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Regulation of mitochondrial free Ca2+ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca2+ concentration ([Ca2+]m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca2+. Cardiac-isolated mitochondria were challenged with repetitive CaCl2 boluses under Na+-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca2+]m levels for an extended period. The preservation of basal [Ca2+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pHm), and mitochondrial membrane potential (ΔΨm). Notably, we found that in PO43− (Pi)-free buffer condition, the CsA-mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of Pi just before pore opening in the Pi-depleted condition reinstated the Ca2+ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.

Calreticulin (CALR) is an endoplasmic reticulum (ER)-resident protein involved in a spectrum of cellular processes. In healthy cells, CALR operates as a chaperone and Ca 2+ buffer to assist correct protein folding within the ER. Besides favoring the maintenance of cellular proteostasis, these cell-intrinsic CALR functions support Ca 2+ -dependent processes, such as adhesion and integrin signaling, and ensure normal antigen presentation on MHC Class I molecules. Moreover, cancer cells succumbing to immunogenic cell death (ICD) expose CALR on their surface, which promotes the uptake of cell corpses by professional phagocytes and ultimately supports the initiation of anticancer immunity. Thus, loss-of-function CALR mutations promote oncogenesis not only as they impair cellular homeostasis in healthy cells, but also as they compromise natural and therapy-driven immunosurveillance. However, the prognostic impact of total or membrane-exposed CALR levels appears to vary considerably with cancer type. For instance, while genetic CALR defects promote pre-neoplastic myeloproliferation, patients with myeloproliferative neoplasms bearing CALR mutations often experience improved overall survival as compared to patients bearing wild-type CALR . Here, we discuss the context-dependent impact of CALR on malignant transformation, tumor progression and response to cancer therapy.

Excitotoxicity is a phenomenon that describes the toxic actions of excitatory neurotransmitters, primarily glutamate, where the exacerbated or prolonged activation of glutamate receptors starts a cascade of neurotoxicity that ultimately leads to the loss of neuronal function and cell death. In this process, the shift between normal physiological function and excitotoxicity is largely controlled by astrocytes since they can control the levels of glutamate on the synaptic cleft. This control is achieved through glutamate clearance from the synaptic cleft and its underlying recycling through the glutamate-glutamine cycle. The molecular mechanism that triggers excitotoxicity involves alterations in glutamate and calcium metabolism, dysfunction of glutamate transporters, and malfunction of glutamate receptors, particularly N-methyl-D-aspartic acid receptors (NMDAR). On the other hand, excitotoxicity can be regarded as a consequence of other cellular phenomena, such as mitochondrial dysfunction, physical neuronal damage, and oxidative stress. Regardless, it is known that the excessive activation of NMDAR results in the sustained influx of calcium into neurons and leads to several deleterious consequences, including mitochondrial dysfunction, reactive oxygen species (ROS) overproduction, impairment of calcium buffering, the release of pro-apoptotic factors, among others, that inevitably contribute to neuronal loss. A large body of evidence implicates NMDAR-mediated excitotoxicity as a central mechanism in the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and epilepsy. In this review article, we explore different causes and consequences of excitotoxicity, discuss the involvement of NMDAR-mediated excitotoxicity and its downstream effects on several neurodegenerative disorders, and identify possible strategies to study new aspects of these diseases that may lead to the discovery of new therapeutic approaches. With the understanding that excitotoxicity is a common denominator in neurodegenerative diseases and other disorders, a new perspective on therapy can be considered, where the targets are not specific symptoms, but the underlying cellular phenomena of the disease.

Synapses enable neurons to communicate with each other and are therefore a prerequisite for normal brain function. Presynaptically, this communication requires energy and generates large fluctuations in calcium concentrations. Mitochondria are optimized for supplying energy and buffering calcium, and they are actively recruited to presynapses. However, not all presynapses contain mitochondria; thus, how might synapses with and without mitochondria differ? Mitochondria are also increasingly recognized to serve additional functions at the presynapse. Here, we discuss the importance of presynaptic mitochondria in maintaining neuronal homeostasis and how dysfunctional presynaptic mitochondria might contribute to the development of disease.

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer’s disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum–mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.

Calcium is used in many cellular processes and is maintained within the cell as free calcium at low concentrations (approximately 100 nM), compared with extracellular (millimolar) concentrations, to avoid adverse effects such as phosphate precipitation. For this reason, cells have adapted buffering strategies by compartmentalizing calcium into mitochondria and the endoplasmic reticulum (ER). In mitochondria, the calcium concentration is in the millimolar range, as it is in the ER. Mitochondria actively contribute to buffering cellular calcium, but if matrix calcium increases beyond physiological demands, it can promote the opening of the mitochondrial permeability transition pore (mPTP) and, consequently, trigger apoptotic or necrotic cell death. The pathophysiological implications of mPTP opening in ischemia-reperfusion, liver, muscle, and lysosomal storage diseases, as well as those affecting the central nervous system, for example, Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) have been reported. In this review, we present an updated overview of the main cellular mechanisms of mitochondrial calcium regulation. We specially focus on neurodegenerative diseases related to imbalances in calcium homeostasis and summarize some proposed therapies studied to attenuate these diseases.

Lanthanides, the 14 4f-block elements plus Lanthanum, have been extensively used to study the structure and biochemical properties of metalloproteins. The characteristics of lanthanides within the lanthanide series are similar, but not identical. The present research offers a systematic investigation of the ability of the entire Ln[sup.3+] series to substitute for Ca[sup.2+] in biological systems. A well-calibrated DFT/PCM protocol is employed in studying the factors that control the metal selectivity in biological systems by modeling typical calcium signaling/buffering binding sites and elucidating the thermodynamic outcome of the competition between the \"alien\" La[sup.3+]/Ln[sup.3+] and \"native\" Ca[sup.2+], and La[sup.3+] − Ln[sup.3+] within the lanthanide series. The calculations performed reveal that the major determinant of the Ca[sup.2+]/Ln[sup.3+] selectivity in calcium proteins is the net charge of the calcium binding pocket; the more negative the charge, the higher the competitiveness of the trivalent Ln[sup.3+] with respect to its Ca[sup.2+] contender. Solvent exposure of the binding site also influences the process; buried active centers with net charge of −4 or −3 are characterized by higher Ln[sup.3+] over Ca[sup.2+] selectivity, whereas it is the opposite for sites with overall charge of −1. Within the series, the competition between La[sup.3+] and its fellow lanthanides is determined by the balance between two competing effects: electronic (favoring heavier lanthanides) and solvation (generally favoring the lighter lanthanides).

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Heart failure (HF) represents one of the leading causes of cardiovascular diseases with high rates of hospitalization, morbidity and mortality worldwide. Ample evidence has consolidated a crucial role for mitochondrial injury in the progression of HF. It is well established that mitochondrial Ca 2+ participates in the regulation of a wide variety of biological processes, including oxidative phosphorylation, ATP synthesis, reactive oxygen species (ROS) generation, mitochondrial dynamics and mitophagy. Nonetheless, mitochondrial Ca 2+ overload stimulates mitochondrial permeability transition pore (mPTP) opening and mitochondrial swelling, resulting in mitochondrial injury, apoptosis, cardiac remodeling, and ultimately development of HF. Moreover, mitochondria possess a series of Ca 2+ transport influx and efflux channels, to buffer Ca 2+ in the cytoplasm. Interaction at mitochondria-associated endoplasmic reticulum membranes (MAMs) may also participate in the regulation of mitochondrial Ca 2+ homeostasis and plays an essential role in the progression of HF. Here, we provide an overview of regulation of mitochondrial Ca 2+ homeostasis in maintenance of cardiac function, in an effort to identify novel therapeutic strategies for the management of HF.

Alzheimer’s disease (AD) is the most frequent cause of age-related neurodegeneration and cognitive impairment, and there are currently no broadly effective therapies. The underlying pathogenesis is complex, but a growing body of evidence implicates mitochondrial dysfunction as a common pathomechanism involved in many of the hallmark features of the AD brain, such as formation of amyloid-beta (Aβ) aggregates (amyloid plaques), neurofibrillary tangles, cholinergic system dysfunction, impaired synaptic transmission and plasticity, oxidative stress, and neuroinflammation, that lead to neurodegeneration and cognitive dysfunction. Indeed, mitochondrial dysfunction concomitant with progressive accumulation of mitochondrial Aβ is an early event in AD pathogenesis. Healthy mitochondria are critical for providing sufficient energy to maintain endogenous neuroprotective and reparative mechanisms, while disturbances in mitochondrial function, motility, fission, and fusion lead to neuronal malfunction and degeneration associated with excess free radical production and reduced intracellular calcium buffering. In addition, mitochondrial dysfunction can contribute to amyloid-β precursor protein (APP) expression and misprocessing to produce pathogenic fragments (e.g., Aβ1-40). Given this background, we present an overview of the importance of mitochondria for maintenance of neuronal function and how mitochondrial dysfunction acts as a driver of cognitive impairment in AD. Additionally, we provide a brief summary of possible treatments targeting mitochondrial dysfunction as therapeutic approaches for AD.