The P2X7 receptor (P2X7R) is an ATP-gated ion channel belonging to the purinergic ligand-gated P2X receptor family. In the central nervous system (CNS), activation of this receptor has been proposed to play a key role in the pathogenesis of various neurodegenerative disorders. Its expression has been clearly demonstrated in microglia, where it regulates numerous cellular processes, including cell activation, cytokine release, and calcium signaling. Recent data show convincing arguments for the presence of P2X7R also in other cell types of the nervous system, such as astrocytes, oligodendrocytes, oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs), although there is still some debate. The most controversial, however, is the presence and role of P2X7R in neurons. In this review, we aim to critically address this question by examining the current literature in the context of the available tools. We revisit the pharmacological regimen required to confirm the functional expression of the receptor and the mouse models that have aided in the investigation of neuronal P2X7R. Finally, we discuss some of the hypothesized contributions of neuronal P2X7R in CNS disorders.

Neuroinflammation and synaptic dysfunction are emerging as early and potentially causative events in Alzheimer’s disease (AD), yet their molecular triggers remain elusive. Here, we identify extracellular ATP (eATP), a major damage-associated molecular pattern, and its purinergic receptor P2X7 (P2X7R) as pivotal drivers of early pathology in AD mice. In vivo bioluminescence imaging revealed a significant cortical accumulation of eATP in AD mice as early as 2 months of age—before amyloid plaque deposition and cognitive impairment. This increase is associated with inflammasome activation, pro-inflammatory cytokine production, microglia reactivity, aberrant synaptic pruning and perineuronal net degradation. Strikingly, genetic deletion of P2X7R rescues these alterations. Two-photon calcium imaging further demonstrates that P2X7R knockout counteracts AD-related neuronal hyperactivity. These findings set the eATP–P2X7R signaling axis as an early driver of AD pathology, linking neuroinflammation to synaptic remodeling and circuit dysfunction, and suggest P2X7R inhibition as a compelling strategy to counteract AD progression. ### Competing Interest Statement The authors have declared no competing interest. Italian Ministry of University and Scientific Research, PRIN2022943TH9, PRIN20225R4Y5, Euro-BioImaging FOE Cure Alzheimer’s Fund, NA European Union – NextGenerationEU, NRRP-National Recovery and Resilience Plan grant, National Centre of Research “Development of gene therapy and drugs with RNA technology,” CN3 “Neurodegenerative Diseases” to P.P. (NextGenerationEU); and CODBAN_000428, CUP J73C24000090007, Spoke 3 – M4C2 – Inv.1.4 Prog. CN00000041, NRRP-National Recovery and Resilience Plan grant, National Centre of Research “Development of gene therapy and drugs with RNA technology,” CN3 “Neurodegenerative Diseases”, D11\_PNRR\_CN3\_S3\_2, Project code IR0000023, Concession Decree No. 101/2022 adopted by the Italian MUR, CUP B53C22001810006, “SEELIFE - Strengthening the Italian Infrastructure of Euro-Bioimaging" European Union’s Horizon 2020 and European Union – NextGenerationEU, Marie Sklodowaka-Curie grant agreement no 101034319 Alzheimer’s Association, E2A-23-1148250

Neurons are bound to maintain a tight control over their cellular homeostasis in order to preserve their many functions, and specifically to precisely regulate ion fluxes for the generation of action potential and neurotransmission. In particular, the energetic balance has to be finely adjusted since its alteration may hamper cellular functionality with consequent cell suffering and death. Mitochondria play a central role in the homeostatic regulation within the cells due to the many vital tasks they perform in terms of energy production, participation in the regulation of Ca 2+ concentration, modulation of intracellular signaling that allow the cell to respond to external solicitation Primary hippocampal neuronal cultures of 14-17 DIV from WT and AD mouse model were challenged with μM concentration of glutamate to induce a mild excitotoxic effect; mitochondrial membrane potential, mitochondrial an cytosolic Ca 2+ transients and cytosolic ATP levels were evaluated with fluorescent probes. Cells were treated also with inhibitors of the permeability transition pore to evaluate its effect on cell functionality We observed that in a model of Familial Alzheimer’s disease, hippocampal neurons are sensitive to a modest excitotoxic signal that produces a transient cytosolic and mitochondrial matrix Ca 2+ with consequent partial loss of mitochondrial membrane potential and delayed recovery of mitochondrial matrix [Ca 2+ ] which might contribute in the activation of the mitochondria permeability transition pore and consequent impairment of mitochondrial and cellular functions. This is accompanied by a reduced cytosolic ATP content that can result in an energy deficit for the cell. A brief treatment with inhibitors of the permeability transition pore is able to reduce these effects and might have protective roles against exogenous insults to the cells and help mitigate the neuronal loss characteristic of Alzheimer’s disease. AD hippocampal neurons are more sensitive to a mild excitotoxic insult that affect mitochondrial functionality and in particular the activation of the permeability transition pore (PTP). Treatment with μM concentrations of two different inhibitors of the PTP prevent mitochondrial impairment.

In Alzheimer's disease (AD), the molecular mechanisms involved in the neurodegeneration are still incompletely defined, though this aspect is crucial for a better understanding of the malady and for devising effective therapies. Mitochondrial dysfunctions and altered Ca2+ signaling have long been implicated in AD, though it is debated whether these events occur early in the course of the pathology, or whether they develop at late stages of the disease and represent consequences of different alterations. Mitochondria are central to many aspects of cellular metabolism providing energy, lipids, reactive oxygen species, signaling molecules for cellular quality control, and actively shaping intracellular Ca2+ signaling, modulating the intensity and duration of the signal itself. Abnormalities in the ability of mitochondria to take up and subsequently release Ca2+ could lead to changes in the metabolism of the organelle, and of the cell as a whole, that eventually result in cell death. We sought to investigate the role of mitochondria and Ca2+ signaling in a model of Familial Alzheimer's disease and found early alterations in mitochondria physiology under stressful condition, namely, reduced maximal respiration, decreased ability to sustain membrane potential, and a slower return to basal matrix Ca2+ levels after a mild excitotoxic stimulus. Treatment with an inhibitor of the permeability transition pore attenuated some of these mitochondrial disfunctions and may represent a promising tool to ameliorate mitochondria and cellular functioning in AD and prevent or slow down cell loss in the disease.

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 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).

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-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.

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.

Herein, we report on the synthesis of a small set of linear precursors of an inosine analogue of cyclic ADP-ribose (cADPR), a second messenger involved in Ca2+ mobilization from ryanodine receptor stores firstly isolated from sea urchin eggs extracts. The synthesized compounds were obtained starting from inosine and are characterized by an N1-alkyl chain replacing the “northern” ribose and a phosphate group attached at the end of the N1-alkyl chain and/or 5′-sugar positions. Preliminary Ca2+ mobilization assays, performed on differentiated C2C12 cells, are reported as well.

Metabolic disorders are severe and chronic impairments of the health of many people and represent a challenge for the society as a whole that has to deal with an ever-increasing number of affected individuals. Among common metabolic disorders are Alzheimer's disease, obesity, and type 2 diabetes. These disorders do not have a univocal genetic cause but rather can result from the interaction of multiple genes, lifestyle, and environmental factors. Mitochondrial alterations have emerged as a feature common to all these disorders, underlining perhaps an impaired coordination between cellular needs and mitochondrial responses that could contribute to their development and/or progression.

Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.

Mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU) is a tightly controlled process that sustains cell functions mainly by fine-tuning oxidative metabolism to cellular needs. The kinetics of Ca2+ fluxes across the mitochondrial membranes have been studied both in vitro and in vivo for many years, and the discovery of the molecular components of the MCU has further clarified that this Ca2+ uptake mechanism is based on a complex system subject to elaborate layers of controls. Alterations in the speed or capacity of the in-and-out pathways can have detrimental consequences for both the organelle and the cell, impairing cellular metabolism and ultimately causing cell death. Here, we report that pretreatment of deenergized mitochondria with low-micromolar Ca2+ concentrations for a few minutes markedly increases the speed of mitochondrial Ca2+ uptake upon re-addition of an oxidizable substrate. We found that this phenomenon is sensitive to alterations in the level of the MCU modulator proteins mitochondrial calcium uptake 1 (MICU1) and 2 (MICU2), and is accompanied by changes in the association of MICU1-MICU2 complexes with MCU. This increased Ca2+ uptake capacity, occurring under conditions mimicking those during ischemia/reperfusion in vivo, could lead to a massive amount of Ca2+ entering the mitochondrial matrix even at relatively low levels of cytosolic Ca2+. We conclude that the phenomenon uncovered here represents a potential threat of mitochondrial Ca2+ overload to the cell.

The privileged physical connection between ER and mitochondria plays an important role in fundamental cellular processes, such as maintenance of Ca2+ and lipid homeostasis, regulation of autophagy and life-or-death decisions. We previously demonstrated that Presenilin-2 (PS2) Familial Alzheimer's disease (FAD)-linked mutants dampen ER [Ca2+] and favor ER-mitochondria physical/functional juxtaposition (Brunello et al. 2009; Zampese et al. 2011). The latter effect is due to the PS2 capacity to bind and inhibit Mitofusin-2, a master regulator of ER-mitochondria tethering (Filadi et al. 2016). Here we investigate the impact of these PS2-mutants-induced effects on cell functionalities, such as mitochondrial activity and dynamics, bioenergetics, cell death and autophagy. FAD-PS2-expressing SH-SY5Y cells, as well as fibroblasts from FAD-PS2 patients or primary cortical/hippocampal neurons from wt or transgenic (tg) FAD-PS2-N141I/APPswe mice were tested for: Oxygen Consumption Rate (OCR) by the Seahorse platform; mitochondrial membrane potential by TMRM measurements; ATP levels (within the nucleus or mitochondrial matrix) by specific genetically encoded ATP probes. Apoptosis sensitivity and autophagy flux were evaluated in SH-SY5Y cells and human FAD fibroblasts by standard biochemical and immunofluorescence-based techniques. Axonal mitochondrial transport was studied in vivo in zebrafish Rohon-Bear sensory neurons expressing a mitochondrial fluorescent protein. Cell respiration and ATP production are reduced in FAD-PS2 experimental models, especially when cells were forced towards high energy requiring pathways. The FAD-PS2-induced Ca2+ dysregulation contributes to these features. These results suggest an unbalanced cell metabolism, potentially connected to impairments in mitochondrial functionality. Moreover, altered apoptosis sensitivity and blocked autophagy are found in FAD-PS2-T122R expressing SH-SY5Y cells and human FAD fibroblasts. The latter pathway results dysfunctional because of a defective processing in the late autophagy phases. Finally, in vivo mitochondrial axonal transport results also affected by FAD-PS2 expression. Our FAD-PS2 models reveal defects in a variety of cell functionalities. Their relationship with the previously described effects of FAD-PS2 expression on Ca2+ homeostasis and ER-mitochondria coupling is suggested. The possibility that the above alterations, more evident in energy-challenged conditions, could be linked to the pathogenicity of FAD-PS2 mutants is discussed.

A typical characteristic of eukaryotic cells compared to prokaryotes is represented by the spatial heterogeneity of the different structural and functional components: for example, most of the genetic material is surrounded by a highly specific membrane structure (the nuclear membrane), continuous with, yet largely different from, the endoplasmic reticulum (ER); oxidative phosphorylation is carried out by organelles enclosed by a double membrane, the mitochondria; in addition, distinct domains, enriched in specific proteins, are present in the plasma membrane (PM) of most cells. Less obvious, but now generally accepted, is the notion that even the concentration of small molecules such as second messengers (Ca2+ and cAMP in particular) can be highly heterogeneous within cells. In the case of most organelles, the differences in the luminal levels of second messengers depend either on the existence on their membrane of proteins that allow the accumulation/release of the second messenger (e.g., in the case of Ca2+, pumps, exchangers or channels), or on the synthesis and degradation of the specific molecule within the lumen (the autonomous intramitochondrial cAMP system). It needs stressing that the existence of a surrounding membrane does not necessarily imply the existence of a gradient between the cytosol and the organelle lumen. For example, the nuclear membrane is highly permeable to both Ca2+ and cAMP (nuclear pores are permeable to solutes up to 50 kDa) and differences in [Ca2+] or [cAMP] between cytoplasm and nucleoplasm are not seen in steady state and only very transiently during cell activation. A similar situation has been observed, as far as Ca2+ is concerned, in peroxisomes.

Dopaminergic neurons of the substantia nigra selectively degenerate over the course of Parkinson's disease. These neurons are also the most heavily pigmented cells of the brain, accumulating the dark pigment neuromelanin over a lifetime. The massive presence of neuromelanin in these brain areas has long been suspected as a key factor involved in the selective vulnerability of neurons. The high concentration of neuromelanin in substantia nigra neurons seems to be linked to the presence of considerable amounts of cytosolic dopamine that have not been sequestered into synaptic vesicles. Over the past few years, studies have uncovered a dual nature of neuromelanin. Intraneuronal neuromelanin can be a protective factor, shielding the cells from toxic effects of redox active metals, toxins, and excess of cytosolic catecholamines. In contrast, neuromelanin released by dying neurons can contribute to the activation of neuroglia triggering the neuroinflammation that characterizes Parkinson's disease. This article reviews recent studies on the molecular aspects of neuromelanin of the human substantia nigra.

We have studied the pathways for Ca(2+) transport in mitochondria of the fruit fly Drosophila melanogaster. We demonstrate the presence of ruthenium red (RR)-sensitive Ca(2+) uptake, of RR-insensitive Ca(2+) release, and of Na(+)-stimulated Ca(2+) release in energized mitochondria, which match well characterized Ca(2+) transport pathways of mammalian mitochondria. Following larger matrix Ca(2+) loading Drosophila mitochondria underwent spontaneous RR-insensitive Ca(2+) release, an event that in mammals is due to opening of the permeability transition pore (PTP). Like the PTP of mammals, Drosophila Ca(2+)-induced Ca(2+) release could be triggered by uncoupler, diamide, and N-ethylmaleimide, indicating the existence of regulatory voltage- and redox-sensitive sites and was inhibited by tetracaine. Unlike PTP-mediated Ca(2+) release in mammals, however, it was (i) insensitive to cyclosporin A, ubiquinone 0, and ADP; (ii) inhibited by P(i), as is the PTP of yeast mitochondria; and (iii) not accompanied by matrix swelling and cytochrome c release even in KCl-based medium. We conclude that Drosophila mitochondria possess a selective Ca(2+) release channel with features intermediate between the PTP of yeast and mammals.

We have studied mitochondrial Ca2+ transport and the permeability transition (PT) in the teleost zebrafish (Danio rerio), a key model system for human diseases. Permeabilized zebrafish embryo cells displayed a mitochondrial energy-dependent Ca2+ uptake system that, like the Ca2+ uniporter of mammals, was inhibited by ruthenium red. Zebrafish mitochondria underwent a Ca2+-dependent PT that displayed Pi-dependent desensitization by cyclosporin A, and responded appropriately to key modulators of the mammalian PT pore (voltage, pH, ubiquinone 0, dithiol oxidants and cross linkers, ligands of the adenine nucleotide translocator, arachidonic acid). Opening of the pore was documented in intact cells, where it led to death that could largely be prevented by cyclosporin A. Our results represent a necessary step toward the use of zebrafish for the screening and validation of PTP inhibitors of potential use in human diseases, as recently shown for collagen VI muscular dystrophy [Telfer et al., 2010].

Blue native gel electrophoresis purification and immunoprecipitation of F(0)F(1)-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F(1) sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.

Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention.