Publications by authors named "Paola Pizzo"

74 Publications

Lighting Up Ca Dynamics in Animal Models.

Cells 2021 Aug 19;10(8). Epub 2021 Aug 19.

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.

Calcium (Ca) signaling coordinates are crucial processes in brain physiology. Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca, and neuronal survival itself relies on Ca-dependent cascades. Indeed, impaired Ca 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, Ca imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.
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http://dx.doi.org/10.3390/cells10082133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392631PMC
August 2021

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

Cells 2021 Aug 3;10(8). Epub 2021 Aug 3.

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.

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 Ca 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 Ca 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.
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http://dx.doi.org/10.3390/cells10081968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394530PMC
August 2021

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

BMC Biol 2021 03 24;19(1):57. Epub 2021 Mar 24.

Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden.

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 (Ca) and Ca-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 Ca-releasing channels inositol 1,4,5-trisphosphate receptors (IPRs) 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.
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http://dx.doi.org/10.1186/s12915-021-00979-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989211PMC
March 2021

Cell calcium: Mitochondria: function and disease.

Authors:
Paola Pizzo

Cell Calcium 2021 06 22;96:102370. Epub 2021 Feb 22.

Department of Biomedical Sciences, University of Padua, Padua, 35131, Italy; Neuroscience Institute, National Research Council (CNR), Padua, 35131, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.ceca.2021.102370DOI Listing
June 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

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

Trends Neurosci 2021 05 16;44(5):342-351. Epub 2021 Feb 16.

Department of Cell and Developmental Biology, University College London, London, UK. Electronic address:

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 Ca 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.
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http://dx.doi.org/10.1016/j.tins.2021.01.001DOI Listing
May 2021

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

Cells 2021 01 21;10(2). Epub 2021 Jan 21.

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.

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 (), presenilin 1 () and presenilin 2 () 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 (Aβ) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently, FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca signaling and mitochondrial function. By exploiting neurons derived from a knock-out (PS2-/-) mouse model, we found that, upon IP-generating stimulation, cytosolic Ca handling is not altered, compared to wild-type cells, while mitochondrial Ca 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.
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http://dx.doi.org/10.3390/cells10020204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909802PMC
January 2021

Mitochondrial bioenergetics and neurodegeneration: a .

Neural Regen Res 2021 Apr;16(4):686-687

Department of Biomedical Sciences, University of Padua; Neuroscience Institute - Italian National Research Council (CNR), Padua, Italy.

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http://dx.doi.org/10.4103/1673-5374.295331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067919PMC
April 2021

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

Cells 2020 09 25;9(10). Epub 2020 Sep 25.

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35131 Padua, Italy.

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-β (Aβ) 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 Ca 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 Ca handling to Ca 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 Ca 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).
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http://dx.doi.org/10.3390/cells9102166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601421PMC
September 2020

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

FEBS J 2021 02 30;288(3):740-755. Epub 2020 Jun 30.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

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.
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http://dx.doi.org/10.1111/febs.15451DOI Listing
February 2021

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

EMBO Rep 2020 07 8;21(7):e49117. Epub 2020 May 8.

Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy.

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 Ca overload caused by Ca release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca entry through plasma membrane. This results in Ca -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.
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http://dx.doi.org/10.15252/embr.201949117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332982PMC
July 2020

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

Cell Rep 2020 02;30(7):2332-2348.e10

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy; Neuroscience Institute - Italian National Research Council (CNR), Padua 35121, Italy. Electronic address:

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 Ca-mediated stimulation of the Krebs cycle and dampened mitochondrial pyruvate uptake. Importantly, this latter event is linked to glycogen-synthase-kinase-3β (GSK-3β) 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-3β 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.
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http://dx.doi.org/10.1016/j.celrep.2020.01.060DOI Listing
February 2020

Intracellular Calcium Dysregulation by the Alzheimer's Disease-Linked Protein Presenilin 2.

Int J Mol Sci 2020 Jan 24;21(3). Epub 2020 Jan 24.

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.

Alzheimer's disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. AD mutations contribute to the generation of toxic amyloid β (Aβ) peptides and the formation of cerebral plaques, leading to the formulation of the amyloid cascade hypothesis for AD pathogenesis. Many drugs have been developed to inhibit this pathway but all these approaches currently failed, raising the need to find additional pathogenic mechanisms. Alterations in cellular calcium (Ca) signaling have also been reported as causative of neurodegeneration. Interestingly, Aβ peptides, mutated presenilin-1 (PS1), and presenilin-2 (PS2) variously lead to modifications in Ca homeostasis. In this contribution, we focus on PS2, summarizing how AD-linked PS2 mutants alter multiple Ca pathways and the functional consequences of this Ca dysregulation in AD pathogenesis.
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http://dx.doi.org/10.3390/ijms21030770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037278PMC
January 2020

Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca Handling.

Front Physiol 2019 20;10:1544. Epub 2019 Dec 20.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an approach involving the manipulation of MT dynamics in by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.
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http://dx.doi.org/10.3389/fphys.2019.01544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933510PMC
December 2019

Calcium Imaging in Drosophila melanogaster.

Adv Exp Med Biol 2020 ;1131:881-900

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca signaling comprises complex pathways that impact on virtually every aspect of cellular physiology. Within such a complex field of study, Drosophila offers the advantages of consolidated molecular and genetic techniques, lack of genetic redundancy and a completely annotated genome since 2000. These and other characteristics provided the basis for the identification of many genes encoding Ca signaling molecules and the disclosure of conserved Ca signaling pathways. In this review, we will analyze the applications of Ca imaging in the fruit fly model, highlighting in particular their impact on the study of normal brain function and pathogenesis of neurodegenerative diseases.
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http://dx.doi.org/10.1007/978-3-030-12457-1_35DOI Listing
October 2019

ER-mitochondria tethering and Ca crosstalk: The IPR team takes the field.

Cell Calcium 2019 Dec 10;84:102101. Epub 2019 Oct 10.

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35121, Padua, Italy; Neuroscience Institute - Italian National Research Council (CNR), Padua, 35121, Italy. Electronic address:

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 (IPRs), in addition to their well-known involvement in ER-mitochondria Ca transfer, are endowed with structural properties at organelles' interface.
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http://dx.doi.org/10.1016/j.ceca.2019.102101DOI Listing
December 2019

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

Aging Clin Exp Res 2021 Jun 12;33(6):1705-1708. Epub 2019 Oct 12.

Neuroscience Institute - Italian National Research Council (CNR), Padua, Italy.

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 (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca 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 Ca signaling. By generating novel tools for measuring Ca in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca signaling and brain network activity, as summarized below.
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http://dx.doi.org/10.1007/s40520-019-01341-0DOI Listing
June 2021

Defective autophagy and Alzheimer's disease: is calcium the key?

Neural Regen Res 2019 Dec;14(12):2081-2082

Department of Biomedical Sciences, University of Padua and Neuroscience Institute - Italian National Research Council (CNR), Padua, Italy.

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http://dx.doi.org/10.4103/1673-5374.262584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788238PMC
December 2019

Glucose dysregulation in pre-clinical Alzheimer's disease.

Aging (Albany NY) 2019 08 4;11(15):5296-5297. Epub 2019 Aug 4.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

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http://dx.doi.org/10.18632/aging.102146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710053PMC
August 2019

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

Biochim Biophys Acta Mol Cell Res 2019 07 26;1866(7):1068-1078. Epub 2018 Oct 26.

Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.

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, Ca 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 Ca signal, thus modulating cell bioenergetics and functionality. Indeed, Ca 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 Ca-dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer.
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http://dx.doi.org/10.1016/j.bbamcr.2018.10.016DOI Listing
July 2019

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

Autophagy 2019 12 27;15(12):2044-2062. Epub 2019 Mar 27.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

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 Aβ peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca 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 Ca content, thus reducing cytosolic Ca response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca 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. Aβ: amyloid β; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A; 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(β-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 Ca transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H-ATPase; WT: wild type.
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http://dx.doi.org/10.1080/15548627.2019.1596489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844518PMC
December 2019

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity.

Acta Neuropathol Commun 2019 03 6;7(1):35. Epub 2019 Mar 6.

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 9RX, UK.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by "tethering proteins" that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson's disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson's disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson's disease and FTD/ALS.
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http://dx.doi.org/10.1186/s40478-019-0688-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402140PMC
March 2019

Familial Alzheimer's disease-linked presenilin mutants and intracellular Ca handling: A single-organelle, FRET-based analysis.

Cell Calcium 2019 05 23;79:44-56. Epub 2019 Feb 23.

Neuroscience Institute - Italian National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35131 Padua, Italy.

An imbalance in Ca homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca concentration within the main intracellular Ca stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca-ATPase) activity. Moreover, by using a cytosolic Ca probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca influx activated by stores depletion (Store-Operated Ca Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca content of intracellular stores yet leading to a complex dysregulation of Ca homeostasis, which represents a common disease phenotype of AD.
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http://dx.doi.org/10.1016/j.ceca.2019.02.005DOI Listing
May 2019

Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer's disease neurons.

Aging Cell 2019 06 21;18(3):e12924. Epub 2019 Feb 21.

Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer's disease (AD). We here combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain (RC), but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the RC. To validate these predictions, we utilized fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, nonintuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity.
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http://dx.doi.org/10.1111/acel.12924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516149PMC
June 2019

Exploiting Cameleon Probes to Investigate Organelles Ca Handling.

Methods Mol Biol 2019 ;1925:15-30

Department of Biomedical Sciences, University of Padua, Padua, Italy.

Calcium ion (Ca) is a ubiquitous intracellular messenger able to generate versatile intracellular signals that modulate a large variety of functions in virtually every cell type. Chemical and genetic biosensors, targeted to different subcellular compartments, have been developed and continuously improved to monitor Ca dynamics in living cells. Here we describe the usage of Förster resonance energy transfer (FRET)-based Cameleon probes to investigate Ca influx across the plasma membrane (PM) or Ca release from the main intracellular Ca store, the endoplasmic reticulum (ER).
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http://dx.doi.org/10.1007/978-1-4939-9018-4_2DOI Listing
June 2019

Mitofusin 2: from functions to disease.

Cell Death Dis 2018 02 28;9(3):330. Epub 2018 Feb 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Mitochondria are highly dynamic organelles whose functions are essential for cell viability. Within the cell, the mitochondrial network is continuously remodeled through the balance between fusion and fission events. Moreover, it dynamically contacts other organelles, particularly the endoplasmic reticulum, with which it enterprises an important functional relationship able to modulate several cellular pathways. Being mitochondria key bioenergetics organelles, they have to be transported to all the specific high-energy demanding sites within the cell and, when damaged, they have to be efficiently removed. Among other proteins, Mitofusin 2 represents a key player in all these mitochondrial activities (fusion, trafficking, turnover, contacts with other organelles), the balance of which results in the appropriate mitochondrial shape, function, and distribution within the cell. Here we review the structural and functional properties of Mitofusin 2, highlighting its crucial role in several cell pathways, as well as in the pathogenesis of neurodegenerative diseases, metabolic disorders, cardiomyopathies, and cancer.
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http://dx.doi.org/10.1038/s41419-017-0023-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832425PMC
February 2018

TOM70 Sustains Cell Bioenergetics by Promoting IP3R3-Mediated ER to Mitochondria Ca Transfer.

Curr Biol 2018 02 27;28(3):369-382.e6. Epub 2018 Jan 27.

Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Novum 5(th) Floor, Karolinska Institutet, 141 57 Huddinge, Sweden. Electronic address:

The mitochondrial translocase of the outer membrane (TOM) is a protein complex that is essential for the post-translational import of nuclear-encoded mitochondrial proteins. Among its subunits, TOM70 and TOM20 are only transiently associated with the core complex, suggesting their possible additional roles within the outer mitochondrial membrane (OMM). Here, by using different mammalian cell lines, we demonstrate that TOM70, but not TOM20, clusters in distinct OMM foci, frequently overlapping with sites in which the endoplasmic reticulum (ER) contacts mitochondria. Functionally, TOM70 depletion specifically impairs inositol trisphosphates (IP3)-linked ER to mitochondria Ca transfer. This phenomenon is dependent on the capacity of TOM70 to interact with IP3-receptors and favor their functional recruitment close to mitochondria. Importantly, the reduced constitutive Ca transfer to mitochondria, observed in TOM70-depleted cells, dampens mitochondrial respiration, affects cell bioenergetics, induces autophagy, and inhibits proliferation. Our data reveal a hitherto unexpected role for TOM70 in pro-survival ER-mitochondria communication, reinforcing the view that the ER-mitochondria signaling platform is a key regulator of cell fate.
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http://dx.doi.org/10.1016/j.cub.2017.12.047DOI Listing
February 2018

The Aging Mitochondria.

Genes (Basel) 2018 Jan 9;9(1). Epub 2018 Jan 9.

Department of Biomedical Sciences, University of Padova, Padova 35121, Italy.

Mitochondrial dysfunction is a central event in many pathologies and contributes as well to age-related processes. However, distinguishing between primary mitochondrial dysfunction driving aging and a secondary mitochondrial impairment resulting from other cell alterations remains challenging. Indeed, even though mitochondria undeniably play a crucial role in aging pathways at the cellular and organismal level, the original hypothesis in which mitochondrial dysfunction and production of free radicals represent the main driving force of cell degeneration has been strongly challenged. In this review, we will first describe mitochondrial dysfunctions observed in aged tissue, and how these features have been linked to mitochondrial reactive oxygen species (ROS)-mediated cell damage and mitochondrial DNA (mtDNA) mutations. We will also discuss the clues that led to consider mitochondria as the starting point in the aging process, and how recent research has showed that the mitochondria aging axis represents instead a more complex and multifactorial signaling pathway. New working hypothesis will be also presented in which mitochondria are considered at the center of a complex web of cell dysfunctions that eventually leads to cell senescence and death.
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http://dx.doi.org/10.3390/genes9010022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793175PMC
January 2018

Highlighting the endoplasmic reticulum-mitochondria connection: Focus on Mitofusin 2.

Pharmacol Res 2018 02 5;128:42-51. Epub 2018 Jan 5.

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy; Neuroscience Institute - Italian National Research Council (CNR), Padua, 35121, Italy. Electronic address:

The endoplasmic reticulum (ER) and the mitochondrial network are two highly interconnected cellular structures. By proteinaceous tethers, specialized membrane domains of the ER are tightly associated with the outer membrane of mitochondria, allowing the assembly of signaling platforms where different cell functions take place or are modulated, such as lipid biosynthesis, Ca homeostasis, inflammation, autophagy and apoptosis. The ER-mitochondria coupling is highly dynamic and contacts between the two organelles can be modified in their number, extension and thickness by different stimuli. Importantly, several pathological conditions, such as cancer, neurodegenerative diseases and metabolic syndromes show alterations in this feature, underlining the key role of ER-mitochondria crosstalk in cell physiology. In this contribution, we will focus on one of the major modulator of ER-mitochondria apposition, Mitofusin 2, discussing the structure of the protein and its debated role on organelles tethering. Moreover, we will critically describe different techniques commonly used to investigate this crucial issue, highlighting their advantages, drawbacks and limits.
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http://dx.doi.org/10.1016/j.phrs.2018.01.003DOI Listing
February 2018

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Cell Death Differ 2018 03 11;25(3):542-572. Epub 2017 Dec 11.

Dpto. de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049, Madrid, Spain.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.
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http://dx.doi.org/10.1038/s41418-017-0020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864235PMC
March 2018
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