Mitochondrial Ca2+ signaling and Alzheimer's disease: Too much or too little?

Alzheimer's disease (AD) is a neurodegenerative disease, caused by poorly known pathogenic mechanisms and aggravated by delayed therapeutic intervention, that still lacks an effective cure. However, it is clear that some important neurophysiological processes are altered years before the onset of clinical symptoms, offering the possibility of identifying biological targets useful for implementation of new therapies. Of note, evidence has been provided suggesting that mitochondria, pivotal organelles in sustaining neuronal energy demand and modulating synaptic activity, are dysfunctional in AD samples. In particular, alterations in mitochondrial Ca2+ signaling have been proposed as causal events for neurodegeneration, although the exact outcomes and molecular mechanisms of these defects, as well as their longitudinal progression, are not always clear. Here, we discuss the importance of a correct mitochondrial Ca2+ handling for neuronal physiology and summarize the latest findings on dysfunctional mitochondrial Ca2+ pathways in AD, analysing possible consequences contributing to the neurodegeneration that characterizes the disease.

Sustained IP3-linked Ca2+ signaling promotes progression of triple negative breast cancer cells by regulating fatty acid metabolism.

Rewiring of mitochondrial metabolism has been described in different cancers as a key step for their progression. Calcium (Ca2+) signaling regulates mitochondrial function and is known to be altered in several malignancies, including triple negative breast cancer (TNBC). However, whether and how the alterations in Ca2+ signaling contribute to metabolic changes in TNBC has not been elucidated. Here, we found that TNBC cells display frequent, spontaneous inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations, which are sensed by mitochondria. By combining genetic, pharmacologic and metabolomics approaches, we associated this pathway with the regulation of fatty acid (FA) metabolism. Moreover, we demonstrated that these signaling routes promote TNBC cell migration in vitro, suggesting they might be explored to identify potential therapeutic targets.

Nerve-dependent distribution of subsynaptic type 1 inositol 1,4,5-trisphosphate receptor at the neuromuscular junction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.

Key Signalling Molecules in Aging and Neurodegeneration.

One of the major challenges of modern medicine is to block or prevent the neurodegenerative processes inevitably associated with different pathological conditions [...].

Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep.

The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs.

Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase.

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.

Loosening ER-Mitochondria Coupling by the Expression of the Presenilin 2 Loop Domain.

Presenilin 2 (PS2), one of the three proteins in which mutations are linked to familial Alzheimer's disease (FAD), exerts different functions within the cell independently of being part of the γ-secretase complex, thus unrelated to toxic amyloid peptide formation. In particular, its enrichment in endoplasmic reticulum (ER) membrane domains close to mitochondria (i.e., mitochondria-associated membranes, MAM) enables PS2 to modulate multiple processes taking place on these signaling hubs, such as Ca2+ handling and lipid synthesis. Importantly, upregulated MAM function appears to be critical in AD pathogenesis. We previously showed that FAD-PS2 mutants reinforce ER-mitochondria tethering, by interfering with the activity of mitofusin 2, favoring their Ca2+ crosstalk. Here, we deepened the molecular mechanism underlying PS2 activity on ER-mitochondria tethering, identifying its protein loop as an essential domain to mediate the reinforced ER-mitochondria connection in FAD-PS2 models. Moreover, we introduced a novel tool, the PS2 loop domain targeted to the outer mitochondrial membrane, Mit-PS2-LOOP, that is able to counteract the activity of FAD-PS2 on organelle tethering, which possibly helps in recovering the FAD-PS2-associated cellular alterations linked to an increased organelle coupling.

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.

Excitotoxicity Revisited: Mitochondria on the Verge of a Nervous Breakdown.

Excitotoxicity is likely to occur in pathological scenarios in which mitochondrial function is already compromised, shaping neuronal responses to glutamate. In fact, mitochondria sustain cell bioenergetics, tune intracellular Ca2+ dynamics, and regulate glutamate availability by using it as metabolic substrate. Here, we suggest the need to explore glutamate toxicity in the context of specific disease models in which it may occur, re-evaluating the impact of mitochondrial dysfunction on glutamate excitotoxicity. Our aim is to signpost new approaches, perhaps combining glutamate and pathways to rescue mitochondrial function, as therapeutic targets in neurological disorders.

Better to keep in touch: investigating inter-organelle cross-talk.

The strategic importance for cellular organelles of being in contact with each other, exchanging messenger molecules, is nowadays well established. Different inter-organelle cross-talk pathways finely regulate multiple physiological cellular mechanisms, and their dysregulation has been found to underlie different pathological conditions. In the last years, a great effort has been made to study such organelle interactions, to understand their functional roles within the cell and the molecules involved in their formation and/or modulation. In this contribution, some examples of organelle cross-talk and their contributions in regulating physiological processes are presented. Moreover, the pro and cons of the available methods for a proper, reliable investigation of membrane contact sites are described.

Familial Alzheimer's disease presenilin-2 mutants affect Ca2+ homeostasis and brain network excitability.

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.

The yin and yang of mitochondrial Ca2+ signaling in cell physiology and pathology.

Mitochondria are autonomous and dynamic cellular organelles orchestrating a diverse range of cellular activities. Numerous cell-signaling pathways target these organelles and Ca2+ is one of the most significant. Mitochondria are able to rapidly and transiently take up Ca2+, thanks to the mitochondrial Ca2+ uniporter complex, as well as to extrude it through the Na+/Ca2+ and H+/Ca2+ exchangers. The transient accumulation of Ca2+ in the mitochondrial matrix impacts on mitochondrial functions and cell pathophysiology. Here we summarize the role of mitochondrial Ca2+ signaling in both physiological (yang) and pathological (yin) processes and the methods that can be used to investigate mitochondrial Ca2+ homeostasis. As an example of the pivotal role of mitochondria in pathology, we described the state of the art of mitochondrial Ca2+ alterations in different pathological conditions, with a special focus on Alzheimer's disease.

Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks.

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).

Sarcoplasmic Reticulum-Mitochondria Kissing in Cardiomyocytes: Ca2+, ATP, and Undisclosed Secrets.

In cardiomyocytes, to carry out cell contraction, the distribution, morphology, and dynamic interaction of different cellular organelles are tightly regulated. For instance, the repetitive close apposition between junctional sarcoplasmic reticulum (jSR) and specialized sarcolemma invaginations, called transverse-tubules (TTs), is essential for an efficient excitation-contraction coupling (ECC). Upon an action potential, Ca2+ microdomains, generated in synchrony at the interface between TTs and jSR, underlie the prompt increase in cytosolic Ca2+ concentration, ultimately responsible for cell contraction during systole. This process requires a considerable amount of energy and the active participation of mitochondria, which encompass ∼30% of the cell volume and represent the major source of ATP in the heart. Importantly, in adult cardiomyocytes, mitochondria are distributed in a highly orderly fashion and strategically juxtaposed with SR. By taking advantage of the vicinity to Ca2+ releasing sites, they take up Ca2+ and modulate ATP synthesis according to the specific cardiac workload. Interestingly, with respect to SR, a biased, polarized positioning of mitochondrial Ca2+ uptake/efflux machineries has been reported, hinting the importance of a strictly regulated mitochondrial Ca2+ handling for heart activity. This notion, however, has been questioned by the observation that, in some mouse models, the deficiency of specific molecules, modulating mitochondrial Ca2+ dynamics, triggers non-obvious cardiac phenotypes. This review will briefly summarize the physiological significance of SR-mitochondria apposition in cardiomyocytes, as well as the pathological consequences of an altered organelle communication, focusing on Ca2+ signaling. We will discuss ongoing debates and propose future research directions.

Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+ -dependent death of cancer cells.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

Defective Mitochondrial Pyruvate Flux Affects Cell Bioenergetics in Alzheimer's Disease-Related Models.

Mitochondria are key organelles for brain health. Mitochondrial alterations have been reported in several neurodegenerative disorders, including Alzheimer's disease (AD), and the comprehension of the underlying mechanisms appears crucial to understand their relationship with the pathology. Using multiple genetic, pharmacological, imaging, and biochemical approaches, we demonstrate that, in different familial AD cell models, mitochondrial ATP synthesis is affected. The defect depends on reduced mitochondrial pyruvate oxidation, due to both lower Ca2+-mediated stimulation of the Krebs cycle and dampened mitochondrial pyruvate uptake. Importantly, this latter event is linked to glycogen-synthase-kinase-3ß (GSK-3ß) hyper-activation, leading, in turn, to impaired recruitment of hexokinase 1 (HK1) to mitochondria, destabilization of mitochondrial-pyruvate-carrier (MPC) complexes, and decreased MPC2 protein levels. Remarkably, pharmacological GSK-3ß inhibition in AD cells rescues MPC2 expression and improves mitochondrial ATP synthesis and respiration. The defective mitochondrial bioenergetics influences glutamate-induced neuronal excitotoxicity, thus representing a possible target for future therapeutic interventions.

ER-mitochondria tethering and Ca2+ crosstalk: The IP3R team takes the field.

Inter-organelle communication represents a booming topic in cell biology research, with endoplasmic reticulum (ER)-mitochondria coupling playing the lion's share. In a recent work, Bartok and colleagues found that inositol trisphosphates receptors (IP3Rs), in addition to their well-known involvement in ER-mitochondria Ca2+ transfer, are endowed with structural properties at organelles' interface.

Calcium, mitochondria and cell metabolism: A functional triangle in bioenergetics.

The versatility of mitochondrial metabolism and its fine adjustments to specific physiological or pathological conditions regulate fundamental cell pathways, ranging from proliferation to apoptosis. In particular, Ca2+ signalling has emerged as a key player exploited by mitochondria to tune their activity according with cell demand. The functional interaction between mitochondria and endoplasmic reticulum (ER) deeply impacts on the correct mitochondrial Ca2+ signal, thus modulating cell bioenergetics and functionality. Indeed, Ca2+ released by the ER is taken up by mitochondria where, both in the intermembrane space and in the matrix, it regulates the activity of transporters, enzymes and proteins involved in organelles' metabolism. In this review, we will briefly summarize Ca2+-dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca2+ signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer.

PSEN2 (presenilin 2) mutants linked to familial Alzheimer disease impair autophagy by altering Ca2+ homeostasis.

PSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aß peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca2+ homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca2+ content, thus reducing cytosolic Ca2+ response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca2+ signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration.Abbreviations: Aß: amyloid ß; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A1; BAPTA-AM: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; CFP: cyan fluorescent protein; EGTA-AM: ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester; ER: endoplasmic reticulum; EGFP-HDQ74: enhanced GFP-huntingtin exon 1 containing 74 polyglutamine repeats; FAD: familial Alzheimer disease; FCS: fetal calf serum; FRET: fluorescence/Förster resonance energy transfer; GFP: green fluorescent protein; IP3: inositol trisphosphate; KD: knockdown; LAMP1: lysosomal associated membrane protein 1; MAP1LC3-II/LC3-II: lipidated microtubule-associated protein 1 light chain 3; MCU: mitochondrial calcium uniporter; MICU1: mitochondrial calcium uptake 1; MEFs: mouse embryonic fibroblasts; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; SQSTM1/p62: sequestosome 1; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB7: RAB7A: member RAS oncogene family; RFP: red fluorescent protein; ATP2A/SERCA: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H+-ATPase; WT: wild type.