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.

Unraveling Presenilin 2 Functions in a Knockout Zebrafish Line to Shed Light into Alzheimer's Disease Pathogenesis.

Mutations in presenilin 2 (PS2) have been causally linked to the development of inherited Alzheimer's disease (AD). Besides its role as part of the γ-secretase complex, mammalian PS2 is also involved, as an individual protein, in a growing number of cell processes, which result altered in AD. To gain more insight into PS2 (dys)functions, we have generated a presenilin2 (psen2) knockout zebrafish line. We found that the absence of the protein does not markedly influence Notch signaling at early developmental stages, suggesting a Psen2 dispensable role in the γ-secretase-mediated Notch processing. Instead, loss of Psen2 induces an exaggerated locomotor response to stimulation in fish larvae, a reduced number of ER-mitochondria contacts in zebrafish neurons, and an increased basal autophagy. Moreover, the protein is involved in mitochondrial axonal transport, since its acute downregulation reduces in vivo organelle flux in zebrafish sensory neurons. Importantly, the expression of a human AD-linked mutant of the protein increases this vital process. Overall, our results confirm zebrafish as a good model organism for investigating PS2 functions in vivo, representing an alternative tool for the characterization of new AD-linked defective cell pathways and the testing of possible correcting drugs.

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.

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 [...].

Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells.

The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca2+ homeostasis, antioxidant cytoprotection and protein-protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca2+ storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca2+ homeostasis.

Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus.

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.

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.

Lighting Up Ca2+ Dynamics in Animal Models.

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.

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.

Calcium Signaling and Mitochondrial Function in Presenilin 2 Knock-Out Mice: Looking for Any Loss-of-Function Phenotype Related to Alzheimer's Disease.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder in which learning, memory and cognitive functions decline progressively. Familial forms of AD (FAD) are caused by mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes. Presenilin 1 (PS1) and its homologue, presenilin 2 (PS2), represent, alternatively, the catalytic core of the γ-secretase complex that, by cleaving APP, produces neurotoxic amyloid beta (Aß) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently, PSEN1 FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to PSEN2 FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca2+ signaling and mitochondrial function. By exploiting neurons derived from a PSEN2 knock-out (PS2-/-) mouse model, we found that, upon IP3-generating stimulation, cytosolic Ca2+ handling is not altered, compared to wild-type cells, while mitochondrial Ca2+ uptake is strongly compromised. Accordingly, PS2-/- neurons show a marked reduction in endoplasmic reticulum-mitochondria apposition and a slight alteration in mitochondrial respiration, whereas mitochondrial membrane potential, and organelle morphology and number appear unchanged. Thus, although some alterations in mitochondrial function appear to be shared between PS2-/- and FAD-PS2-expressing neurons, the mechanisms leading to these defects are quite distinct between the two models. Taken together, our data appear to be difficult to reconcile with the proposal that FAD-PS2 mutants are loss-of-function, whereas the concept that PS2 plays a key role in sustaining mitochondrial function is here confirmed.

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.

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).

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.