mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo

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 F\uf6rster 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

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Exploring the role of astrocytic Ca2+ signaling in Alzheimer's Disease

Alzheimer's disease (AD) is a chronic incurable neurodegenerative disease, characterized by severe and progressive memory loss and cognitive dysfunctions. The molecular and cellular mechanisms of AD pathogenesis and the early events that anticipate the cognitive decline remain poorly understood. Over the last decades research on neurodegenerative diseases has always been conducted by starting from the aetiology of the disease and proceeding then by looking at the effect of the different hallmarks mainly on neuronal physiology. Noteworthy, a fundamental albeit neglected aspect is the accumulating evidence of the existence in the brain of dynamic interactions between neurons and astrocytes. Astrocytes are the most abundant type of glial cells in the central nervous system, they are electrically unexcitable but express a form of excitability based on variations of the intracellular concentration of Ca2+ ions. It is now largely recognized that astrocytes play crucial roles in brain function, from the control of tissue homeostasis to the modulation of both neurovascular coupling and synaptic transmission. Considering the variety of functions exerted by astrocytes in the brain, it is reasonable to hypothesize an involvement of this cell type in the pathogenesis of brain disorders, including AD. The main goal of my PhD project is to shed light onto the relationship between astrocytic Ca2+ signaling and AD. We focused on astrocytic Ca2+ dysfunctions in mouse models of AD along the progression of the disease, by clarifying whether astrocytic Ca2+ signal dysfunctions precede or follow Abeta plaque deposition. We evaluated the nature of these alterations in terms of signaling pathways, astrocytic compartments, i.e. soma, proximal processes and microdomains, and brain areas involved. To this aim, we employed three FAD mouse models, PS2.30H and APPSwe, which express the human PS2-N141I mutation and the human APP Swedish mutation alone respectively, and the PS2APP (B6.152H) model that expresses both mutants. All the experiments were carried out in mice at 3 and 6 months of age, before and after, respectively, the onset of plaque deposition in the PS2APP model. To investigate astrocytic activity, I carried out Ca2+ imaging experiments in brain slice and in vivo preparations from somato-sensory cortex (SSCx) and hippocampal brain slices by using the genetically encoded Ca2+ indicator (GECI) GCaMP6f which allows to study Ca2+ signals with high spatial resolution at different astrocytic compartments, including Ca2+ microdomains at the thin processes in close contact with the synapses. We found that along with the progression of AD, astrocyte Ca2+ activity in the SSCx exhibits a sequence of changes: in 3-month-old PS2APP mice, spontaneous activity is significantly increased, while in 6-month-old PS2APP mice, i.e. after the appearance of amyloid plaques, both spontaneous activity and the responsiveness to different metabotropic agonists are drastically reduced in all astrocytic territories. The decrease in evoked responses is likely due to a general IP3-dependent mechanism. Moreover, we verified that these alterations are not present in APPSwe and PS2.30H mice, demonstrating that the expression of APP or PS2 mutant alone is not sufficient to fully recapitulate the astrocytic Ca2+ defects observed in PS2APP mice. Although these defects start in concomitance with Aβ plaque deposition, astrocytic hypoactivity is unrelated to plaque proximity. We are currently investigating whether these alterations are maintained in the real in vivo context. Preliminary experiments reveal that, as in brain slices, spontaneous activity is drastically reduced in the SSCx of anesthetized mice. In conclusion, astrocytic Ca2+ activity is strongly affected in AD. Additional experiments are currently under development, to assess the relevance of these astrocytic Ca2+ defects in the disturbances of synaptic plasticity and learning/memory functions characterizing AD.Il morbo di Alzheimer è una malattia neurodegenerativa, ancora oggi incurabile, che rappresenta circa il 60% dei casi di demenza. I pazienti AD mostrano un lento e progressivo declino delle funzioni cognitive. I meccanismi molecolari e cellulari alla base del morbo di Alzheimer sono ancora largamente ignoti. Negli ultimi anni la ricerca scientifica si è focalizzata sullo studio degli effetti dei principali marcatori della malattia, i.e. placche amiloidi e grovigli neurofibrillari, sulla fisiologia neuronale. Un aspetto cruciale, sebbene a lungo trascurato,è l'esistenza di complesse interazioni fra i neuroni e gli astrociti, le cellule gliali più abbondanti del nostro cervello. Al contrario dei neuroni, gli astrociti sono cellule elettricamente non eccitabili, che tuttavia posseggono una forma diversa di eccitabilità  basata su variazioni della concentrazione intracellulare dello ione Ca2+. Gli astrociti svolgono una grande varietà  di funzioni che vanno dal controllo dell'omeostasi cerebrale alla modulazione del coupling neurovascolare e della trasmissione sinaptica. L' obiettivo della mia tesi di dottorato è quello di esplorare il possibile ruolo di queste cellule nel morbo di Alzheimer. In particolare, la nostra attenzione si è focalizzata sullo studio di possibili disfunzioni del segnale Ca2+ astrocitario in modelli murini del morbo di Alzheimer, studiando i cambiamenti dell' attività  Ca2+ negli astrociti durante la progressione della malattia, verificando quindi se le disfunzioni astrocitarie siano precedenti o successive alla deposizione delle placche amiloidi. Abbiamo caratterizzato queste disfunzioni in termini di vie di segnale, compartimenti astrocitari, i.e. soma, processi prossimali e microdomini, e aree cerebrali coinvolte. Nello specifico, abbiamo usato tre modelli murini che presentano mutazioni correlate con le forme familiari di morbo di Alzheimer. I modelli PS2.30H e APPSwe esprimono rispettivamente una forma umana mutata di PS2 (PS2-N141I) e di APP (APPSwe). Il terzo modello, PS2APP (B6.152H), esprime entrambe le proteine mutate. Gli esperimenti sono stati condotti a 3 e 6 mesi, rispettivamente prima e dopo la comparsa delle placche amiloidi nel modello PS2APP. Per studiare l'attività astrocitaria abbiamo condotto esperimenti di Ca2+ imaging in preparazioni ex vivo ed in vivo di corteccia somato-sensoriale e in fettine ippocampali. Per studiare il segnale Ca2+ astrocitario a livello del soma e dei processi, inclusi quelli più a contatto con le sinapsi, abbiamo usato un indicatore genetico per il Ca2+, il GCaMP6f. Dai nostri risultati si evince che durante la progressione della patologia nei topi PS2APP l' attività  degli astrociti subisce diversi cambiamenti: a 3 mesi l'attività  spontanea è significativamente aumentata, mentre a 6 mesi, dopo la comparsa delle placche amiloidi, sia l'attività spontanea che quella evocata dall' attivazione di diversi recettori metabotropici sono drasticamente ridotte in tutti i compartimenti astrocitari. La riduzione della risposta evocata sembra essere dovuta ad un meccanismo generale dipendente dal segnale IP3. Queste alterazioni non sono presenti negli altri due modelli murini di Alzheimer usati, dimostrando che l'espressione dell'APP o della PS2 mutata da sola non è sufficiente a ricapitolare i difetti astrocitari del segnale Ca2+ presenti nel modello PS2APP. Sebbene nel modello PS2APP le alterazioni si presentino in concomitanza con le placche amiloidi, l'poattività  astrocitaria non è influenzata dalla vicinanza delle placche. Inoltre, esperimenti preliminari in vivo confermano che nei topi PS2APP l'attività astrocitaria a 6 mesi è drasticamente ridotta. In conclusione, nel modello PS2APP l'attività Ca2+ degli astrociti è severamente alterata. Ulteriori esperimenti, anche di tipo comportamentale, atti ad investigare le conseguenze di queste alterazioni del segnale Ca2+ astrocitario sono attualmente in sviluppo

Two decades of astrocytes in neurovascular coupling

: The brain is a highly energy demanding organ, which accounts in humans for the 20% of total energy consumption at resting state although comprising only 2% of the body mass. The necessary delivery of nutrients to brain parenchyma is ensured by the cerebral circulatory system, through the exchange of glucose and oxygen (O2) at the capillary level. Notably, a tight spatial and temporal correlation exists between local increases in neuronal activity and the subsequent changes in regional cerebral blood flow. The recognized concept of neurovascular coupling (NVC), also named functional hyperemia, expresses this close relationship and stands at the basis of the modern functional brain imaging techniques. Different cellular and molecular mechanisms have been proposed to mediate this tight coupling. In this context, astrocytes are ideally positioned to act as relay elements that sense neuronal activity through their perisynaptic processes and release vasodilator agents at their endfeet in contact with brain parenchymal vessels. Two decades after the astrocyte involvement in neurovascular coupling has been proposed, we here review the experimental evidence that contributed to unraveling the molecular and cellular mechanisms underlying cerebral blood flow regulation. While traveling through the different controversies that moved the research in this field, we keep a peculiar focus on those exploring the role of astrocytes in neurovascular coupling and conclude with two sections related to methodological aspects in neurovascular research and to some pathological conditions resulting in altered neurovascular coupling

Dynamic interactions between GABAergic and astrocytic networks

Brain network activity derives from the concerted action of different cell populations. Together with interneurons, astrocytes play fundamental roles in shaping the inhibition in brain circuitries and modulating neuronal transmission. In this review, we summarize past and recent findings that reveal in neural networks the importance of the interaction between GABAergic signaling and astrocytes and discuss its physiological and pathological relevance

Biofilm Removal and Bacterial Re-Colonization Inhibition of a Novel Erythritol/Chlorhexidine Air-Polishing Powder on Titanium Disks

Air-polishing with low abrasiveness powders is fast arising as a valid and mini-invasive instrument for the management of biofilm colonizing dental implants. In general, the reported advantage is the efficient removal of plaque with respect to the titanium integrity. In the present study, we evaluated the in situ plaque removal and the preventive efficacy in forestalling further infection of an innovative erythritol/chlorhexidine air-polishing powder and compared it with sodium bicarbonate. Accordingly, two peri-implantitis-linked biofilm formers, strains Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, were selected and used to infect titanium disks before and after the air-polishing treatment to test its ability in biofilm removal and re-colonization inhibition, respectively. Biofilm cell numbers and viability were assayed by colony-forming unit (CFU) count and metabolic-colorimetric (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) (XTT) assay. Results demonstrated that air-polishing performed with either sodium bicarbonate or erythritol/chlorhexidine was effective in reducing bacteria biofilm viability and number on pre-infected specimens, thus showing a similar ability in counteracting existing infection in situ; on the other hand, when air-polished pre-treated disks were infected, only erythritol/chlorhexidine powder showed higher post-treatment biofilm re-growth inhibition. Finally, surface analysis via mechanical profilometry failed to show an increase in titanium roughness, regardless of the powder selected, thus excluding any possible surface damage due to the use of either sodium bicarbonate or erythritol/chlorhexidine

Biofilm Removal and Bacterial Re-Colonization Inhibition of a Novel Erythritol/Chlorhexidine Air-Polishing Powder on Titanium Disks

Air-polishing with low abrasiveness powders is fast arising as a valid and mini-invasive instrument for the management of biofilm colonizing dental implants. In general, the reported advantage is the efficient removal of plaque with respect to the titanium integrity. In the present study, we evaluated the in situ plaque removal and the preventive efficacy in forestalling further infection of an innovative erythritol/chlorhexidine air-polishing powder and compared it with sodium bicarbonate. Accordingly, two peri-implantitis-linked biofilm formers, strains Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, were selected and used to infect titanium disks before and after the air-polishing treatment to test its ability in biofilm removal and re-colonization inhibition, respectively. Biofilm cell numbers and viability were assayed by colony-forming unit (CFU) count and metabolic-colorimetric (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) (XTT) assay. Results demonstrated that air-polishing performed with either sodium bicarbonate or erythritol/chlorhexidine was effective in reducing bacteria biofilm viability and number on pre-infected specimens, thus showing a similar ability in counteracting existing infection in situ; on the other hand, when air-polished pre-treated disks were infected, only erythritol/chlorhexidine powder showed higher post-treatment biofilm re-growth inhibition. Finally, surface analysis via mechanical profilometry failed to show an increase in titanium roughness, regardless of the powder selected, thus excluding any possible surface damage due to the use of either sodium bicarbonate or erythritol/chlorhexidine

Mitochondrial Ca2+ Signaling and Bioenergetics in Alzheimer’s Disease

Alzheimer’s disease (AD) is a hereditary and sporadic neurodegenerative illness defined by the gradual and cumulative loss of neurons in specific brain areas. The processes that cause AD are still under investigation and there are no available therapies to halt it. Current progress puts at the forefront the “calcium (Ca2+) hypothesis” as a key AD pathogenic pathway, impacting neuronal, astrocyte and microglial function. In this review, we focused on mitochondrial Ca2+ alterations in AD, their causes and bioenergetic consequences in neuronal and glial cells, summarizing the possible mechanisms linking detrimental mitochondrial Ca2+ signals to neuronal death in different experimental AD models

Calcium Signals in Astrocyte Microdomains, a Decade of Great Advances.

The glial cells astrocytes have long been recognized as important neuron-supporting elements in brain development, homeostasis, and metabolism. After the discovery that the reciprocal communication between astrocytes and neurons is a fundamental mechanism in the modulation of neuronal synaptic communication, over the last two decades astrocytes became a hot topic in neuroscience research. Crucial to their functional interactions with neurons are the cytosolic Ca(2+) elevations that mediate gliotransmission. Large attention has been posed to the so-called Ca(2+)microdomains, dynamic Ca(2+) changes spatially restricted to fine astrocytic processes including perisynaptic astrocytic processes (PAPs). With presynaptic terminals and postsynaptic neuronal membranes, PAPs compose the tripartite synapse. The distinct spatial-temporal features and functional roles of astrocyte microdomain Ca(2+) activity remain poorly defined. However, thanks to the development of genetically encoded Ca(2+) indicators (GECIs), advanced microscopy techniques, and innovative analytical approaches, Ca(2+) transients in astrocyte microdomains were recently studied in unprecedented detail. These events have been observed to occur much more frequently (∼50–100-fold) and dynamically than somatic Ca(2+) elevations with mechanisms that likely involve both IP(3)-dependent and -independent pathways. Further progress aimed to clarify the complex, dynamic machinery responsible for astrocytic Ca(2+) activity at microdomains is a crucial step in our understanding of the astrocyte role in brain function and may also reveal astrocytes as novel therapeutic targets for different brain diseases. Here, we review the most recent studies that improve our mechanistic understanding of the essential features of astrocyte Ca(2+) microdomains