Drosophila melanogaster , Retículo Endoplásmico/fisiología , Proteínas de Unión al GTP/genética , Proteínas de la Membrana/genética , Neuronas Motoras/fisiología , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Paraplejía Espástica Hereditaria/genética , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Células Cultivadas , Humanos , Transducción de Señal , Sinapsis , Transmisión Sináptica/genéticaRESUMEN
Calcium ion (Ca(2+)) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca(2+) store and the free Ca(2+) concentration ([Ca(2+)]) within its lumen ([Ca(2+)]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca(2+) sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca(2+) probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca(2+), low dynamic range, and an affinity for the cation that is too high for the levels of [Ca(2+)] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca(2+) affinity and dynamic range for monitoring [Ca(2+)] variations within the ER. As an example, resting [Ca(2+)]ER have been evaluated in a known paradigm of altered ER Ca(2+) homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca(2+) affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca(2+) content in cells expressing mutated PS2, compared to controls.
RESUMEN
Establishment and maintenance of proper architecture is essential for endoplasmic reticulum (ER) function. Homotypic membrane fusion is required for ER biogenesis and maintenance, and has been shown to depend on GTP hydrolysis. Here we demonstrate that Drosophila Atlastin--the fly homologue of the mammalian GTPase atlastin 1 involved in hereditary spastic paraplegia--localizes on ER membranes and that its loss causes ER fragmentation. Drosophila Atlastin embedded in distinct membranes has the ability to form trans-oligomeric complexes and its overexpression induces enlargement of ER profiles, consistent with excessive fusion of ER membranes. In vitro experiments confirm that Atlastin autonomously drives membrane fusion in a GTP-dependent fashion. In contrast, GTPase-deficient Atlastin is inactive, unable to form trans-oligomeric complexes owing to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Atlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity that is required for ER homotypic fusion.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Dinaminas , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Fusión de Membrana , Animales , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retículo Endoplásmico/patología , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , Células HeLa , Humanos , Transporte de Proteínas , Proteolípidos/metabolismoRESUMEN
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.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , AMP Cíclico/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Humanos , Microscopía Fluorescente/métodosRESUMEN
The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Guanosina Trifosfato/fisiología , Proteínas de la Membrana/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Drosophila , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , HidrólisisRESUMEN
Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer's disease (AD) linking astrocyte Ca2+ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.
Asunto(s)
Enfermedad de Alzheimer , Ratones , Femenino , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Astrocitos/metabolismo , Estudios Longitudinales , Plasticidad Neuronal/fisiología , Trastornos de la Memoria/metabolismo , Señalización del Calcio/fisiología , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The endoplasmic reticulum (ER) is the most abundant and widespread organelle in cells. Its peculiar membrane architecture, formed by an intricate network of tubules and cisternae, is critical to its multifaceted function. Regulation of ER morphology is coordinated by a few ER-specific membrane proteins and is thought to be particularly important in neurons, where organized ER membranes are found even in the most distant neurite terminals. Mutation of ER-shaping proteins has been implicated in the neurodegenerative disease hereditary spastic paraplegia (HSP). In this review we discuss the involvement of these proteins in the pathogenesis of HSP, focusing on the experimental evidence linking their molecular function to disease onset. Although the precise biochemical activity of some ER-related HSP proteins has been elucidated, the pathological mechanism underlying ER-linked HSP is still undetermined and needs to be further investigated.
Asunto(s)
Retículo Endoplásmico/patología , Paraplejía Espástica Hereditaria/patología , Aparato de Golgi/metabolismo , Humanos , Microtúbulos/metabolismo , Biogénesis de Organelos , Proteínas/metabolismo , Paraplejía Espástica Hereditaria/genéticaRESUMEN
Ca2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca2+ concentration [Ca2+] in living cells. Synthetic fluorescent Ca2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca2+ indicator named mt-fura-2 , obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca2+ indicator fura-2 .The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca2+ probes . More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca2+ probes .
Asunto(s)
Quelantes del Calcio/química , Calcio/análisis , Fura-2/química , Mitocondrias/química , Ciclosporina , Citosol/química , Células HeLa , Humanos , Microscopía FluorescenteRESUMEN
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+]).
Asunto(s)
Mitocondrias , Orgánulos , Ratones , Animales , Ratones Transgénicos , Mitocondrias/genética , Proteínas Fluorescentes Verdes/genética , Orgánulos/metabolismo , Señalización del Calcio , Calcio de la Dieta/metabolismoRESUMEN
Calcium (Ca2+) signaling coordinates are crucial processes in brain physiology. Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca2+, and neuronal survival itself relies on Ca2+-dependent cascades. Indeed, impaired Ca2+ homeostasis has been reported in aging as well as in the onset and progression of neurodegeneration. Understanding the physiology of brain function and the key processes leading to its derangement is a core challenge for neuroscience. In this context, Ca2+ imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca2+ sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca2+ imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.
Asunto(s)
Calcio/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Animales , Señalización del Calcio/fisiología , Humanos , Neuronas/metabolismoRESUMEN
The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Sp astic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. Here we have examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. We found that all pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism.
RESUMEN
Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-ß (Aß) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).
Asunto(s)
Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Presenilina-1/genética , Presenilina-2/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Encéfalo/patología , Calcio/metabolismo , Señalización del Calcio/genética , Membrana Celular/genética , Flavina-Adenina Dinucleótido/genética , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Mutantes/genética , Presenilina-2/metabolismoRESUMEN
The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.
RESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Orgánulos/metabolismo , Presenilina-1/genética , Presenilina-2/genética , Calcio/análisis , Humanos , Mutación , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Células Tumorales CultivadasRESUMEN
The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. We show here that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. Our findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases. Abbreviations: ALP: autophagy-lysosome pathway; ARE: antioxidant response element; Atg8a: autophagy-related 8a; ATPsynß: ATP synthase, ß subunit; C-L: caspase-like proteasomal activity; cncC: cap-n-collar isoform-C; CT-L: chymotrypsin-like proteasomal activity; Drp1: dynamin related protein 1; ER: endoplasmic reticulum; foxo: forkhead box, sub-group O; GLU: glucose; GFP: green fluorescent protein; GLY: glycogen; Hsf: heat shock factor; Hsp: Heat shock protein; Keap1: kelch-like ECH-associated protein 1; Marf: mitochondrial assembly regulatory factor; NFE2L2/Nrf2: nuclear factor, erythroid 2 like 2; Opa1: optic atrophy 1; PN: proteostasis network; RNAi: RNA interference; ROS: reactive oxygen species; ref(2)P: refractory to sigma P; SQSTM1: sequestosome 1; SdhA: succinate dehydrogenase, subunit A; T-L: trypsin-like proteasomal activity; TREH: trehalose; UAS: upstream activation sequence; Ub: ubiquitin; UPR: unfolded protein response; UPP: ubiquitin-proteasome pathway.