The NADase CD38 may not dictate NAD levels in brain mitochondria of aged mice but regulates hydrogen peroxide generation.

Aging is a time-related functional decline that affects many species. One of the hallmarks of aging is mitochondrial dysfunction, which leads to metabolic decline. The NAD decline during aging, in several tissues, correlates with increase in NADase activity of CD38. Knock out or pharmacological inhibition of CD38 activity can rescue mitochondrial function in several tissues, however, the role of CD38 in controlling NAD levels and metabolic function in the aging brain is unknown. In this work, we investigated CD38 NADase activity controlling NAD levels and mitochondrial function in mice brain with aging. We demonstrate that NADase activity of CD38 does not dictate NAD total levels in brain of aging mice and does not control mitochondrial oxygen consumption nor other oxygen parameters markers of mitochondrial dysfunction. However, for the first time we show that CD38 regulates hydrogen peroxide (H2O2) generation, one of the reactive oxygen species (ROS) in aging brain, through regulation of pyruvate dehydrogenase and alfa-ketoglutarate dehydrogenase, as mitochondria H2O2 leakage sites. The effect may be related to mitochondrial calcium handling differences in CD38 absence. Our study highlights a novel role of CD38 in brain energy metabolism and aging.

Physical exercise improves mitochondrial function in ovariectomized rats.

Estrogen deficiency causes metabolic disorders in humans and rodents, including in part due to changes in energy expenditure. We have shown previously that skeletal muscle mitochondrial function is compromised in ovariectomized (Ovx) rats. Since physical exercise is a powerful strategy to improve skeletal muscle mitochondrial content and function, we hypothesize that exercise training would counteract the deficiency-induced skeletal muscle mitochondrial dysfunction in Ovx rats. We report that exercised Ovx rats, at 60-65% of maximal exercise capacity for 8 weeks, exhibited less fat accumulation and body weight gain compared with sedentary controls. Treadmill exercise training decreased muscle lactate production, indicating a shift to mitochondrial oxidative metabolism. Furthermore, reduced soleus muscle mitochondrial oxygen consumption confirmed that estrogen deficiency is detrimental to mitochondrial function. However, exercise restored mitochondrial oxygen consumption in Ovx rats, achieving similar levels as in exercised control rats. Exercise-induced skeletal muscle peroxisome proliferator-activated receptor-γ coactivator-1α expression was similar in both groups. Therefore, the mechanisms by which exercise improves mitochondrial oxygen consumption appears to be different in Ovx-exercised and sham-exercised rats. While there was an increase in mitochondrial content in sham-exercised rats, demonstrated by a greater citrate synthase activity, no induction was observed in Ovx-exercised rats. Normalizing mitochondrial respiratory capacity by citrate synthase activity indicates a better oxidative phosphorylation efficiency in the Ovx-exercised group. In conclusion, physical exercise sustains mitochondrial function in ovarian hormone-deficient rats through a non-conventional mitochondrial content-independent manner.

Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca2+ Handling and Ca2+-Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion.

Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca2+ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca2+ (Ca2+ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca2+-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca2+ load and t1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury-thus favoring Ca2+ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle-which feed the mitochondrial complexes with the electrons removed from different substrates-as the result of accentuated stimulus of citrate synthase activity by BMMC.

Coupling of GABA Metabolism to Mitochondrial Glucose Phosphorylation.

Glucose and oxygen (O2) are vital to the brain. Glucose metabolism and mitochondria play a pivotal role in this process, culminating in the increase of reactive O2 species. Hexokinase (HK) is a key enzyme on glucose metabolism and is coupled to the brain mitochondrial redox modulation by recycling ADP for oxidative phosphorylation (OXPHOS). GABA shunt is an alternative pathway to GABA metabolism that increases succinate levels, a Krebs cycle intermediate. Although glucose and GABA metabolisms are intrinsically connected, their interplay coordinating mitochondrial function is poorly understood. Here, we hypothesize that the HK and the GABA shunt interact to control mitochondrial metabolism differently in the cortex and the hypothalamus. The GABA shunt stimulated mitochondrial O2 consumption and H2O2 production higher in hypothalamic synaptosomes (HSy) than cortical synaptosomes (CSy). The GABA shunt increased the HK coupled to OXPHOS activity in both population of synaptosomes, but the rate of activation was higher in HSy than CSy. Significantly, malonate and vigabatrin blocked the effects of the GABA shunt in the HK activity coupled to OXPHOS. It indicates that the glucose phosphorylation is linked to GABA and Krebs cycle reactions. Together, these data shed light on the HK and SDH role on the metabolism of each region fed by GABA turnover, which depends on the neurons' metabolic route.

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells.

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D1R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H2O2 and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.

3-Bromopyruvate: A new strategy for inhibition of glycolytic enzymes in Leishmania amazonensis.

The compound 3-bromopyruvate (3-BrPA) is well-known and studies from several researchers have demonstrated its involvement in tumorigenesis. It is an analogue of pyruvic acid that inhibits ATP synthesis by inhibiting enzymes from the glycolytic pathway and oxidative phosphorylation. In this work, we investigated the effect of 3-BrPA on energy metabolism of L. amazonensis. In order to verify the effect of 3-BrPA on L. amazonensis glycolysis, we measured the activity level of three glycolytic enzymes located at different points of the pathway: (i) glucose kinases, step 1, (ii) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), step 6, and (iii) enolase, step 9. 3-BrPA, in a dose-dependent manner, significantly reduced the activity levels of all the enzymes. In addition, 3-BrPA treatment led to a reduction in the levels of phosphofruto-1-kinase (PFK) protein, suggesting that the mode of action of 3-BrPA involves the downregulation of some glycolytic enzymes. Measurement of ATP levels in promastigotes of L. amazonensis showed a significant reduction in ATP generation. The O2 consumption was also significantly inhibited in promastigotes, confirming the energy depletion effect of 3-BrPA. When 3-BrPA was added to the cells at the beginning of growth cycle, it significantly inhibited L. amazonensis proliferation in a dose-dependent manner. Furthermore, the ability to infect macrophages was reduced by approximately 50% when promastigotes were treated with 3-BrPA. Taken together, these studies corroborate with previous reports which suggest 3-BrPA as a potential drug against pathogenic microorganisms that are reliant on glucose catabolism for ATP supply.

Mitotherapy: Unraveling a Promising Treatment for Disorders of the Central Nervous System and Other Systemic Conditions.

Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer's disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.

Mortality of septic shock patients is associated with impaired mitochondrial oxidative coupling efficiency in lymphocytes: a prospective cohort study.

BACKGROUND: Septic shock is a life-threatening condition that challenges immune cells to reprogram their mitochondrial metabolism towards to increase ATP synthesis for building an appropriate immunity. This could print metabolic signatures in mitochondria whose association with disease progression and clinical outcomes remain elusive. METHOD: This is a single-center prospective cohort study performed in the ICU of one tertiary referral hospital in Brazil. Between November 2017 and July 2018, 90 consecutive patients, aged 18 years or older, admitted to the ICU with septic shock were enrolled. Seventy-five patients had Simplified Acute Physiology Score (SAPS 3) assessed at admission, and Sequential Organ Failure Assessment (SOFA) assessed on the first (D1) and third (D3) days after admission. Mitochondrial respiration linked to complexes I, II, V, and biochemical coupling efficiency (BCE) were assessed at D1 and D3 and Δ (D3-D1) in isolated lymphocytes. Clinical and mitochondrial endpoints were used to dichotomize the survival and death outcomes. Our primary outcome was 6-month mortality, and secondary outcomes were ICU and hospital ward mortality. RESULTS: The mean SAPS 3 and SOFA scores at septic shock diagnosis were 75.8 (± 12.9) and 8 (± 3) points, respectively. The cumulative ICU, hospital ward, and 6-month mortality were 32 (45%), 43 (57%), and 50 (66%), respectively. At the ICU, non-surviving patients presented elevated arterial lactate (2.8 mmol/L, IQR, 2-4), C-reactive protein (220 mg/L, IQR, 119-284), and capillary refill time (5.5 s, IQR, 3-8). Respiratory rates linked to CII at D1 and D3, and ΔCII were decreased in non-surviving patients. Also, the BCE at D1 and D3 and the ΔBCE discriminated patients who would evolve to death in the ICU, hospital ward, and 6 months after admission. After adjusting for possible confounders, the ΔBCE value but not SOFA scores was independently associated with 6-month mortality (RR 0.38, CI 95% 0.18-0.78; P = 0.009). At a cut-off of - 0.002, ΔBCE displayed 100% sensitivity and 73% specificity for predicting 6-month mortality CONCLUSIONS: The ΔBCE signature in lymphocytes provided an earlier recognition of septic shock patients in the ICU at risk of long-term deterioration of health status.

Mesenchymal Stromal Cells From Emphysematous Donors and Their Extracellular Vesicles Are Unable to Reverse Cardiorespiratory Dysfunction in Experimental Severe Emphysema.

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) from patients with chronic obstructive pulmonary disease (COPD) appear to be phenotypically and functionally similar to BM-MSCs from healthy sources in vitro, the impact of COPD on MSC metabolism and mitochondrial function has not been evaluated. In this study, we aimed to comparatively characterize MSCs from healthy and emphysematous donors (H-MSCs and E-MSCs) in vitro and to assess the therapeutic potential of these MSCs and their extracellular vesicles (H-EVs and E-EVs) in an in vivo model of severe emphysema. For this purpose, C57BL/6 mice received intratracheal porcine pancreatic elastase once weekly for 4 weeks to induce emphysema; control animals received saline under the same protocol. Twenty-four hours after the last instillation, animals received saline, H-MSCs, E-MSCs, H-EVs, or E-EVs intravenously. In vitro characterization demonstrated that E-MSCs present downregulation of anti-inflammatory (TSG-6, VEGF, TGF-ß, and HGF) and anti-oxidant (CAT, SOD, Nrf2, and GSH) genes, and their EVs had larger median diameter and lower average concentration. Compared with H-MSC, E-MSC mitochondria also exhibited a higher respiration rate, were morphologically elongated, expressed less dynamin-related protein-1, and produced more superoxide. When co-cultured with alveolar macrophages, both H-MSCs and E-MSCs induced an increase in iNOS and arginase-1 levels, but only H-MSCs and their EVs were able to enhance IL-10 levels. In vivo, emphysematous mice treated with E-MSCs or E-EVs demonstrated no amelioration in cardiorespiratory dysfunction. On the other hand, H-EVs, but not H-MSCs, were able to reduce the neutrophil count, the mean linear intercept, and IL-1ß and TGF-ß levels in lung tissue, as well as reduce pulmonary arterial hypertension and increase the right ventricular area in a murine model of elastase-induced severe emphysema. In conclusion, E-MSCs and E-EVs were unable to reverse cardiorespiratory dysfunction, whereas H-EVs administration was associated with a reduction in cardiovascular and respiratory damage in experimental severe emphysema.

NOD Mice Recapitulate the Cardiac Disturbances Observed in Type 1 Diabetes.

This work aimed at testing the hypothesis that NOD/ShiLtJ mice (NOD) recapitulate the cardiac disturbances observed on type 1 diabetes (T1D). NOD mice were studied 4 weeks after the onset of hyperglycemia, and NOR/Lt mice matched as control. Cardiac function was evaluated by echocardiography and electrocardiography (ECG). Action potentials (AP) and Ca2+ transients were evaluated at whole heart level. Heart mitochondrial function was evaluated by high-resolution respirometry and H2O2 release. NOD mice presented a reduction in hearth weight. Mitochondrial oxygen fluxes and H2O2 release were similar between NOD and NOR mice. ECG revealed a QJ interval prolongation in NOD mice. Furthermore, AP duration at 30% of repolarization was increased, and it depicted slower Ca2+ transient kinetics. NOD mice presented greater number/severity of ventricular arrhythmias both in vivo and in vitro. In conclusion, NOD mice evoked cardiac electrical and calcium handling disturbances similar to the observed in T1D. Graphical Abstract .

Mitochondrial pyruvate carrier as a key regulator of fever and neuroinflammation.

The mitochondrial pyruvate carrier (MPC) is an inner-membrane transporter that facilitates pyruvate uptake from the cytoplasm into mitochondria. We previously reported that MPC1 protein levels increase in the hypothalamus of animals during fever induced by lipopolysaccharide (LPS), but how this increase contributes to the LPS responses remains to be studied. Therefore, we investigated the effect of UK 5099, a classical MPC inhibitor, in a rat model of fever, on hypothalamic mitochondrial function and neuroinflammation in LPS-stimulated preoptic area (POA) primary microcultures. Intracerebroventricular administration of UK 5099 reduced the LPS-induced fever. High-resolution respirometry revealed an increase in oxygen consumption and oxygen flux related to ATP synthesis in the hypothalamic homogenate from LPS-treated animals linked to mitochondrial complex I plus II. Preincubation with UK 5099 prevented the LPS-induced increase in oxygen consumption, ATP synthesis and spare capacity only in complex I-linked respiration and reduced mitochondrial H2O2 production. In addition, treatment of rat POA microcultures with UK 5099 reduced the secretion of the proinflammatory and pyrogenic cytokines TNFα and IL-6 as well as the immunoreactivity of inflammatory transcription factors NF-κB and NF-IL6 four hours after LPS stimulation. These results suggest that the regulation of mitochondrial pyruvate metabolism through MPC inhibition may be effective in reducing neuroinflammation and fever.

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.

Hyperglycemia in a type 1 Diabetes Mellitus model causes a shift in mitochondria coupled-glucose phosphorylation and redox metabolism in rat brain.

Hyperglycemia associated with Diabetes Mellitus type 1 (DM1) comorbidity may cause severe complications in several tissues that lead to premature death. These dysfunctions are related, among others, to redox imbalances caused by the uncontrolled cellular levels of reactive oxygen species (ROS). Brain is potentially prone to develop diabetes complications because of its great susceptibility to oxidative stress. In addition to antioxidant enzymes, mitochondria-coupled hexokinase (mt-HK) plays an essential role in maintaining high flux of oxygen and glucose to control the mitochondrial membrane and redox potential in brain. This redox control is critical for healthy conditions in brain and in the pathophysiological progression of DM1. The mitochondrial and mt-HK contribution in this process is essential to understand the relationship between DM1 complications and the management of the cellular redox balance. Using a rat model of one month of hyperglycemia induced by a single administration intraperitoneally of streptozotocin, we showed in the present work that, in rat brain mitochondria, there is a specifically reduction of the mitochondrial complex I (CI) activity and an increase in the activity of the antioxidant enzyme thioredoxin reductase, which are related to decreased hydrogen peroxide generation, oxygen consumption and mt-HK coupled-to-OxPhos activity via mitochondrial CI. Surprisingly, DM1 increases respiratory parameters and mt-HK activity via mitochondrial complex II (CII). This way, for the first time, we provide evidence that early progression of hyperglycemia, in brain tissue, changes the coupling of glucose phosphorylation at the level of mitochondria by rearranging the oxidative machinery of brain mitochondria towards CII dependent electron harvest. In addition, DM1 increased the production of H2O2 by α-ketoglutarate dehydrogenase without causing oxidative stress. Finally, DM1 increased the oxidation status of PTEN and decreased the activation of NF-kB in DM1. These results indicate that this reorganization of glucose-oxygen-ROS axis in mitochondria may impact turnover of glucose, brain amino acids, redox and inflammatory signaling. In addition, this reorganization may be involved in early protection mechanisms against the development of cognitive degeneration and neurodegenerative disease, widely associated to mitochondrial CI deficits.

Guanosine Neuroprotection of Presynaptic Mitochondrial Calcium Homeostasis in a Mouse Study with Amyloid-ß Oligomers.

Amyloid-ß oligomers (AßOs) toxicity causes mitochondrial dysfunction, leading to synaptic failure in Alzheimer's disease (AD). Considering presynaptic high energy demand and tight Ca2+ regulation, impairment of mitochondrial function can lead to deteriorated neural activity and cell death. In this study, an AD mouse model induced by ICV (intracerebroventricular) injection of AßOs was used to investigate the toxicity of AßOs on presynaptic function. As a therapeutic approach, GUO (guanosine) was given by oral route to evaluate the neuroprotective effects on this AD model. Following 24 h and 48 h from the model induction, behavioral tasks and biochemical analyses were performed, respectively. AßOs impaired object recognition (OR) short-term memory and reduced glutamate uptake and oxidation in the hippocampus. Moreover, AßOs decreased spare respiratory capacity, reduced ATP levels, impaired Ca2+ handling, and caused mitochondrial swelling in hippocampal synaptosomes. Guanosine crossed the BBB, recovered OR short-term memory, reestablished glutamate uptake, recovered mitochondrial Ca2+ homeostasis, and partially prevented mitochondrial swelling. Therefore, this endogenous purine presented a neuroprotective effect on presynaptic mitochondria and should be considered for further studies in AD models.

Type 2 diabetes mellitus alters cardiac mitochondrial content and function in a non-obese mice model.

Type 2 diabetes mellitus (T2DM) is associated with an increase of premature appearance of several disorders such as cardiac complications. Thus, we test the hypothesis that a combination of a high fat diet (HFD) and low doses of streptozotocin (STZ) recapitulate a suitable mice model of T2DM to study the cardiac mitochondrial disturbances induced by this disease. Animals were divided in 2 groups: the T2DM group was given a HFD and injected with 2 low doses of STZ, while the CNTRL group was given a standard chow and a buffer solution. The combination of HFD and STZ recapitulate the T2DM metabolic profile showing higher blood glucose levels in T2DM mice when compared to CNTRL, and also, insulin resistance. The kidney structure/function was preserved. Regarding cardiac mitochondrial function, in all phosphorylative states, the cardiac mitochondria from T2DM mice presented reduced oxygen fluxes when compared to CNTRL mice. Also, mitochondria from T2DM mice showed decreased citrate synthase activity and lower protein content of mitochondrial complexes. Our results show that in this non-obese T2DM model, which recapitulates the classical metabolic alterations, mitochondrial function is impaired and provides a useful model to deepen study the mechanisms underlying these alterations.

Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant.

Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.

Neuroprotection from optic nerve injury and modulation of oxidative metabolism by transplantation of active mitochondria to the retina.

Mitochondrial dysfunctions are linked to a series of neurodegenerative human conditions, including Parkinson's disease, schizophrenia, optic neuropathies, and glaucoma. Recently, a series of studies have pointed mitotherapy - exogenous mitochondria transplant - as a promising way to attenuate the progression of neurologic disorders; however, the neuroprotective and pro-regenerative potentials of isolated mitochondria in vivo have not yet been elucidated. In this present work, we tested the effects of transplants of active (as well-coupled organelles were named), liver-isolated mitochondria on the survival of retinal ganglion cells and axonal outgrowth after optic nerve crush. Our data show that intravitreally transplanted, full active mitochondria incorporate into the retina, improve its oxidative metabolism and electrophysiological activity at 1 day after transplantation. Moreover, mitotherapy increases cell survival in the ganglion cell layer at 14 days, and leads to a higher number of axons extending beyond the injury site at 28 days; effects that are dependent on the organelles' structural integrity. Together, our findings support mitotherapy as a promising approach for future therapeutic interventions upon central nervous system damage.

The yeast protein Ubx4p contributes to mitochondrial respiration and lithium-galactose-mediated activation of the unfolded protein response.

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes.

Zika virus (ZIKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZIKV. However, the consequences of ZIKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZIKV infection in iPSC-derived astrocytes are presented. Our results show ROS imbalance, mitochondrial defects and DNA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the Northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit.

Energization by multiple substrates and calcium challenge reveal dysfunctions in brain mitochondria in a model related to acute psychosis.

Schizophrenia etiology is unknown, nevertheless imbalances occurring in an acute psychotic episode are important to its development, such as alterations in cellular energetic state, REDOX homeostasis and intracellular Ca2+ management, all of which are controlled primarily by mitochondria. However, mitochondrial function was always evaluated singularly, in the presence of specific respiratory substrates, without considering the plurality of the electron transport system. In this study, mitochondrial function was analyzed under conditions of isolated or multiple respiratory substrates using brain mitochondria isolated from MK-801-exposed mice. Results showed a high H2O2 production in the presence of pyruvate/malate, with no change in oxygen consumption. In the condition of multiple substrates, however, this effect is lost. The analysis of Ca2+ retention capacity revealed a significant change in the uptake kinetics of this ion by mitochondria in MK-801-exposed animals. Futhermore, when mitochondria were exposed to calcium, a total loss of oxidative phosphorylation and an impressive increase in H2O2 production were observed in the condition of multiple substrates. There was no alteration in the activity of the antioxidant enzymes analyzed. The data demonstrate for the first time, in an animal model of psychosis, two important aspects (1) mitochondria may compensate deficiencies in a single mitochondrial complex when they oxidize several substrates simultaneously, (2) Ca2+ handling is compromised in MK-801-exposed mice, resulting in a loss of phosphorylative capacity and an increase in H2O2 production. These data favor the hypothesis that disruption of key physiological roles of mitochondria may be a trigger in acute psychosis and, consequently, schizophrenia.