Mitochondrial Respiratory Deficits Research Articles

Tamoxifen-induced knockdown of the mitochondrial calcium uniporter in Thy1-expressing neurons protects mice from hypoxic/ischemic brain injury

The mitochondrial calcium uniporter (MCU) mediates high-capacity mitochondrial calcium uptake that stimulates energy production. However, excessive MCU activity can cause ischemic heart injury. To examine if the MCU is also involved in hypoxic/ischemic (HI) brain injury, we have generated conditional MCU knockout mice by tamoxifen (TMX) administration to adult MCU-floxed (MCUfl/fl) mice expressing a construct encoding Thy1-cre/ERT2-eYFP. Relative to TMX/Thy1-cre/ERT2-eYFP controls, HI-induced sensorimotor deficits, forebrain neuron loss and mitochondrial damage were decreased for conditional MCU knockout mice. MCU knockdown by siRNA-induced silencing in cortical neuron cultures also reduced cell death and mitochondrial respiratory deficits following oxygen-glucose deprivation. Furthermore, MCU silencing did not produce metabolic abnormalities in cortical neurons observed previously for global MCU nulls that increased reliance on glycolysis for energy production. Based on these findings, we propose that brain-penetrant MCU inhibitors have strong potential to be well-tolerated and highly-efficacious neuroprotectants for the acute management of ischemic stroke.

A chlorogenic acid-phospholipid complex ameliorates post-myocardial infarction inflammatory response mediated by mitochondrial reactive oxygen species in SAMP8 mice

Mitochondrial reactive oxygen species (mtROS) directly stimulate the inflammatory cytokines cascades and participate in age-related changes of cardiovascular diseases. Application of small molecule targeting the mtROS is significant towards development of better therapy to combat inflammatory response after myocardial infarction (MI) in the aging heart. Chlorogenic acid (CGA) is a well-known natural compound while the clinical potential is largely stifled by its poor oral absorption. In the present study, we tested the protective effect of a novel chlorogenic acid-phospholipid complex (CGA-PC) against acute post-MI inflammation in aged senescence accelerated mouse model. 10-month-old SAMP8 mice were treated with CGA-PC (equivalent of CGA 10 or 20 mg/kg body weight) or phospholipid randomly by gavage on a daily basis for 2 weeks. mtROS, lipid peroxidation, H2O2 production and oxygen consumption were evaluated in hearts subjected to ischemia reperfusion (I/R) induced by left anterior descending artery ligation. CGA-PC significantly reduced pro-inflammatory cytokines and myocardial necrosis, accompanied by decreased oxidative stress and mitochondrial respiratory deficits. p-JNK, MnSOD and soluble cytochrome c were up-regulated in the necrotic heart tissue, while CGA-PC treatment increased the expression of MKP-1 and inhibited the downstream activation of JNK. Our study indicated that CGA-PC ameliorated post-MI inflammatory response in aging heart and that it might be a promising candidate for the clinical development of CGA.

Mitochondrial Respiration Is Impaired during Late-Stage Hamster Prion Infection.

Mitochondria are crucial to proper neuronal function and overall brain health. Mitochondrial dysfunction within the brain has been observed in many neurodegenerative diseases, including prion disease. Several markers of decreased mitochondrial activity during prion infection have been reported, yet the bioenergetic respiratory status of mitochondria from prion-infected animals is unknown. Here we show that clinically ill transgenic mice overexpressing hamster prion protein (Tg7) infected with the hamster prion strain 263K suffer from a severe deficit in mitochondrial oxygen consumption in response to the respiratory complex II substrate succinate. Characterization of the mitochondrial proteome of purified brain mitochondria from infected and uninfected Tg7 mice showed significant differences in the relative abundance of key mitochondrial electron transport proteins in 263K-infected animals relative to that in controls. Our results suggest that at clinical stages of prion infection, dysregulation of respiratory chain proteins may lead to impairment of mitochondrial respiration in the brain.IMPORTANCE Mitochondrial dysfunction is present in most major neurodegenerative diseases, and some studies have suggested that mitochondrial processes may be altered during prion disease. Here we show that hamster prion-infected transgenic mice overexpressing the hamster prion protein (Tg7 mice) suffer from mitochondrial respiratory deficits. Tg7 mice infected with the 263K hamster prion strain have little or no signs of mitochondrial dysfunction at the disease midpoint but suffer from a severe deficit in mitochondrial respiration at the clinical phase of disease. A proteomic analysis of the isolated brain mitochondria from clinically affected animals showed that several proteins involved in electron transport, mitochondrial dynamics, and mitochondrial protein synthesis were dysregulated. These results suggest that mitochondrial dysfunction, possibly exacerbated by prion protein overexpression, occurs at late stages during 263K prion disease and that this dysfunction may be the result of dysregulation of mitochondrial proteins.

High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington's disease expansion mutation carriers shows normal mitochondrial respiratory function.

Alterations in mitochondrial respiration are an important hallmark of Huntington’s disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.

Diminished force production and mitochondrial respiratory deficits are strain-dependent myopathies of subacute limb ischemia

Reduced skeletal muscle mitochondrial function might be a contributing mechanism to the myopathy and activity based limitations that typically plague patients with peripheral arterial disease (PAD). We hypothesized that mitochondrial dysfunction, myofiber atrophy, and muscle contractile deficits are inherently determined by the genetic background of regenerating ischemic mouse skeletal muscle, similar to how patient genetics affect the distribution of disease severity with clinical PAD. Genetically ischemia protected (C57BL/6) and susceptible (BALB/c) mice underwent either unilateral subacute hind limb ischemia (SLI) or myotoxic injury (cardiotoxin) for 28days. Limbs were monitored for blood flow and tissue oxygen saturation and tissue was collected for the assessment of histology, muscle contractile force, gene expression, mitochondrial content, and respiratory function. Despite similar tissue O2 saturation and mitochondrial content between strains, BALB/c mice suffered persistent ischemic myofiber atrophy (55.3% of C57BL/6) and muscle contractile deficits (approximately 25% of C57BL/6 across multiple stimulation frequencies). SLI also reduced BALB/c mitochondrial respiratory capacity, assessed in either isolated mitochondria (58.3% of C57BL/6 at SLI on day (d)7, 59.1% of C57BL/6 at SLI d28 across multiple conditions) or permeabilized myofibers (38.9% of C57BL/6 at SLI d7; 76.2% of C57BL/6 at SLI d28 across multiple conditions). SLI also resulted in decreased calcium retention capacity (56.0% of C57BL/6) in BALB/c mitochondria. Nonischemic cardiotoxin injury revealed similar recovery of myofiber area, contractile force, mitochondrial respiratory capacity, and calcium retention between strains. Ischemia-susceptible BALB/c mice suffered persistent muscle atrophy, impaired muscle function, and mitochondrial respiratory deficits during SLI. Interestingly, parental strain susceptibility to myopathy appears specific toregenerative insults including an ischemic component. Our findings indicate that the functional deficits that plague PAD patients could include mitochondrial respiratory deficits genetically inherent to the regenerating muscle myofibers.

Dimethyl fumarate alters microglia phenotype and protects neurons against proinflammatory toxic microenvironments

Delayed-release dimethyl fumarate (DMF) is an approved treatment for multiple sclerosis (MS). Microglia are considered central to MS pathophysiology, however the effects of DMF and the primary metabolite monomethyl fumarate (MMF) on microglia are not well characterized. We demonstrated that DMF and MMF altered transcriptional responses in primary microglia related to the nuclear factor (erythroid-derived 2)-like 2 pathway. Additionally, through an NRF2 independent manner, DMF, but not MMF significantly reduced production of proinflammatory mediators in classically activated microglia, and further rescued mitochondrial respiratory deficits in primary cortical neurons that were induced by activated microglia. These data suggest the mechanism of action of DMF may involve modulation of microglia inflammatory responses and attenuation of neurotoxicity.

Abstract 4782: Cisplatin induces mitochondrial damage and hippocampal neurotoxicity: a potential mechanism for chemotherapy-related cognitive impairment

Abstract Advances in cancer treatment, chemotherapy in particular, have substantially increased the number of long-term cancer survivors. However, these drugs often have neurotoxic effects that impair cognitive function, thereby diminishing the quality of life of millions of cancer survivors. Chemotherapy-related cognitive impairment (CRCI, chemo-brain) is commonly reported following the administration of chemotherapeutic agents and comprises a wide variety of neurological problems. Cisplatin is used to treat breast cancer and advanced ovarian cancer among other malignancies. Notably, more than 30% of advanced ovarian cancer patients develop CRCI during and after cisplatin-based chemotherapy. A plausible explanation for CRCI is that cisplatin might impair the structure and functions of neurons in brain regions involved in learning and memory, such as the hippocampus. We have recently identified mitochondrial dysfunction and increased oxidative stress as a mechanism through which cisplatin causes hippocampal cell death, and severe dendritic damage in surviving neurons. The aims of this study were to examine the effect of the antioxidant N-acetylcysteine (NAC) in mitigating cisplatin-induced hippocampal damage and assesse the effect of cisplatin on cognitive performance in a rat model. At a high dose, cisplatin (1μM) induced ∼35% increase in caspase-9 activation in primary rat hippocampal neurons, whereas at a substantially lower dose, cisplatin (0.1μM) induced non-reversible damage to dendritic spines and branches. Both doses produced severe mitochondrial respiratory deficits and significant ROS production. Delayed treatment with NAC partially mitigated neuronal apoptosis and ameliorated cisplatin induced dendritic spine loss. When administered to adult Sprague Dawley rats, cisplatin (3 mg/kg) administered for two consecutive days caused ∼40% reduction in the number of dendritic spines in CA1 and CA3 hippocampal neurons. Lastly, cognitive testing of rats treated with a chronic cisplatin regimen, revealed significant deficits in hippocampus-dependent tasks. Rats were given weekly cisplatin (5mg/kg, i.p.) or saline injections for 4 weeks and then trained in Context-Object Discrimination, 6 weeks later (n = 7,8). Cisplatin-treated rats were impaired in discriminating between the out-of-context and in-context object. Mitochondrial dysfunction provokes free radical production, with resulting loss of dendritic spines. When administered to rats, cisplatin causes hippocampal neuronal and mitochondrial damage, as well as cognitive deficits, supporting that role of mitochondrial toxicity in the mechanisms of cisplatin-induced CRCI. Importantly the data demonstrates that these processes can be potentially mitigated with administration of the clinically available antioxidant, NAC. The effect of NAC in ameliorating cisplatin induced CRCI in rats is being evaluated. Citation Format: Naomi Lomeli, Jennifer Czerniawski, Kaijun Di, John Guzowski, Daniela Bota. Cisplatin induces mitochondrial damage and hippocampal neurotoxicity: a potential mechanism for chemotherapy-related cognitive impairment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4782.

Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model.

BackgroundMitochondrial dysfunction is a hallmark of neurodegenerative diseases including Alzheimer’s disease (AD), with morphological and functional abnormalities limiting the electron transport chain and ATP production. A contributing factor of mitochondrial abnormalities is loss of nicotinamide adenine dinucleotide (NAD), an important cofactor in multiple metabolic reactions. Depletion of mitochondrial and consequently cellular NAD(H) levels by activated NAD glycohydrolases then culminates in bioenergetic failure and cell death. De Novo NAD+ synthesis from tryptophan requires a multi-step enzymatic reaction. Thus, an alternative strategy to maintain cellular NAD+ levels is to administer NAD+ precursors facilitating generation via a salvage pathway. We administered nicotinamide mononucleotide (NMN), an NAD+ precursor to APP(swe)/PS1(ΔE9) double transgenic (AD-Tg) mice to assess amelioration of mitochondrial respiratory deficits. In addition to mitochondrial respiratory function, we examined levels of full-length mutant APP, NAD+-dependent substrates (SIRT1 and CD38) in homogenates and fission/fusion proteins (DRP1, OPA1 and MFN2) in mitochondria isolated from brain. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN.MethodsMitochondrial oxygen consumption rates were examined in N2A neuroblastoma cells and non-synaptic brain mitochondria isolated from mice (3 months). Western blotting was utilized to assess APP, SIRT1, CD38, DRP1, OPA1 and MFN2 in brain of transgenic and non-transgenic mice (3–12 months). Mitochondrial morphology was assessed with confocal microscopy. One-way or two-way analysis of variance (ANOVA) and post-hoc Holm-Sidak method were used for statistical analyses of data. Student t-test was used for direct comparison of two groups.ResultsWe now demonstrate that mitochondrial respiratory function was restored in NMN-treated AD-Tg mice. Levels of SIRT1 and CD38 change with age and NMN treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN-treated mice.ConclusionsThis is the first study to directly examine amelioration of NAD+ catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound. This might lead to well-defined physiologic abnormalities that can serve an important role in the validation of promising agents such as NMN that target NAD+ catabolism preserving mitochondrial function.

Mitochondrial oxygen consumption deficits in skeletal muscle isolated from an Alzheimer’s disease-relevant murine model

BackgroundAge is considered a primary risk factor for neurodegenerative diseases including Alzheimer’s disease (AD). It is also now well understood that mitochondrial function declines with age. Mitochondrial deficits have been previously assessed in brain from both human autopsy tissue and disease-relevant transgenic mice. Recently it has been recognized that abnormalities of muscle may be an intrinsic aspect of AD and might contribute to the pathophysiology. However, deficits in mitochondrial function have yet to be clearly assessed in tissues outside the central nervous system (CNS). In the present study, we utilized a well-characterized AD-relevant transgenic mouse strain to assess mitochondrial respiratory deficits in both brain and muscle. In addition to mitochondrial function, we assessed levels of transgene-derived amyloid precursor protein (APP) in homogenates isolated from brain and muscle of these AD-relevant animals.ResultsWe now demonstrate that skeletal muscles isolated from these animals have differential levels of mutant full-length APP depending on muscle type. Additionally, isolated muscle fibers from young transgenic mice (3 months) have significantly decreased maximal mitochondrial oxygen consumption capacity compared to non-transgenic, age-matched mice, with similar deficits to those previously described in brain.ConclusionsThis is the first study to directly examine mitochondrial function in skeletal muscle from an AD-relevant transgenic murine model. As with brain, these deficits in muscle are an early event, occurring prior to appearance of amyloid plaques.

MicroRNA Let-7f Mediates Mitochondrial Respiratory Deficit Induced by Repeated Ethanol Exposure and Withdrawal in HT22 Cells

Aims: The expression of gene and gene product is typically inhibited by a small noncoding RNA (microRNA) or DNA methylation. The aim of this study is to investigate mechanisms involving microRNA let-7f by which the repeated cycles of ethanol exposure and withdrawal provoke mitochondrial respiratory damage. Study Design: The rat or cell model of repeated withdrawal from a high dose of ethanol exposure was used to mimic human alcoholics who repeat the cycles of heavy drinking and unsuccessful attempts at abstaining. Place and Duration of Study: Department of Pharmacology and Neuroscience University of North Texas Health Science Center at Fort Worth, between June 2011 and March 2014. Methodology: Male adult rats received an ethanol program, consisting of two cycles of ethanol exposure (4 weeks) and withdrawal (2 weeks). At the end of the ethanol program, one hemisphere of each rat was used to measure the level of let-7f using TaqMan let-7f primers and qPCR. The other hemisphere was used to measure the methylation of cytosine in let-7f gene using bisulfite conversion and pyrosequencing. Separately, HT22 cells (mouse hippocampal cells) were exposed to an ethanol program, consisting of two cycles of ethanol exposure (20 hours) and withdrawal (4 hours). During the entire ethanol program, the cells were treated with let-7f antagomir (inhibitor) or a Short Communication International Neuropsychiatric Disease Journal, 2(6): 303-315, 2014 304 methylation-inducing methyl-donor. The role of let-7f in mitochondria was assessed by quantifying a mitochondrial enzyme, cytochrome c oxidase-IV (COX subunit IV) and realtime mitochondrial respiration using an immunoblot method and XF respirometry, respectively. Results: The level of let-7f increased (2.4±0.5 fold increase), whereas the methylation of let-7f gene decreased in the brain of rats that underwent repeated ethanol exposure and withdrawal (called “repeated-ethanol/withdrawal”). The methyl-donor treatment completely abolished the increase in let-7f induced by repeated-ethanol/withdrawal. let-7f antagomir treatment also abolished the inhibiting effect of repeated-ethanol/withdrawal on COX-IV and mitochondrial respiration. Conclusion: These data suggest that repeated-ethanol/withdrawal provokes the dysregulation of let-7f, thereby damaging brain mitochondria. Mitochondria-associated microRNA may be a potential research and drug target to manage alcoholism.