Peculiarities of ion homeostasis in neurons containing calcium-permeable AMPA receptors.

Glutamate excitotoxicity accompanies numerous brain pathologies, including traumatic brain injury, ischemic stroke, and epilepsy. Disturbances of the ion homeostasis, mitochondria dysfunction, and further cell death are considered the main detrimental consequences of excitotoxicity. It is well known that neurons demonstrate different vulnerability to pathological exposures. In this regard, neurons containing calcium-permeable AMPA receptors (CP-AMPARs) may show higher susceptibility to excitotoxicity due to an additional pathway of Ca2+ influx. Here, we demonstrate that neurons containing CP-AMPARs are characterized by the higher amplitude of the glutamate-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and slower restoration of [Ca2+]i level compared to non-CP-AMPA neurons. Moreover, we have found that NASPM, an antagonist of CP-AMPARs, significantly decreases the amplitude of the [Ca2+]i elevation induced by glutamate or selective AMPARs agonist, 5-fluorowillardiine. In contrast, the antagonists of NMDARs or KARs affect insignificantly. We have also described some peculiarities of Na+, K+, and H+ intracellular dynamics in neurons containing CP-AMPARs. In particular, the amplitude of [Na+]i elevation was lower compared to non-CP-AMPA neurons, whereas the amplitude of [K+]i decrease was higher. We have shown the significant inverse correlation between [K+]i and [Ca2+]i and between intracellular pH and [Na+]i in CP-AMPARs-containing and non-CP-AMPA neurons upon glutamate excitotoxicity. Our data indicate that CP-AMPARs-mediated Ca2+ influx and slow removal of Ca2+ from the cytosol may underlie the vulnerability of the CP-AMPARs-containing neurons to glutamate excitotoxicity. Further studies of the mechanisms mediating the disturbances in ion homeostasis are crucial for developing new approaches for protecting these neurons at brain pathologies.

Impaired Mitochondrial Network Morphology and Reactive Oxygen Species Production in Fibroblasts from Parkinson's Disease Patients.

The mitochondrial network (MN) is a dynamic structure undergoing constant remodeling in the cell. It is assumed that perturbations to the MN may be associated with various pathologies, including Parkinson's disease (PD). Using automatic image analysis and super-resolution microscopy, we have assessed the MN parameters in fibroblasts from patients with established hereditary PD mutations (associated with PINK1, LRRK2, and α-synuclein, as well as PINK1 and Parkin proteins simultaneously) under normal conditions and after hydrogen peroxide-induced stress. Fibroblasts with the Pink1/Parkin mutation are most different in morphology to fibroblasts obtained from conditionally healthy donors: the MN is larger, and it contains longer mitochondria and accumulated individual mitochondria. In addition to MN, we evaluated other cellular parameters, such as cytosolic and mitochondrial ROS production and mitochondrial membrane potential. It has been shown that mitochondria of fibroblasts with mutations in genes encoding PINK1, α-synuclein, and Pink/Parkin tend towards hyperpolarization and cytosolic ROS overproduction, while mitochondrial ROS production was higher only in fibroblasts with PINK1 and α-synuclein mutations.

TR-57 Treatment of SUM159 Cells Induces Mitochondrial Dysfunction without Affecting Membrane Potential.

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones, a new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accompanied 90, 94, 88 and 87% decreases in the content of Complex I, II, III and IV proteins, respectively. The content of the FOF1-ATPase subunits decreased sharply by approximately 35% after 24 h and remained unchanged up to 72 h of incubation with TR-57. At the same time, a disappearance of the ATPIF1, the natural inhibitor of mitochondrial FOF1-ATPase, was observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin did not affect the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 h retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that although TR-57 treatment leads to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of FOF1-ATPase supports mitochondrial polarization.

Dependence of state transitions on illumination time in arabidopsis and barley plants.

Solar energy absorbed by plants can be redistributed between photosystems in the process termed "state transitions" (ST). ST represents a reversible transition of a part of the PSII light harvesting complex (L-LHCII) between photosystem II (PSII) and photosystem I (PSI) in response to the change in light spectral composition. The present work demonstrates a slower development of the state 1 to state 2 transition, i.e., L-LHCII transition from PSII to PSI, in the leaves of dicotyledonous arabidopsis (Arabidopsis thaliana) than in the leaves of monocotyledonous barley (Hordeum vulgare) plants that was assessed by the measurement of chlorophyll a fluorescence at 77 K and of chlorophyll a fluorescence at room temperature. It is known that the first step of the state 1 to state 2 transition is phosphorylation of Lhcb1 and Lhcb2 proteins; however, we detected no difference in the rate of accumulation of these phosphorylated proteins in the studied plants. Therefore, the parameters, which possibly affect the second step of this transition, i.e., the migration of L-LHCII complexes along the thylakoid membrane, were evaluated. Spin-probe EPR measurements demonstrated that the thylakoid membranes viscosity in arabidopsis was higher compared to that in barley. Moreover, confocal microscopy data evidenced the different size of chloroplasts in the leaves of the studied species being larger in arabidopsis. The obtained results suggest that the observed deference in the development of the state 1 to state 2 transition in arabidopsis and barley is caused by the slower L-LHCII migration rate in arabidopsis than in barley plants rather than by the difference in the Lhcb1 and Lhcb2 phosphorylation.

Behaviour of FITC-Labeled Polyallylamine in Polyelectrolyte Microcapsules.

There are many studies devoted to the application of polyelectrolyte microcapsules (PMC) in various fields; however, there are significantly fewer studies devoted to the study of the polyelectrolyte microcapsules themselves. The study examined the mutual arrangement of the polyelectrolytes in 13-layered PMC capsules composed of (PAH/PSS)6PAH. The research showed that different layers of the polyelectrolyte microcapsules dissociate equally, as in the case of 13-layered PMC capsules composed of (PAH/PSS)6PAH with a well-defined shell, and in the case of 7-layered PMC capsules composed of (PAH/PSS)3PAH, where the shell is absent. The study showed that polyallylamine layers labeled with FITC migrate to the periphery of the microcapsule regardless of the number of layers. This is due to an increase in osmotic pressure caused by the rapid flow of ions from the interior of the microcapsule into the surrounding solution. In addition, FITC-polyallylamine has a lower charge density and less interaction with polystyrene sulfonate in the structure of the microcapsule. Meanwhile, the hydrophilicity of FITC-polyallylamine does not change or decreases slightly. The results suggest that this effect promotes the migration of labeled polyallylamine to a more hydrophilic region of the microcapsule, towards its periphery.

Effects of Cobra Cardiotoxins on Intracellular Calcium and the Contracture of Rat Cardiomyocytes Depend on Their Structural Types.

Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca2+ concentration and an assessment of the rat cardiomyocytes' shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed for Naja oxiana cobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.

Regulation of Papillary Muscle Contractility by NAD and Ammonia Interplay: Contribution of Ion Channels and Exchangers.

Various models, including stem cells derived and isolated cardiomyocytes with overexpressed channels, are utilized to analyze the functional interplay of diverse ion currents involved in cardiac automaticity and excitation-contraction coupling control. Here, we used ß-NAD and ammonia, known hyperpolarizing and depolarizing agents, respectively, and applied inhibitory analysis to reveal the interplay of several ion channels implicated in rat papillary muscle contractility control. We demonstrated that: 4 mM ß-NAD, having no strong impact on resting membrane potential (RMP) and action potential duration (APD90) of ventricular cardiomyocytes, evoked significant suppression of isometric force (F) of paced papillary muscle. Reactive blue 2 restored F to control values, suggesting the involvement of P2Y-receptor-dependent signaling in ß-NAD effects. Meantime, 5 mM NH4Cl did not show any effect on F of papillary muscle but resulted in significant RMP depolarization, APD90 shortening, and a rightward shift of I-V relationship for total steady state currents in cardiomyocytes. Paradoxically, NH4Cl, being added after ß-NAD and having no effect on RMP, APD, and I-V curve, recovered F to the control values, indicating ß-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca2+-activated K+ channels (SK, IK, and BK), or NCX exchanger reverse mode prevented this effect, indicating consistent cooperation of all currents mediated by these channels and NCX. We suggest that the activation of Kir2.x and HCN channels by extracellular K+, that creates positive and negative feedback, and known ammonia and K+ resemblance, may provide conditions required for the activation of all the chain of channels involved in the interplay. Here, we present a mechanistic model describing an interplay of channels and second messengers, which may explain discovered antagonism of ß-NAD and ammonia on rat papillary muscle contractile activity.

«One Small Step for Mouse¼: High CO2 Inhalation as a New Therapeutic Strategy for Parkinson's Disease.

Parkinson's disease (PD) is a ubiquitous neurodegenerative disorder for which no effective treatment strategies are available. Existing pharmacotherapy is aimed only at correcting symptoms and slowing the progression of the disease, mainly by replenishing dopamine deficiency. It is assumed that mitochondrial dysfunction plays a key role in the pathogenesis of PD. It has been suggested that activation of specific degradation of damaged mitochondria (mitophagy) may prevent cell death. An almost exclusive way to initiate mitophagy is acidification of intracellular pH. We attempted to implement transient brain acidification using two experimental therapy strategies: forced moderate physical activity and high CO2 inhalation. The beneficial effects of CO2 supplementation on behavioral aspects were demonstrated in a rotenone-induced PD model. Mice treated with CO2 restored their exploratory behavior and total locomotor activity lost after rotenone administration. Additionally, this treatment enabled the removal of impaired coordination. We have illustrated this therapeutic strategy using histological studies of brain sections to confirm the survival of nigrostriatal areas. These findings suggest that high CO2 inhalation presumably initiates mitophagy via transient brain acidification, and can treat PD-like symptoms in a rodent rotenone model of PD.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

Lactate and Pyruvate Activate Autophagy and Mitophagy that Protect Cells in Toxic Model of Parkinson's Disease.

Intracellular quality control regulated by autophagy process is important for maintenance of cellular homeostasis. Deregulation of autophagy and more specifically mitophagy leads to accumulation of the misfolded proteins and damaged mitochondria that in turn leads to the cell loss. Alteration of autophagy and mitophagy has shown to be involved in the number of disorders including neurodegenerative diseases. Autophagy and mitophagy could be activated by short-time acidification of the cytosol; however, most of the compounds which can induce it are toxic. Here, we tested several organic compounds which are involved in cellular metabolism on their ability to change intracellular pH and induce mitophagy/autophagy. We have found that lactate and pyruvate are able to reduce intracellular pH in non-toxic concentrations. Short-term (2 h) and long-term (24 h) incubation of the cells with lactate and pyruvateinduced mitophagy and autophagy. Incubation of the SH-SY5Y cells or primary neurons and astrocytes with lactate or pyruvate also activated mitophagy and autophagy after MPP + treatment that led to recovery of mitochondrial function and protection of these cells against apoptotic and necrotic death. Thus, pyruvate- or lactate-induced acidification of cytosol activates cell protective mitophagy and autophagy.

Metabolically induced intracellular pH changes activate mitophagy, autophagy, and cell protection in familial forms of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder induced by the loss of dopaminergic neurons in midbrain. The mechanism of neurodegeneration is associated with aggregation of misfolded proteins, oxidative stress, and mitochondrial dysfunction. Considering this, the process of removal of unwanted organelles or proteins by autophagy is vitally important in neurons, and activation of these processes could be protective in PD. Short-time acidification of the cytosol can activate mitophagy and autophagy. Here, we used sodium pyruvate and sodium lactate to induce changes in intracellular pH in human fibroblasts with PD mutations (Pink1, Pink1/Park2, α-synuclein triplication, A53T). We have found that both lactate and pyruvate in millimolar concentrations can induce a short-time acidification of the cytosol in these cells. This induced activation of mitophagy and autophagy in control and PD fibroblasts and protected against cell death. Importantly, application of lactate to acute brain slices of WT and Pink1 KO mice also induced a reduction of pH in neurons and astrocytes that increased the level of mitophagy. Thus, acidification of the cytosol by compounds, which play an important role in cell metabolism, can also activate mitophagy and autophagy and protect cells in the familial form of PD.

Dopamine controls neuronal spontaneous calcium oscillations via astrocytic signal.

Dopamine is a neuromodulator and neurotransmitter responsible for a number of physiological processes. Dysfunctions of the dopamine metabolism and signalling are associated with neurological and psychiatric diseases. Here we report that in primary co-culture of neurons and astrocytes dopamine-induces calcium signal in astrocytes and suppress spontaneous synchronous calcium oscillations (SSCO) in neurons. Effect of dopamine on SSCO in neurons was dependent on calcium signal in astrocytes and could be modified by inhibition of dopamine-induced calcium signal or by stimulation of astrocytic calcium rise with ATP. Ability of dopamine to suppress SSCO in neurons was independent on D1- or D2- like receptors but dependent on GABA and alpha-adrenoreceptors. Inhibitor of monoaminoxidase bifemelane blocked effect of dopamine on astrocytes but also inhibited the effect dopamine on SSCO in neurons. These findings suggest that dopamine-induced calcium signal may stimulate release of neuromodulators such as GABA and adrenaline and thus suppress spontaneous calcium oscillations in neurons.

Dissecting Cellular Mechanisms of Long-Chain Acylcarnitines-Driven Cardiotoxicity: Disturbance of Calcium Homeostasis, Activation of Ca2+-Dependent Phospholipases, and Mitochondrial Energetics Collapse.

Long-chain acylcarnitines (LCAC) are implicated in ischemia-reperfusion (I/R)-induced myocardial injury and mitochondrial dysfunction. Yet, molecular mechanisms underlying involvement of LCAC in cardiac injury are not sufficiently studied. It is known that in cardiomyocytes, palmitoylcarnitine (PC) can induce cytosolic Ca2+ accumulation, implicating L-type calcium channels, Na+/Ca2+ exchanger, and Ca2+-release from sarcoplasmic reticulum (SR). Alternatively, PC can evoke dissipation of mitochondrial potential (ΔΨm) and mitochondrial permeability transition pore (mPTP). Here, to dissect the complex nature of PC action on Ca2+ homeostasis and oxidative phosphorylation (OXPHOS) in cardiomyocytes and mitochondria, the methods of fluorescent microscopy, perforated path-clamp, and mitochondrial assays were used. We found that LCAC in dose-dependent manner can evoke Ca2+-sparks and oscillations, long-living Ca2+ enriched microdomains, and, finally, Ca2+ overload leading to hypercontracture and cardiomyocyte death. Collectively, PC-driven cardiotoxicity involves: (I) redistribution of Ca2+ from SR to mitochondria with minimal contribution of external calcium influx; (II) irreversible inhibition of Krebs cycle and OXPHOS underlying limited mitochondrial Ca2+ buffering; (III) induction of mPTP reinforced by PC-calcium interplay; (IV) activation of Ca2+-dependent phospholipases cPLA2 and PLC. Based on the inhibitory analysis we may suggest that simultaneous inhibition of both phospholipases could be an effective strategy for protection against PC-mediated toxicity in cardiomyocytes.

Correction: Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation.

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of ß-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Role of DJ-1 in the mechanism of pathogenesis of Parkinson's disease.

DJ-1 protein has multiple specific mechanisms to protect dopaminergic neurons against neurodegeneration in Parkinson's disease. Wild type DJ-1 can acts as oxidative stress sensor and as an antioxidant. DJ-1 exhibits the properties of molecular chaperone, protease, glyoxalase, transcriptional regulator that protects mitochondria from oxidative stress. DJ-1 increases the expression of two mitochondrial uncoupling proteins (UCP 4 and UCP5), that decrease mitochondrial membrane potential and leads to the suppression of ROS production, optimizes of a number of mitochondrial functions, and is regarded as protection for the neuronal cell survival. We discuss also the stabilizing interaction of DJ-1 with the mitochondrial Bcl-xL protein, which regulates the activity of (Inositol trisphosphate receptor) IP3R, prevents the cytochrome c release from mitochondria and inhibits the apoptosis activation. Upon oxidative stress DJ-1 is able to regulate various transcription factors including nuclear factor Nrf2, PI3K/PKB, and p53 signal pathways. Stress-activated transcription factor Nrf2 regulates the pathways to protect cells against oxidative stress and metabolic pathways initiating the NADPH and ATP production. DJ-1 induces the Nrf2 dissociation from its inhibitor Keap1 (Kelch-like ECH-associated protein 1), promoting Nrf2 nuclear translocation and binding to antioxidant response elements. DJ-1 is shown to be a co-activator of the transcription factor NF-kB. Under nitrosative stress, DJ-1 may regulate PI3K/PKB signaling through PTEN transnitrosylation, which leads to inhibition of phosphatase activity. DJ-1 has a complex modulating effect on the p53 pathway: one side DJ-1 directly binds to p53 to restore its transcriptional activity and on the other hand DJ-1 can stimulate deacylation and suppress p53 transcriptional activity. The ability of the DJ-1 to induce activation of different transcriptional factors and change redox balance protect neurons against aggregation of α-synuclein and oligomer-induced neurodegeneration.

α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease.

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.

Interaction of misfolded proteins and mitochondria in neurodegenerative disorders.

The number of the people affected by neurodegenerative disorders is growing dramatically due to the ageing of population. The major neurodegenerative diseases share some common pathological features including the involvement of mitochondria in the mechanism of pathology and misfolding and the accumulation of abnormally aggregated proteins. Neurotoxicity of aggregated ß-amyloid, tau, α-synuclein and huntingtin is linked to the effects of these proteins on mitochondria. All these misfolded aggregates affect mitochondrial energy metabolism by inhibiting diverse mitochondrial complexes and limit ATP availability in neurones. ß-Amyloid, tau, α-synuclein and huntingtin are shown to be involved in increased production of reactive oxygen species, which can be generated in mitochondria or can target this organelle. Most of these aggregated proteins are capable of deregulating mitochondrial calcium handling that, in combination with oxidative stress, lead to opening of the mitochondrial permeability transition pore. Despite some of the common features, aggregated ß-amyloid, tau, α-synuclein and huntingtin have diverse targets in mitochondria that can partially explain neurotoxic effect of these proteins in different brain regions.