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BACKGROUND & AIMS: The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. METHODS: Six-week-old, male, C57Bl/6 J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with in vivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. RESULTS: Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein in vivo. In vitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C in vitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. CONCLUSIONS: KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. IMPACT AND IMPLICATIONS: Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.
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Carnitina O-Palmitoiltransferasa , Hígado , Masculino , Ratones , Animales , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/farmacología , Acetilación , Hígado/metabolismo , Obesidad/metabolismo , Glucosa/metabolismo , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Fructosa/metabolismo , Fructoquinasas/genética , Fructoquinasas/metabolismoRESUMEN
Pioglitazone, an FDA-approved compound, has been shown to target the novel mitochondrial protein mitoNEET and produce short-term neuroprotection and functional benefits following traumatic brain injury. To expand on these findings, we now investigate the dose- and time-dependent effects of pioglitazone administration on mitochondrial function after experimental traumatic brain injury. We then hypothesize that optimal pioglitazone dosing will lead to ongoing neuroprotection and cognitive benefits that are dependent on pioglitazone-mitoNEET signalling pathways. We show that delayed intervention is significantly more effective than early intervention at improving acute mitochondrial bioenergetics in the brain after traumatic brain injury. In corroboration, we demonstrate that mitoNEET is more heavily expressed, especially near the cortical contusion, in the 18 h following traumatic brain injury. To explore whether these findings relate to ongoing pathological and behavioural outcomes, mice received controlled cortical impact followed by initiation of pioglitazone treatment at either 3 or 18 h post-injury. Mice with treatment initiation at 18 h post-injury exhibited significantly improved behaviour and tissue sparing compared to mice with pioglitazone initiated at 3 h post-injury. Further using mitoNEET knockout mice, we show that this therapeutic effect is dependent on mitoNEET. Finally, we demonstrate that delayed pioglitazone treatment improves serial motor and cognitive performance in conjunction with attenuated brain atrophy after traumatic brain injury. This study illustrates that mitoNEET is the critical target for delayed pioglitazone intervention after traumatic brain injury, mitochondrial-targeting is highly time-dependent after injury and there is an extended therapeutic window to effectively treat mitochondrial dysfunction after brain injury.
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Lesiones Traumáticas del Encéfalo , Proteínas de Unión a Hierro/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Pioglitazona/farmacología , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
It was hypothesized that the catalyst nanoceria can increase inflammation/oxidative stress from the basal and reduce it from the elevated state. Macrophages clear nanoceria. To test the hypothesis, M0 (non-polarized), M1- (classically activated, pro-inflammatory), and M2-like (alternatively activated, regulatory phenotype) RAW 264.7 macrophages were nanoceria exposed. Inflammatory responses were quantified by IL-1ß level, arginase activity, and RT-qPCR and metabolic changes and oxidative stress by the mito and glycolysis stress tests (MST and GST). Morphology was determined by light microscopy, macrophage phenotype marker expression, and a novel three-dimensional immunohistochemical method. Nanoceria blocked IL-1ß and arginase effects, increased M0 cell OCR and GST toward the M2 phenotype and altered multiple M1- and M2-like cell endpoints toward the M0 level. M1-like cells had greater volume and less circularity/roundness. M2-like cells had greater volume than M0 macrophages. The results are overall consistent with the hypothesis.
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Arginasa , Nanoestructuras , Arginasa/metabolismo , Cerio , Humanos , Inflamación , Estrés OxidativoRESUMEN
Traumatic brain injury (TBI) leads to acute necrosis at the site of injury followed by a sequence of secondary events lasting from hours to weeks and often years. Targeting mitochondrial impairment following TBI has shown improvements in brain mitochondrial bioenergetics and neuronal function. Recently formoterol, a highly selective ß2-adrenoreceptor agonist, was found to induce mitochondrial biogenesis (MB) via Gßγ-Akt-eNOS-sGC pathway. Activation of MB is a novel approach that has been shown to restore mitochondrial function in several disease and injury models. We hypothesized that activation of MB as a target of formoterol after TBI would mitigate mitochondrial dysfunction, enhance neuronal function and improve behavioral outcomes. TBI-injured C57BL/6 male mice were injected (i.p.) with vehicle (normal saline) or formoterol (0.3 mg/kg) at 15 min, 8 h, 16 h, 24 h and then daily after controlled cortical impact (CCI) until euthanasia. After CCI, mitochondrial copy number and bioenergetic function were decreased in the ipsilateral cortex of the CCI-vehicle group. Compared to CCI-vehicle, cortical and hippocampal mitochondrial respiration rates as well as cortical mitochondrial DNA copy number were increased in the CCI-formoterol group. Mitochondrial Ca2+ buffering capacity in the hippocampus was higher in the CCI-formoterol group compared to CCI-vehicle group. Both assessments of cognitive performance, novel object recognition (NOR) and Morris water maze (MWM), decreased following CCI and were restored in the CCI-formoterol group. Although no changes were seen in the amount of cortical tissue spared between CCI-formoterol and CCI-vehicle groups, elevated levels of hippocampal neurons and improved white matter sparing in the corpus callosum were observed in CCI-formoterol group. Collectively, these results indicate that formoterol-mediated MB activation may be a potential therapeutic target to restore mitochondrial bioenergetics and promote functional recovery after TBI.
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Lesiones Traumáticas del Encéfalo/complicaciones , Cognición/efectos de los fármacos , Fumarato de Formoterol/farmacología , Mitocondrias/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Fumarato de Formoterol/uso terapéutico , Hipocampo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Biogénesis de Organelos , Sustancia Blanca/efectos de los fármacosRESUMEN
Mitochondrial activity is highly dynamic in the healthy brain, and it can reflect both the signaling potential and the signaling history of neural circuits. Recent studies spanning invertebrates to mammals have highlighted a role for neural mitochondrial dynamics in learning and memory processes as well as behavior. In the current study, we investigate the interplay between biogenic amine signaling and neural energetics in the honey bee, Apis mellifera. In this species, aggressive behaviors are regulated by neural energetic state and biogenic amine titers, but it is unclear how these mechanisms are linked to impact behavioral expression. We show that brain mitochondrial number is highest in aggression-relevant brain regions and in individual bees that are most responsive to aggressive cues, emphasizing the importance of energetics in modulating this phenotype. We also show that the neural energetic response to alarm pheromone, an aggression inducing social cue, is activity dependent, modulated by the "fight or flight" insect neurotransmitter octopamine. Two other neuroactive compounds known to cause variation in aggression, dopamine, and serotonin, also modulate neural energetic state in aggression-relevant regions of the brain. However, the effects of these compounds on respiration at baseline and following alarm pheromone exposure are distinct, suggesting unique mechanisms underlying variation in mitochondrial respiration in these circuits. These results motivate new explanations for the ways in which biogenic amines alter sensory perception in the context of aggression. Considering neural energetics improves predictions about the regulation of complex and context-dependent behavioral phenotypes.
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Agresión/fisiología , Abejas/fisiología , Aminas Biogénicas/metabolismo , Señales (Psicología) , Mitocondrias/metabolismo , Neuronas/metabolismo , Animales , Citrato (si)-Sintasa/metabolismo , Dopamina/metabolismo , Femenino , Octopamina/metabolismo , Consumo de Oxígeno , Feromonas/administración & dosificación , Serotonina/metabolismoRESUMEN
Traumatic brain injury (TBI) results in cognitive impairment, which can be long-lasting after moderate to severe TBI. Currently, there are no FDA-approved therapeutics to treat the devastating consequences of TBI and improve recovery. This study utilizes a prodrug of 2,4-dinitrophenol, MP201, a mitochondrial uncoupler with extended elimination time, that was administered after TBI to target mitochondrial dysfunction, a hallmark of TBI. Using a model of cortical impact in male C57/BL6 mice, MP201 (80 mg/kg) was provided via oral gavage 2-hr post-injury and daily afterwards. At 25-hr post-injury, mice were euthanized and the acute rescue of mitochondrial bioenergetics was assessed demonstrating a significant improvement in both the ipsilateral cortex and ipsilateral hippocampus after treatment with MP201. Additionally, oxidative markers, 4-hydroxyneneal and protein carbonyls, were reduced compared to vehicle animals after MP201 administration. At 2-weeks post-injury, mice treated with MP201 post-injury (80 mg/kg; q.d.) displayed significantly increased cortical sparing (p = .0059; 38% lesion spared) and improved cognitive outcome (p = .0133) compared to vehicle-treated mice. Additionally, vehicle-treated mice had significantly lower (p = .0019) CA3 neuron count compared to sham while MP201-treated mice were not significantly different from sham levels. These results suggest that acute mitochondrial dysfunction can be targeted to impart neuroprotection from reactive oxygen species, but chronic administration may have an added benefit in recovery. This study highlights the potential for safe, effective therapy by MP201 to alleviate negative outcomes of TBI.
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2,4-Dinitrofenol/farmacología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Profármacos/farmacología , Desacopladores/farmacología , Animales , Lesiones Traumáticas del Encéfalo/inducido químicamente , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacosRESUMEN
Neuronal function demands high-level energy production, and as such, a decline in mitochondrial respiration characterizes brain injury and disease. A growing number of studies, however, link brain mitochondrial function to behavioral modulation in non-diseased contexts. In the honey bee, we show for the first time that an acute social interaction, which invokes an aggressive response, may also cause a rapid decline in brain mitochondrial bioenergetics. The degree and speed of this decline has only been previously observed in the context of brain injury. Furthermore, in the honey bee, age-related increases in aggressive tendency are associated with increased baseline brain mitochondrial respiration, as well as increased plasticity in response to metabolic fuel type in vitro Similarly, diet restriction and ketone body feeding, which commonly enhance mammalian brain mitochondrial function in vivo, cause increased aggression. Thus, even in normal behavioral contexts, brain mitochondria show a surprising degree of variation in function over both rapid and prolonged time scales, with age predicting both baseline function and plasticity in function. These results suggest that mitochondrial function is integral to modulating aggression-related neuronal signaling. We hypothesize that variation in function reflects mitochondrial calcium buffering activity, and that shifts in mitochondrial function signal to the neuronal soma to regulate gene expression and neural energetic state. Modulating brain energetic state is emerging as a critical component of the regulation of behavior in non-diseased contexts.
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Abejas/fisiología , Encéfalo/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Agresión/fisiología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Abejas/metabolismo , Conducta Animal/fisiología , Encéfalo/fisiología , Cuerpos Cetónicos , Neuronas/metabolismoRESUMEN
Mitochondrial function analysis is a well-established method used in preclinical and clinical investigations to assess pathophysiological changes in various disease states, including traumatic brain injury (TBI). Although there are multiple approaches to assess mitochondrial function, one common method involves respirometric assays utilizing either Clark-type oxygen electrodes or fluorescent-based Seahorse analysis (Agilent). However, these functional analysis methods are typically limited to the availability of freshly isolated tissue samples due to the compromise of the electron transport chain (ETC) upon storage, caused by freeze-thaw-mediated breakdown of mitochondrial membranes. In this study, we propose and refine a method for evaluating electron flux through the ETC, encompassing complexes I, II, and IV, in frozen homogenates or mitochondrial samples within a single well of a Seahorse plate. Initially, we demonstrate the impact of TBI on freshly isolated mitochondria using the conventional oxidative phosphorylation protocol (OxPP), followed by a comparison with ETC analysis conducted on frozen tissue samples within the context of a controlled cortical impact (CCI) model of TBI. Additionally, we explore the effects of mitochondrial isolation from fresh versus snap-frozen brain tissues and their storage at -80°C, assessing its impact on electron transport chain protocol (ETCP) activity. Our findings indicate that while both sets of samples were frozen at a single time point, mitochondria from snap-frozen tissues exhibited reduced injury effects compared to preparations from fresh tissues, which were either homogenized or isolated into mitochondria and subsequently frozen for later use. Thus, we demonstrate that the preparation of homogenates or isolated mitochondria can serve as an appropriate method for storing brain samples, allowing for later analysis of mitochondrial function, following TBI using ETCP.
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Neonatal intraventricular hemorrhage (IVH) releases blood products into the lateral ventricles and brain parenchyma. There are currently no medical treatments for IVH and surgery is used to treat a delayed effect of IVH, post-hemorrhagic hydrocephalus. However, surgery is not a cure for intrinsic brain injury from IVH, and is performed in a subacute time frame. Like many neurological diseases and injuries, innate immune activation is implicated in the pathogenesis of IVH. Innate immune activation is a pharmaceutically targetable mechanism to reduce brain injury and post-hemorrhagic hydrocephalus after IVH. Here, we tested the macrolide antibiotic azithromycin, which has immunomodulatory properties, to reduce innate immune activation in an in vitro model of microglial activation using the blood product hemoglobin (Hgb). We then utilized azithromycin in our in vivo model of IVH, using intraventricular blood injection into the lateral ventricle of post-natal day 5 rat pups. In both models, azithromycin modulated innate immune activation by several outcome measures including mitochondrial bioenergetic analysis, cytokine expression and flow cytometric analysis. This suggests that azithromycin, which is safe for neonates, could hold promise for modulating innate immune activation after IVH.
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Lesiones Encefálicas , Hidrocefalia , Ratas , Animales , Azitromicina/farmacología , Encéfalo/patología , Hemorragia Cerebral/patología , Hidrocefalia/etiología , Lesiones Encefálicas/patología , Hemoglobinas/farmacologíaRESUMEN
Apolipoprotein ε4 (APOE4) carriers develop brain metabolic dysfunctions decades before the onset of Alzheimer's disease (AD). A goal of the study is to identify if rapamycin, an inhibitor for the mammalian target of rapamycin (mTOR) inhibitor, would enhance synaptic and mitochondrial function in asymptomatic mice with human APOE4 gene (E4FAD) before they showed metabolic deficits. A second goal is to determine whether there may be genetic-dependent responses to rapamycin when compared to mice with human APOE3 alleles (E3FAD), a neutral AD genetic risk factor. We fed asymptomatic E4FAD and E3FAD mice with control or rapamycin diets for 16 weeks from starting from 3 months of age. Neuronal mitochondrial oxidative metabolism and excitatory neurotransmission rates were measured using in vivo 1H-[13C] proton-observed carbon-edited magnetic resonance spectroscopy, and isolated mitochondrial bioenergetic measurements using Seahorse. We found that rapamycin enhanced neuronal mitochondrial function, glutamate-glutamine cycling, and TCA cycle rates in the asymptomatic E4FAD mice. In contrast, rapamycin enhances glycolysis, non-neuronal activities, and inhibitory neurotransmission of the E3FAD mice. These findings indicate that rapamycin might be able to mitigate the risk for AD by enhancing brain metabolic functions for cognitively intact APOE4 carriers, and the responses to rapamycin are varied by APOE genotypes. Consideration of precision medicine may be needed for future rapamycin therapeutics.
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Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane that metabolizes amine substrates like serotonin, norepinephrine and dopamine. MAO inhibitors (MAOIs) are frequently utilized to treat disorders such as major depression or Parkinson's disease (PD), though their effects on brain mitochondrial bioenergetics are unclear. These studies measured bioenergetic activity in mitochondria isolated from the mouse cortex in the presence of inhibitors of either MAO-A, MAO-B, or both isoforms. We found that only 10 µM clorgyline, the selective inhibitor of MAO-A and not MAO-B, increased mitochondrial oxygen consumption rate in State V(CI) respiration compared to vehicle treatment. We then assessed mitochondrial bioenergetics, reactive oxygen species (ROS) production, and Electron Transport Chain (ETC) complex function in the presence of 0, 5, 10, 20, 40, or 80 µM of clorgyline to determine if this change was dose-dependent. The results showed increased oxygen consumption rates across the majority of respiration states in mitochondria treated with 5, 10, or 20 µM with significant bioenergetic inhibition at 80 µM clorgyline. Next, we assessed mitochondrial ROS production in the presence of the same concentrations of clorgyline in two different states: high mitochondrial membrane potential (ΔΨm) induced by oligomycin and low ΔΨm induced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). There were no changes in ROS production in the presence of 5, 10, 20, or 40 µM clorgyline compared to vehicle after the addition of oligomycin or FCCP. There was a significant increase in mitochondrial ROS in the presence of 80 µM clorgyline after FCCP addition, as well as reduced Complex I and Complex II activities, which are consistent with inhibition of bioenergetics seen at this dose. There were no changes in Complex I, II, or IV activities in mitochondria treated with low doses of clorgyline. These studies shed light on the direct effect of MAO-A inhibition on brain mitochondrial bioenergetic function, which may be a beneficial outcome for those taking these medications.
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Inhibidores de la Monoaminooxidasa , Monoaminooxidasa , Ratones , Animales , Monoaminooxidasa/metabolismo , Clorgilina/farmacología , Clorgilina/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona , Especies Reactivas de Oxígeno/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Mitocondrias/metabolismo , RespiraciónRESUMEN
Traumatic brain injury (TBI) is caused by an impact or penetrating injury to the head resulting in abnormal brain function. Mitochondrial dysfunction is an important hallmark of TBI and has been thoroughly studied in male rodent models of brain injury, but relatively little is known about these outcomes in females. These studies were designed to examine sex as a biological variable for mitochondria-related outcomes after the severe controlled cortical impact (CCI) mouse model of TBI. Synaptic and non-synaptic mitochondria were isolated from the sham- or CCI-injured cortex as well as the hippocampus ipsilateral to the craniotomy 3, 12, 24, or 48 h post-surgery, and then bioenergetics were measured. Subtle variations were observed in the timeline of mitochondrial dysfunction between sexes. Non-synaptic cortical mitochondria from injured females showed early impairment at 12 h post-CCI compared to mitochondria from injured males at 24 h post-CCI. Contrastingly, in the synaptic fraction, mitochondria from injured males showed early impairment at 12 h post-CCI, whereas mitochondria from injured females showed impairment at 24 h post-CCI. Based on bioenergetic impairments at 24 h post-CCI, synaptic and non-synaptic mitochondrial calcium loading was also measured at this time point. Consistent with bioenergetic data at 24 h, non-synaptic mitochondria from injured males had increased calcium loading compared to uninjured control, but this effect was not observed in females. Finally, histological assessment of cortical tissue sparing in each sex was measured at 7 days post-injury. There was a lack of sex-based differences in cortical tissue sparing after severe CCI. Overall, there were some subtle sex differences in mitochondrial outcomes after CCI, but these findings were not statistically significant. This study highlights the importance of utilizing both sexes when measuring mitochondrial function after severe CCI.
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Pioglitazone interacts through the mitochondrial protein mitoNEET to improve brain bioenergetics following traumatic brain injury. To provide broader evidence regarding the therapeutic effects of pioglitazone after traumatic brain injury, the current study is focused on immediate and delayed therapy in a model of mild brain contusion. To assess pioglitazone therapy on mitochondrial bioenergetics in cortex and hippocampus, we use a technique to isolate subpopulations of total, glia-enriched and synaptic mitochondria. Pioglitazone treatment was initially administered at either 0.25, 3, 12 or 24â h following mild controlled cortical impact. At 48â h post-injury, ipsilateral cortex and hippocampus were dissected and mitochondrial fractions were isolated. Maximal mitochondrial respiration injury-induced deficits were observed in total and synaptic fractions, and 0.25â h pioglitazone treatment following mild controlled cortical impact was able to restore respiration to sham levels. While there are no injury-induced deficits in hippocampal fractions, we do find that 3â h pioglitazone treatment after mild controlled cortical impact can significantly increase maximal mitochondrial bioenergetics compared to vehicle-treated mild controlled cortical impact group. However, delayed pioglitazone treatment initiated at either 3 or 24â h after mild brain contusion does not improve spared cortical tissue. We demonstrate that synaptic mitochondrial deficits following mild focal brain contusion can be restored with early initiation of pioglitazone treatment. Further investigation is needed to determine functional improvements with pioglitazone beyond that of overt cortical tissue sparing following mild contusion traumatic brain injury.
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Mild traumatic brain injury (mTBI) results in impairment of brain metabolism, which is propagated by mitochondrial dysfunction in the brain. Mitochondrial dysfunction has been identified as a pathobiological therapeutic target to quell cellular dyshomeostasis. Further, therapeutic approaches targeting mitochondrial impairments, such as mild mitochondrial uncoupling, have been shown to alleviate behavioral alterations after TBI. To examine how mild mitochondrial uncoupling modulates acute mitochondrial outcomes in a military-relevant model of mTBI, we utilized repeated blast overpressure of 11 psi peak overpressure to model repeated mild blast traumatic brain injury (rmbTBI) in rats followed by assessment of mitochondrial respiration and mitochondrial-related oxidative damage at 2 days post-rmbTBI. Treatment groups were administered 8 or 80 mg/kg MP201, a prodrug of 2,4 dinitrophenol (DNP) that displays improved pharmacokinetics compared with its metabolized form. Synaptic and glia-enriched mitochondria were isolated using fractionated a mitochondrial magnetic separation technique. There was a consistent physiological response, decreased heart rate, following mbTBI among experimental groups. Although there was a lack of injury effect in mitochondrial respiration of glia-enriched mitochondria, there were impairments in mitochondrial respiration in synaptic mitochondria isolated from the prefrontal cortex (PFC) and the amygdala/entorhinal/piriform cortex (AEP) region. Impairments in synaptic mitochondrial respiration were rescued by oral 80 mg/kg MP201 treatment after rmbTBI, which may be facilitated by increases in complex II and complex IV activity. Mitochondrial oxidative damage in glia-enriched mitochondria was increased in the PFC and hippocampus after rmbTBI. MP201 treatment alleviated elevated glia-enriched mitochondrial oxidative damage following rmbTBI. However, there was a lack of injury-associated differences in oxidative damage in synaptic mitochondria. Overall, our report demonstrates that rmbTBI results in mitochondrial impairment diffusely throughout the brain and mild mitochondrial uncoupling can restore mitochondrial bioenergetics and oxidative balance.
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Traumatismos por Explosión , Conmoción Encefálica , Lesiones Traumáticas del Encéfalo , Profármacos , Ratas , Animales , Profármacos/farmacología , Mitocondrias , Encéfalo , Estrés OxidativoRESUMEN
We developed a new method to isolate small extracellular vesicles (sEVs) from male and female wild-type and 5xFAD mouse brains to investigate the sex-specific functions of sEVs in Alzheimer's disease (AD). A mass spectrometric analysis revealed that sEVs contained proteins critical for EV formation and Aß. ExoView analysis showed that female mice contained more GFAP and Aß-labeled sEVs, suggesting that a larger proportion of sEVs from the female brain is derived from astrocytes and/or more likely to bind to Aß. Moreover, sEVs from female brains had more acid sphingomyelinase (ASM) and ceramide, an enzyme and its sphingolipid product important for EV formation and Aß binding to EVs, respectively. We confirmed the function of ASM in EV formation and Aß binding using co-labeling and proximity ligation assays, showing that ASM inhibitors prevented complex formation between Aß and ceramide in primary cultured astrocytes. Finally, our study demonstrated that sEVs from female 5xFAD mice were more neurotoxic than those from males, as determined by impaired mitochondrial function (Seahorse assays) and LDH cytotoxicity assays. Our study suggests that sex-specific sEVs are functionally distinct markers for AD and that ASM is a potential target for AD therapy.
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Enfermedad de Alzheimer , Vesículas Extracelulares , Ratones , Masculino , Femenino , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo , Ceramidas/metabolismoRESUMEN
The brain undergoes oxidative stress and mitochondrial dysfunction following physiological insults such as Traumatic brain injury (TBI), ischemia-reperfusion, and stroke. Pharmacotherapeutics targeting mitochondria (mitoceuticals) against oxidative stress include antioxidants, mild uncouplers, and enhancers of mitochondrial biogenesis, which have been shown to improve pathophysiological outcomes after TBI. However, to date, there is no effective treatment for TBI. Studies have suggested that the deletion of LDL receptor-related protein 1 (LRP1) in adult neurons or glial cells could be beneficial and promote neuronal health. In this study, we used WT and LRP1 knockout (LKO) mouse embryonic fibroblast cells to examine mitochondrial outcomes following exogenous oxidative stress. Furthermore, we developed a novel technique to measure mitochondrial morphometric dynamics using transgenic mitochondrial reporter mice mtD2g (mitochondrial-specific Dendra2 green) in a TBI model. We found that oxidative stress increased the quantity of fragmented and spherical-shaped mitochondria in the injury core of the ipsilateral cortex following TBI, whereas rod-like elongated mitochondria were seen in the corresponding contralateral cortex. Critically, LRP1 deficiency significantly decreased mitochondrial fragmentation, preserving mitochondrial function and cell growth following exogenous oxidative stress. Collectively, our results show that targeting LRP1 to improve mitochondrial function is a potential pharmacotherapeutic strategy against oxidative damage in TBI and other neurodegenerative diseases.
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Lesiones Traumáticas del Encéfalo , Fibroblastos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Estrés Oxidativo , Animales , Ratones , Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Fibroblastos/metabolismo , Ratones Transgénicos , Mitocondrias/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genéticaRESUMEN
In Alzheimer's disease (AD), reactive astrocytes produce extracellular vesicles (EVs) that affect mitochondria in neurons. Here, we show that Aß-induced generation of the sphingolipid ceramide by acid sphingomyelinase (A-SMase) triggered proinflammatory cytokine (C1q, TNF-α, IL-1α) release by microglia, which induced the reactive astrocytes phenotype and secretion of EVs enriched with ceramide. These EVs impeded the capacity of neurons to respond to energy demand. Inhibition of A-SMase with Arc39 and Imipramine reduced the secretion of cytokines from microglia, prompting us to test the effect of Imipramine on EV secretion and AD pathology in the 5xFAD mouse model. Brain derived-EVs from 5xFAD mice treated with Imipramine contained reduced levels of the astrocytic marker GFAP, ceramide, and Aß and did not impair mitochondrial respiration when compared to EVs derived from untreated 5xFAD brain. Consistently, Imipramine-treated 5xFAD mice showed reduced AD pathology. Our study identifies A-SMase inhibitors as potential AD therapy by preventing cyotokine-elicited secretion of mitotoxic EVs from astrocytes.
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Enfermedad de Alzheimer , Vesículas Extracelulares , Animales , Ratones , Enfermedad de Alzheimer/tratamiento farmacológico , Astrocitos , Esfingomielina Fosfodiesterasa , Imipramina/farmacología , CeramidasRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a progressive age-dependent disorder whose risk is affected by genetic factors. Better models for investigating early effects of risk factors such as apolipoprotein E (APOE) genotype are needed. OBJECTIVE: To determine whether APOE genotype produces neuropathologies in an AD-susceptible neural system, we compared effects of human APOE É3 (E3) and APOE É4 (E4) alleles on the mouse olfactory epithelium. METHODS: RNA-Seq using the STAR aligner and DESeq2, immunohistochemistry for activated caspase-3 and phosphorylated histone H3, glucose uptake after oral gavage of 2-[1,2-3H (N)]-deoxy-D-glucose, and Seahorse Mito Stress tests on dissociated olfactory mucosal cells. RESULTS: E3 and E4 olfactory mucosae show 121 differentially abundant mRNAs at age 6 months. These do not indicate differences in cell type proportions, but effects on 17 odorant receptor mRNAs suggest small differences in tissue development. Ten oxidoreductases mRNAs important for cellular metabolism and mitochondria are less abundant in E4 olfactory mucosae but this does not translate into differences in cellular respiration. E4 olfactory mucosae show lower glucose uptake, characteristic of AD susceptibility and consistent with greater expression of the glucose-sensitive gene, Asns. Olfactory sensory neuron apoptosis is unaffected at age 6 months but is greater in E4 mice at 10 months. CONCLUSION: Effects of human APOE alleles on mouse olfactory epithelium phenotype are apparent in early adulthood, and neuronal loss begins to increase by middle age (10 months). The olfactory epithelium is an appropriate model for the ability of human APOE alleles to modulate age-dependent effects associated with the progression of AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Mucosa Olfatoria/patología , Olfato/genética , Adulto , Alelos , Animales , Apolipoproteínas E , Encéfalo/patología , Femenino , Genotipo , Humanos , Masculino , RatonesRESUMEN
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function. We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation (i.e., miR-142-3p, miR-142-5p, and miR-146a) are altered in the hippocampus at 3-12 hours following a severe traumatic brain injury. In the present study, we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats. In addition, several inflammatory genes were analyzed in the cytosol fractions. The analysis showed that while elevated levels were observed in cytoplasm, the mitochondria-enriched miRNAs, miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury. Although not statistically significant, miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury, and recovered by 7 days. In contrast, miRNAs that are not enriched in mitochondria, including miR-124a, miR-150, miR-19b, miR-155, and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days. The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury. No major alterations were observed in sham operated animals. This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers. These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling. To test this, we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappaB inflammatory modulators in the injured brain. Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), two important modulators of the nuclear factor-kappaB (NF-κB) pro-inflammatory pathway. Moreover, injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels. Taken together, our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain. All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee (IACUC protocol # 2014-1300) on August 17, 2017.
RESUMEN
The extent that age-dependent mitochondrial dysfunction drives neurodegeneration is not well understood. This study tested the hypothesis that mitochondria contribute to spinal cord injury (SCI)-induced neurodegeneration in an age-dependent manner by using 2,4-dinitrophenol (DNP) to uncouple electron transport, thereby increasing cellular respiration and reducing reactive oxygen species (ROS) production. We directly compared the effects of graded DNP doses in 4- and 14-month-old (MO) SCI-mice and found DNP to have increased efficacy in mitochondria isolated from 14-MO animals. In vivo, all DNP doses significantly exacerbated 4-MO SCI neurodegeneration coincident with worsened recovery. In contrast, low DNP doses (1.0-mg/kg/day) improved tissue sparing, reduced ROS-associated 3-nitrotyrosine (3-NT) accumulation, and improved anatomical and functional recovery in 14-MO SCI-mice. By directly comparing the effects of DNP between ages we demonstrate that mitochondrial contributions to neurodegeneration diverge with age after SCI. Collectively, our data indicate an essential role of mitochondria in age-associated neurodegeneration.