Ncx3-induced mitochondrial dysfunction in midbrain leads to neuroinflammation in striatum of A53t-α-synuclein transgenic old mice

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson’s disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging

AMOXICILLIN AND CLAVULANIC ACID VS CEFTAZIDIME IN THE SURGICAL EXTRACTION OF IMPACTED THIRD MOLAR: A COMPARATIVE STUDY

The objective of this work is to compare the effectiveness and the side effects of two different drugs, amoxicillin and clavulanic acid vs ceftazidime, used as antibiotic prophylaxis in the surgical extraction of third molars and to demonstrate that the use of second choice antibiotic has no significant advantages in comparison with a first choice antibiotic. One hundred and seven patients with impacted third molar were selected and divided into two groups: amoxicillin and clavulanic acid were administered to group 1 and ceftazidime to group 2 for five days after surgery and we observed the postoperative period. The statistical analysis showed no differences between the two groups which lead to the conclusion that there is no indication to routinely administrate intramuscular second-choice antibiotic prophylactic therapy (ceftazidime) in case of surgical extraction of the third molar

Nuclear-encoded NCX3 and AKAP121: Two novel modulators of mitochondrial calcium efflux in normoxic and hypoxic neurons

Mitochondria are highly dynamic organelles extremely important for cell survival. Their structure resembles that of prokaryotic cells since they are composed with two membranes, the inner (IMM) and the outer mitochondrial membrane (OMM) delimitating the intermembrane space (IMS) and the matrix which contains mitochondrial DNA (mtDNA). This structure is strictly related to mitochondrial function since they produce the most of the cellular ATP through the oxidative phosphorylation which generate the electrochemical gradient at the two sides of the inner mitochondrial membrane an essential requirement for mitochondrial function. Cells of highly metabolic demand like those composing muscle, liver and brain, are particularly dependent on mitochondria for their activities. Mitochondria undergo to continual changes in morphology since, they fuse and divide, branch and fragment, swell and extend. Importantly, they move throughout the cell to deliver ATP and other metabolites where they are mostly required. Along with the capability to control energy metabolism, mitochondria play a critical role in the regulation of many physiological processes such as programmed cell death, autophagy, redox signalling, and stem cells reprogramming. All these phenomena are regulated by Ca2+ ions within this organelle. This review will discuss the molecular mechanisms regulating mitochondrial calcium cycling in physiological and pathological conditions with particular regard to their impact on mitochondrial dynamics and function during ischemia. Particular emphasis will be devoted to the role played by NCX3 and AKAP121 as new molecular targets for mitochondrial function and dysfunction

Inhibition of autophagy by chloroquine prevents resistance to PI3K/AKT inhibitors and potentiates their antitumor effect in combination with paclitaxel in triple negative breast cancer models

Background: Triple negative breast cancer (TNBC) is an aggressive disease characterized by high risk of relapse and development of resistance to different chemotherapy agents. Several targeted therapies have been investigated in TNBC with modest results in clinical trials. Among these, PI3K/AKT inhibitors have been evaluated in addition to standard therapies, yielding conflicting results and making attempts on elucidating inherent mechanisms of resistance of great interest. Increasing evidences suggest that PI3K/AKT inhibitors can induce autophagy in different cancers. Autophagy represents a supposed mechanism of drug-resistance in aggressive tumors, like TNBC. We, therefore, investigated if two PI3K/AKT inhibitors, ipatasertib and taselisib, could induce autophagy in breast cancer models, and whether chloroquine (CQ), a well known autophagy inhibitor, could potentiate ipatasertib and taselisib anti-cancer effect in combination with conventional chemotherapy. Methods: The induction of autophagy after ipatasertib and taselisib treatment was evaluated in MDAMB231, MDAM468, MCF7, SKBR3 and MDAB361 breast cancer cell lines by assaying LC3-I conversion to LC3-II through immunoblotting and immunofluorescence. Other autophagy-markers as p62/SQSTM1 and ATG5 were evaluated by immunoblotting. Synergistic antiproliferative effect of double and triple combinations of ipatasertib/taselisib plus CQ and/or paclitaxel were evaluated by SRB assay and clonogenic assay. Anti-apoptotic effect of double combination of ipatasertib/taselisib plus CQ was evaluated by increased cleaved-PARP by immunoblot and by Annexin V- flow cytometric analysis. In vivo experiments were performed on xenograft model of MDAMB231 in NOD/SCID mice. Results: Our results suggested that ipatasertib and taselisib induce increased autophagy signaling in different breast cancer models. This effect was particularly evident in PI3K/AKT resistant TNBC cells, where the inhibition of autophagy by CQ potentiates the therapeutic effect of PI3K/AKT inhibitors in vitro and in vivo TNBC models, synergizing with taxane-based chemotherapy. Conclusion: These data suggest that inhibition of authophagy with CQ could overcome mechanism of drug resistance to PI3K/AKT inhibitors plus paclitaxel in TNBC making the evaluation of such combinations in clinical trials warranted

Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging

20The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.nonePunzo, Daniela; Errico, Francesco; Cristino, Luigia; Sacchi, Silvia; Keller, Simona; Belardo, Carmela; Luongo, Livio; Nuzzo, Tommaso; Imperatore, Roberta; Florio, Ermanno; De Novellis, Vito; Affinito, Ornella; Migliarini, Sara; Maddaloni, Giacomo; Sisalli, Maria Josè; Pasqualetti, Massimo; Pollegioni, Loredano; Maione, Sabatino; Chiariotti, Lorenzo; Usiello, AlessandroPunzo, Daniela; Errico, Francesco; Cristino, Luigia; Sacchi, Silvia; Keller, Simona; Belardo, Carmela; Luongo, Livio; Nuzzo, Tommaso; Imperatore, Roberta; Florio, Ermanno; De Novellis, Vito; Affinito, Ornella; Migliarini, Sara; Maddaloni, Giacomo; Sisalli, Maria Josè; Pasqualetti, Massimo; Pollegioni, Loredano; Maione, Sabatino; Chiariotti, Lorenzo; Usiello, Alessandr

Kv7.4 channels regulate potassium permeability in neuronal mitochondria

Mitochondrial K+ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia–reperfusion injury. Kv7.4 channels have been recently shown to regulate K+ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca2+ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons. In coupled mitochondria isolated from F11 cells, K+-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBKCa channel blocker iberiotoxin and only partially inhibited by the mitoKATP blockers glyburide or ATP. Interestingly, K+-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca2+ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K+ permeability of neuronal mitochondria

Tracking the evolution of epialleles during neural differentiation and brain development: D-Aspartate oxidase as a model gene

We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases

Involvement of the sodium–calcium exchanger 3 (NCX3) in ziram-induced calcium dysregulation and toxicity

Ziram is a dimethyldithiocarbamate fungicide which can cause intraneuronal calcium (Ca(2+)) dysregulation and subsequently neuronal death. The signaling mechanisms underlying ziram-induced Ca(2+) dyshomeostasis and neurotoxicity are not fully understood. NCX3 is the third isoform of the sodium-calcium exchanger (NCX) family and plays an important role in regulating Ca(2+) homeostasis in excitable cells. We previously generated a mouse model deficient for the sodium-calcium exchanger 3 and showed that NCX3 is protective against ischemic damage. In the present study, we aim to examine whether NCX3 exerts a similar role against toxicological injury caused by the pesticide ziram. Our data show baby hamster kidney (BHK) cells stably transfected with NCX3 (BHK-NCX3) are more susceptible to ziram toxicity than cells transfected with the empty vector (BHK-WT). Increased toxicity in BHK-NCX3 was associated with a rapid rise in cytosolic Ca(2+) concentration [Ca(2+)(i)] induced by ziram. Profound mitochondrial dysfunction and ATP depletion were also observed in BHK-NCX3 cells following treatment with ziram. Lastly, primary dopaminergic neurons lacking NCX3 (NCX3(−/−)) were less sensitive to ziram neurotoxicity than wildtype control dopaminergic neurons. These results demonstrate that NCX3 genetic deletion protects against ziram-induced neurotoxicity and suggest NCX3 and its downstream molecular pathways as key factors involved in ziram toxicity. Our study identifies new molecular events through which pesticides (e.g. ziram) can lead to pathological features of degenerative diseases such as Parkinson’s disease and indicates new targets to slow down neuronal degeneration