High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Overexpression of RCAN1, a Gene on Human Chromosome 21, Alters Cell Redox and Mitochondrial Function in Enamel Cells.

The regulator of calcineurin (RCAN1) has been implicated in the pathogenesis of Down syndrome (DS). Individuals with DS show dental abnormalities for unknown reasons, and RCAN1 levels have been found to be elevated in several tissues of DS patients. A previous microarray analysis comparing cells of the two main formative stages of dental enamel, secretory and maturation, showed a significant increase in RCAN1 expression in the latter. Because the function of RCAN1 during enamel formation is unknown, there is no mechanistic evidence linking RCAN1 with the dental anomalies in individuals with DS. We investigated the role of RCAN1 in enamel by overexpressing RCAN1 in the ameloblast cell line LS8 (LS8+RCAN1). We first confirmed that RCAN1 is highly expressed in maturation stage ameloblasts by qRT-PCR and used immunofluorescence to show its localization in enamel-forming ameloblasts. We then analyzed cell redox and mitochondrial bioenergetics in LS8+RCAN1 cells because RCAN1 is known to impact these processes. We show that LS8+RCAN1 cells have increased reactive oxygen species (ROS) and decreased mitochondrial bioenergetics without changes in the expression of the complexes of the electron transport chain, or in NADH levels. However, LS8+RCAN1 cells showed elevated mitochondrial Ca2+ uptake and decreased expression of several enamel genes essential for enamel formation. These results provide insight into the role of RCAN1 in enamel and suggest that increased RCAN1 levels in the ameloblasts of individuals with DS may impact enamel formation by altering both the redox environment and mitochondrial function, as well as decreasing the expression of enamel-specific genes.

Mitochondria modulate ameloblast Ca2+ signaling.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.

Calcium Transport in Specialized Dental Epithelia and Its Modulation by Fluoride.

Most cells use calcium (Ca2+) as a second messenger to convey signals that affect a multitude of biological processes. The ability of Ca2+ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca2+ (cCa2+) concentration is maintained low at ~100 nM so that the impact of elevations in cCa2+ is readily sensed and transduced by cells. However, such elevations in cCa2+ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of cCa2+ including Ca2+ pumps, exchangers and sequestering Ca2+ within intracellular organelles. This Ca2+ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca2+. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca2+ uptake into ameloblasts is mainly regulated by the store operated Ca2+ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca2+ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.

Mitochondrial Function in Enamel Development.

Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of AmelX and Enam genes that are essential for enamel formation. The ameloblast cell line LS8 treated with H2O2 to promote ROS elicited significant expression changes in AmelX and Enam. Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.

TRPM7 activation potentiates SOCE in enamel cells but requires ORAI.

Calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediated by STIM1/2 and ORAI (ORAI1-3) proteins form the dominant store-operated Ca2+ entry (SOCE) pathway in a wide variety of cells. Among these, the enamel-forming cells known as ameloblasts rely on CRAC channel function to enable Ca2+ influx, which is important for enamel mineralization. This key role of the CRAC channel is supported by human mutations and animal models lacking STIM1 and ORAI1, which results in enamel defects and hypomineralization. A number of recent reports have highlighted the role of the chanzyme TRPM7 (transient receptor potential melastanin 7), a transmembrane protein containing an ion channel permeable to divalent cations (Mg2+, Ca2+), as a modulator of SOCE. This raises the question as to whether TRPM7 should be considered an alternative route for Ca2+ influx, or if TRPM7 modifies CRAC channel activity in enamel cells. To address these questions, we monitored Ca2+ influx mediated by SOCE using the pharmacological TRPM7 activator naltriben and the inhibitor NS8593 in rat primary enamel cells and in the murine ameloblast cell line LS8 cells stimulated with thapsigargin. We also measured Ca2+ dynamics in ORAI1/2-deficient (shOrai1/2) LS8 cells and in cells with siRNA knock-down of Trpm7. We found that primary enamel cells stimulated with the TRPM7 activator potentiated Ca2+ influx via SOCE compared to control cells. However, blockade of TRPM7 with NS8593 did not decrease the SOCE peak. Furthermore, activation of TRPM7 in shOrai1/2 LS8 cells lacking SOCE failed to elicit Ca2+ influx, and Trpm7 knock-down had no effect on SOCE. Taken together, our data suggest that TRPM7 is a positive modulator of SOCE potentiating Ca2+ influx in enamel cells, but its function is fully dependent on the prior activation of the ORAI channels.

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Differential regulation of Ca2+ influx by ORAI channels mediates enamel mineralization.

Store-operated Ca2+ entry (SOCE) channels are highly selective Ca2+ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca2+ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2-/- mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca2+ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca2+ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury.

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N1-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N1-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N1-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N1-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N1-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.

Protein Localization at Mitochondria-ER Contact Sites in Basal and Stress Conditions.

Mitochondria-endoplasmic reticulum (ER) contacts (MERCs) are sites at which the outer mitochondria membrane and the Endoplasmic Reticulum surface run in parallel at a constant distance. The juxtaposition between these organelles determines several intracellular processes such as to name a few, Ca2+ and lipid homeostasis or autophagy. These specific tasks can be exploited thanks to the enrichment (or re-localization) of dedicated proteins at these interfaces. Recent proteomic studies highlight the tissue specific composition of MERCs, but the overall mechanisms that control MERCs plasticity remains unclear. Understanding how proteins are targeted at these sites seems pivotal to clarify such contextual function of MERCs. This review aims to summarize the current knowledge on protein localization at MERCs and the possible contribution of the mislocalization of MERCs components to human disorders.

Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington's Disease.

Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.