Deficits In Mitochondrial Respiration Research Articles

Glial swip-10 controls systemic mitochondrial function, oxidative stress, and neuronal viability via copper ion homeostasis

Cuprous copper [Cu(I)] is an essential cofactor for enzymes that support many fundamental cellular functions including mitochondrial respiration and suppression of oxidative stress. Neurons are particularly reliant on mitochondrial production of ATP, with many neurodegenerative diseases, including Parkinson's disease, associated with diminished mitochondrial function. The gene MBLAC1 encodes a ribonuclease that targets pre-mRNA of replication-dependent histones, proteins recently found in yeast to reduce Cu(II) to Cu(I), and when mutated disrupt ATP production, elevates oxidative stress, and severely impacts cell growth. Whether this process supports neuronal and/or systemic physiology in higher eukaryotes is unknown. Previously, we identified swip-10, the putative Caenorhabditis elegans ortholog of MBLAC1, establishing a role for glial swip-10 in limiting dopamine (DA) neuron excitability and sustaining DA neuron viability. Here, we provide evidence from computational modeling that SWIP-10 protein structure mirrors that of MBLAC1 and locates a loss of function coding mutation at a site expected to disrupt histone RNA hydrolysis. Moreover, we find through genetic, biochemical, and pharmacological studies that deletion of swip-10 in worms negatively impacts systemic Cu(I) levels, leading to deficits in mitochondrial respiration and ATP production, increased oxidative stress, and neurodegeneration. These phenotypes can be offset in swip-10 mutants by the Cu(I) enhancing molecule elesclomol and through glial expression of wildtype swip-10. Together, these studies reveal a glial-expressed pathway that supports systemic mitochondrial function and neuronal health via regulation of Cu(I) homeostasis, a mechanism that may lend itself to therapeutic strategies to treat devastating neurodegenerative diseases.

From Young to Old: Mimicking Neuronal Aging in Directly Converted Neurons from Young Donors.

A substantial challenge in human brain aging is to find a suitable model to mimic neuronal aging in vitro as accurately as possible. Using directly converted neurons (iNs) from human fibroblasts is considered a promising tool in human aging since it retains the aging-associated mitochondrial donor signature. Still, using iNs from aged donors can pose certain restrictions due to their lower reprogramming and conversion efficacy than those from younger individuals. To overcome these limitations, our study aimed to establish an in vitro neuronal aging model mirroring features of in vivo aging by acute exposure on young iNs to either human stress hormone cortisol or the mitochondrial stressor rotenone, considering stress as a trigger of in vivo aging. The impact of rotenone was evident in mitochondrial bioenergetic properties by showing aging-associated deficits in mitochondrial respiration, cellular ATP, and MMP and a rise in glycolysis, mitochondrial superoxide, and mitochondrial ROS; meanwhile, cortisol only partially induced an aging-associated mitochondrial dysfunction. To replicate the in vivo aging-associated mitochondrial dysfunctions, using rotenone, a mitochondrial complex I inhibitor, proved to be superior to the cortisol model. This work is the first to use stress on young iNs to recreate aging-related mitochondrial impairments.

Mitochondrial Mechanisms of Endothelial Cell Damage in Alzheimer’s Disease and Cerebral Amyloid Angiopathy

AbstractBackgroundCerebrovascular endothelial cells (EC) are crucial regulators of cerebral homeostasis and function through the maintenance of the blood‐brain barrier (BBB). ECs damage and/or dysfunction may result in defects in brain clearance and perfusion, microhemorrhages, inflammation, and neurodegeneration. Damage to the ECs may result from cardiovascular risk factors (CVRF) such as high levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy). In Alzheimer’s Disease (AD) and Cerebral Amyloid Angiopathy (CAA), the presence of a CVRF may further exacerbate cerebrovascular function and neurodegeneration. EC do not rely highly on mitochondrial ATP production as they are glycolytic cells. Instead, EC mitochondria serve mostly as signaling organelles, which sense cellular damage and mediate downstream pathways such as inflammation. In this study, we aim to understand the mechanistic nature of endothelial mitochondrial dysfunction and its elicited pathways in Aβ‐ and mixed (Aβ+HHcy)‐mediated vascular pathologies. Understanding the mechanisms by which the mitochondria mediate EC inflammation, may pave the way to new roads for therapeutic advancements in the field.MethodHuman brain microvascular ECs were challenged with Aβ, Hcy, or the combination. Mitochondrial metabolic function, inflammatory cascades and signals, and EC barrier function were assessed.ResultThe presence of Aβ, but not Hcy, induced deficits in mitochondrial respiration and ATP production. In addition, there is evidence of mitochondrial fragmentation and the expression of fission proteins. Preliminary results further suggest a release of mitochondrial danger associated molecular patterns (mtDAMPs) that can promote an inflammatory cascade. The mitochondrial damage and subsequent inflammatory response are associated with an increase in EC barrier permeability and cytokine release, which is exacerbated in the presence of Hcy.ConclusionThe EC dysfunction associated with Aβ is accompanied by severe deficits in mitochondrial metabolism and the release of mtDAMPS. The presence of Hcy exacerbates some of the detrimental effects of Aβ, particularly on mtDAMPS and BBB function, but Hcy does not appear to affect mitochondrial metabolic changes. Overall, our results reveal a novel role for mtDAMPs in vascular dysfunction in the presence of Aβ and/or Hcy.

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT.

The recognition that cytosolic mitochondrial DNA (mtDNA) activates cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

High fat diet feeding induces brain mitochondrial dysfunction in female Ossabaw swine

Study objective: Emerging evidence now suggest reduced mitochondrial function as a key initiator in Alzheimer’s disease (AD)-like pathology increasing the need for models of AD that display mitochondrial dysfunction. Dyslipidemia is a common aliment from overconsumption of a high fat diet (HFD) that increases the risk of AD and may contribute to mitochondrial dysfunction. Rodent studies utilize HFDs to induce brain pathology resembling that of AD. Unsurprisingly, rodent studies are rarely recapitulated in humans, which leads to a very low clinical success rate and highlights the need for more translatable models to study AD-like pathology. Swine seem to share many characteristics of humans and might offer a more translatable model to study HFD induced pathology. Thus, the objective of this study was to determine if HFD feeding in swine could offer a more translatable model to study mitochondrial dysfunction during neurodegeneration. Hypothesis: 6-months of HFD feeding will decrease indices of mitochondrial function and biogenesis in female Ossabaw swine. Methods: To characterize the effects of a HFD on brain mitochondrial health in a highly translatable swine model, high resolution respirometry measurements were taken from isolated brain mitochondria from in female Ossabaw swine fed a HFD for 6-months. Additionally, indices of mitochondrial biogenesis in whole tissue was measured (transcriptional markers of biogenesis and citrate synthase activity). Results: HFD feeding significantly increased body weight (p<0.05) with trending increases in blood glucose (p<0.10). Maximal uncoupled mitochondrial respiration was significantly decreased (p<0.05) with state 3-complex I and state 3 complex I & II trending down in the dorsal hippocampus (DH) after HFD diet feeding (p<0.10). Additionally, state 2 respiration was significantly decreased in the prefrontal cortex (PFC) after HFD feeding (p<0.05). HFD feeding increased transcriptional markers of mitochondrial biogenesis in the DH and PFC (p<0.05), without altering whole homogenate citrate synthase activity in either brain region. Conclusions: These data indicate HFD feeding in Ossabaw pigs could offer a potential model to study pathologies relating to mitochondrial dysfunction during neurodegenerative process. Additionally, the DH was found to be more vulnerable to HFD-induced perturbations, evident by the exacerbated deficits in mitochondrial respiration in this brain region. Finally, these data suggest HFD-induced increase in transcriptional markers of mitochondrial biogenesis may be a compensatory response to HFD-induced mitochondrial respiration deficits. However, citrate synthase activity was not different between groups indicating this upregulation of transcriptional markers was unable to translate to an increase in mitochondrial content in the DH and PFC. The project was supported by the MU Office of Postdoctoral Education Research Grant Program. Additional funding was also generously provided Scott Rector. Images in figure 2 adapted and modified from “Krebs cycle template”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates . This work was also supported with resources and the use of facilities at the University of Missouri and Harry S. Truman Memorial Veterans Hospital in Columbia, Missouri. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Disease-specific differences in gene expression, mitochondrial function and mitochondria-endoplasmic reticulum interactions in iPSC-derived cerebral organoids and cortical neurons in schizophrenia and bipolar disorder

We compared transcriptomic profiles of cerebral organoids differentiated from induced pluripotent stem cells of eight schizophrenia and eight bipolar disorder patients to identify genes that were differentially expressed in cerebral organoids between two disorders. Gene ontology analysis showed relative up-regulation in schizophrenia organoids of genes related to response to cytokines, antigen binding and clathrin-coated vesicles, while showing up-regulation in bipolar disorder of genes involved in calcium binding. Gene set enrichment analysis revealed enrichment in schizophrenia of genes involved in mitochondrial and oxidative phosphorylation while showing enrichment in bipolar disorder of genes involved in long term potentiation and neuro-transporters. We compared mitochondrial function in cerebral organoids from schizophrenia and bipolar disorder subjects and found that while schizophrenia organoids showed deficits in basal oxygen consumption rate and ATP production when compared to healthy control organoids, while bipolar disorder organoids did not show these deficits. Gene ontology analyses also revealed enrichment in bipolar disorder of genes in ion binding and regulation of transport. Experiments examining the interaction between mitochondria and endoplasmic reticulum in cortical neurons from bipolar disorder subjects showed a significantly lower number of contact sites between mitochondria and endoplasmic reticulum when compared to cortical neurons from schizophrenia patients. These results point to disease-specific deficits in mitochondrial respiration in schizophrenia and in mitochondrial-endoplasmic reticulum interactions in bipolar disorder.

Increasing plasma L-kynurenine impairs mitochondrial oxidative phosphorylation prior to the development of atrophy in murine skeletal muscle: A pilot study.

Introduction: L-Kynurenine (L-Kyn), a product of tryptophan (Trp) catabolism, has been linked with impairments in walking speed, muscle strength/size, and physical function. The purpose of this pilot study was to develop a dietary model that elevates plasma L-Kyn levels in mice and characterize its impact on muscle health and function. Methods: Four-month-old C57BL6J male mice were randomized to either a L-Kyn supplemented (150 mg/kg) or chow diet for 10 weeks. Plasma L-Kyn and Trp levels were measured via mass spectrometry. Primary outcomes included assessments of muscle weights, myofiber cross-sectional area (CSA), nerve-stimulated contractile performance, and mitochondrial oxidative phosphorylation (OXPHOS) and hydrogen peroxide (H2O2) production. Additional experiments in cultured myotubes explored the impact of enhancing L-Kyn metabolism. Results: Mice randomized to the L-Kyn diet displayed significant increases in plasma L-Kyn levels (p = 0.0028) and the L-Kyn/Trp ratio (p = 0.011) when compared to chow fed mice. Food intake and body weights were not different between groups. There were no detectable differences in muscle weights, myofiber CSA, or contractile performance. L-Kyn fed mice displayed reductions in mitochondrial OXPHOS (p = 0.05) and maximal ADP-stimulated respiration (p = 0.0498). In cultured myotubes, overexpression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha prevented atrophy and proteolysis, as well as deficits in mitochondrial respiration with L-Kyn treatment. Conclusion: Dietary feeding of L-Kyn increases plasma L-Kyn levels and the L-Kyn/Trp ratio in healthy male mice. Mitochondrial impairments in muscle were observed in mice with elevated L-Kyn without changes in muscle size or function. Enhancing L-Kyn metabolism can protect against these effects in culture myotubes.

Perinatal versus adult loss of ULK1 and ULK2 distinctly influences cardiac autophagy and function

ABSTRACT Impairments in macroautophagy/autophagy, which degrades dysfunctional organelles as well as long-lived and aggregate proteins, are associated with several cardiomyopathies; however, the regulation of cardiac autophagy remains insufficiently understood. In this regard, ULK1 and ULK2 are thought to play primarily redundant roles in autophagy initiation, but whether their function is developmentally determined, potentially having an impact on cardiac integrity and function remains unknown. Here, we demonstrate that perinatal loss of ULK1 or ULK2 in cardiomyocytes (cU1-KO and cU2-KO mice, respectively) enhances basal autophagy without altering autophagy machinery content while preserving cardiac function. This increased basal autophagy is dependent on the remaining ULK protein given that perinatal loss of both ULK1 and ULK2 in cU1/2-DKO mice impaired autophagy causing age-related cardiomyopathy and reduced survival. Conversely, adult loss of cardiac ULK1, but not of ULK2 (i.e., icU1-KO and icU2-KO mice, respectively), led to a rapidly developing cardiomyopathy, heart failure and early death. icU1-KO mice had impaired autophagy with robust deficits in mitochondrial respiration and ATP synthesis. Trehalose ameliorated autophagy impairments in icU1-KO hearts but did not delay cardiac dysfunction suggesting that ULK1 plays other critical, autophagy-independent, functions in the adult heart. Collectively, these results indicate that cardiac ULK1 and ULK2 are functionally redundant in the developing heart, while ULK1 assumes a more unique, prominent role in the adult heart. Abbreviations: ATG4: autophagy related 4, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG13: autophagy related 13; CYCS: Cytochrome C; DNM1L, dynamin 1-like; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MFN1: mitofusin 1; MFN2: mitofusin 2; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MYH: myosin, heavy polypeptide; NBR1: NBR1 autophagy cargo receptor; NDUFA9: NADH:ubiquinone oxidoreductase subunit A9; OPA1: OPA1, mitochondrial dynamin like GTPase; PPARGC1A, peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; SDHA: succinate dehydrogenase complex, subunit A, flavoprotein (Fp); SQSTM1: sequestosome 1; ULK1: unc-51 like kinase 1; ULK2: unc-51 like kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1

Lower citrate synthase activity, mitochondrial complex expression, and fewer oxidative myofibers characterize skeletal muscle from growth-restricted fetal sheep.

Skeletal muscle from the late gestation sheep fetus with intrauterine growth restriction (IUGR) has evidence of reduced oxidative metabolism. Using a sheep model of placental insufficiency and IUGR, we tested the hypothesis that by late gestation, IUGR fetal skeletal muscle has reduced capacity for oxidative phosphorylation because of intrinsic deficits in mitochondrial respiration. We measured mitochondrial respiration in permeabilized muscle fibers from biceps femoris (BF) and soleus (SOL) from control and IUGR fetal sheep. Using muscles including BF, SOL, tibialis anterior (TA), and flexor digitorum superficialis (FDS), we measured citrate synthase (CS) activity, mitochondrial complex subunit abundance, fiber type distribution, and gene expression of regulators of mitochondrial biosynthesis. Ex vivo mitochondrial respiration was similar in control and IUGR muscle. However, CS activity was lower in IUGR BF and TA, indicating lower mitochondrial content, and protein expression of individual mitochondrial complex subunits was lower in IUGR TA and BF in a muscle-specific pattern. IUGR TA, BF, and FDS also had lower expression of type I oxidative fibers. Fiber-type shifts that support glycolytic instead of oxidative metabolism may be advantageous for the IUGR fetus in a hypoxic and nutrient-deficient environment, whereas these adaptions may be maladaptive in postnatal life.

Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is currently infecting millions of people worldwide and is causing drastic changes in people’s lives. Recent studies have shown that neurological symptoms are a major issue for people infected with SARS-CoV-2. However, the mechanism through which the pathological effects emerge is still unclear. Brain endothelial cells (ECs), one of the components of the blood–brain barrier, are a major hurdle for the entry of pathogenic or infectious agents into the brain. They strongly express angiotensin converting enzyme 2 (ACE2) for its normal physiological function, which is also well-known to be an opportunistic receptor for SARS-CoV-2 spike protein, facilitating their entry into host cells. First, we identified rapid internalization of the receptor-binding domain (RBD) S1 domain (S1) and active trimer (Trimer) of SARS-CoV-2 spike protein through ACE2 in brain ECs. Moreover, internalized S1 increased Rab5, an early endosomal marker while Trimer decreased Rab5 in the brain ECs. Similarly, the permeability of transferrin and dextran was increased in S1 treatment but decreased in Trimer, respectively. Furthermore, S1 and Trimer both induced mitochondrial damage including functional deficits in mitochondrial respiration. Overall, this study shows that SARS-CoV-2 itself has toxic effects on the brain ECs including defective molecular delivery and metabolic function, suggesting a potential pathological mechanism to induce neurological signs in the brain.

Pronounced α-Synuclein Pathology in a Seeding-Based Mouse Model Is Not Sufficient to Induce Mitochondrial Respiration Deficits in the Striatum and Amygdala

Increasing evidence suggests that cross talk between α-synuclein pathology formation and mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson’s disease (PD) and related synucleinopathies. While mitochondrial dysfunction is a well-studied phenomenon in the substantia nigra, which is selectively vulnerable in PD and some models thereof, less information is available in other brain regions that are also affected by synuclein pathology. Therefore, we sought to test the hypothesis that early α-synuclein pathology causes mitochondrial dysfunction and that this effect might be exacerbated in conditions of increased vulnerability in affected brain regions, such as the amygdala. We combined a model of intracerebral α-synuclein pathology seeding with chronic glucocorticoid treatment, which models non-motor symptoms of PD and affects amygdala physiology. We measured mitochondrial respiration, reactive oxygen species (ROS) generation and protein abundance as well as α-synuclein pathology in male mice. Chronic corticosterone administration induced mitochondrial hyperactivity in the amygdala. Although injection of α-synuclein preformed fibrils (PFFs) into the striatum resulted in pronounced α-synuclein pathology in both striatum and amygdala, mitochondrial respiration in these brain regions was not compromised, regardless of corticosterone treatment. Our results suggest that early stage α-synuclein pathology does not influence mitochondrial respiration in the striatum and amygdala, even in corticosterone-induced respirational hyperactivity. We discuss our findings in light of relevant literature, warn of a potential publication bias and encourage scientists to report their negative results within the framework of this model.

Animal Models of Metabolic Epilepsy and Epilepsy Associated Metabolic Dysfunction: A Systematic Review.

Epilepsy is a serious neurological disorder affecting around 70 million people globally and is characterized by spontaneous recurrent seizures. Recent evidence indicates that dysfunction in metabolic processes can lead to the alteration of neuronal and network excitability, thereby contributing to epileptogenesis. Developing a suitable animal model that can recapitulate all the clinical phenotypes of human metabolic epilepsy (ME) is crucial yet challenging. The specific environment of many symptoms as well as the primary state of the applicable neurobiology, genetics, and lack of valid biomarkers/diagnostic tests are the key factors that hinder the process of developing a suitable animal model. The present systematic review summarizes the current state of available animal models of metabolic dysfunction associated with epileptic disorders. A systematic search was performed by using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) model. A range of electronic databases, including google scholar, Springer, PubMed, ScienceDirect, and Scopus, were scanned between January 2000 and April 2020. Based on the selection criteria, 23 eligible articles were chosen and are discussed in the current review. Critical analysis of the selected literature delineated several available approaches that have been modeled into metabolic epilepsy and pointed out several drawbacks associated with the currently available models. The result describes available models of metabolic dysfunction associated with epileptic disorder, such as mitochondrial respiration deficits, Lafora disease (LD) model-altered glycogen metabolism, causing epilepsy, glucose transporter 1 (GLUT1) deficiency, adiponectin responsive seizures, phospholipid dysfunction, glutaric aciduria, mitochondrial disorders, pyruvate dehydrogenase (PDH) α-subunit gene (PDHA1), pyridoxine dependent epilepsy (PDE), BCL2-associated agonist of cell death (BAD), Kcna1 knock out (KO), and long noncoding RNAs (lncRNA) cancer susceptibility candidate 2 (lncRNA CASC2). Finally, the review highlights certain focus areas that may increase the possibilities of developing more suitable animal models and underscores the importance of the rationalization of animal models and evaluation methods for studying ME. The review also suggests the pressing need of developing precise robust animal models and evaluation methods for investigating ME.

Allopregnanolone Reverses Bioenergetic Deficits in Female Triple Transgenic Alzheimer's Mouse Model.

Previously, we reported that the neurosteroid allopregnanolone (Allo) promoted neural stem cell regeneration, restored cognitive function, and reduced Alzheimer's Disease (AD) pathology in the triple transgenic Alzheimer's mouse model (3xTgAD). To investigate the underlying systems biology of Allo action in AD models in vivo, we assessed the regulation of Allo on the bioenergetic system of the brain. Outcomes of these analysis indicated that Allo significantly reversed deficits in mitochondrial respiration and biogenesis and key mitochondrial enzyme activity and reduced lipid peroxidation in the 3xTgAD mice in vivo. To explore the mechanisms by which Allo regulates the brain metabolism, we conducted targeted transcriptome analysis. These data further confirmed that Allo upregulated genes involved in glucose metabolism, mitochondrial bioenergetics, and signaling pathways while simultaneously downregulating genes involved in Alzheimer's pathology, fatty acid metabolism, and mitochondrial uncoupling and dynamics. Upstream regulatory pathway analysis predicted that Allo induced peroxisome proliferator-activated receptor gamma (PPARG) and coactivator 1-alpha (PPARGC1A) pathways while simultaneously inhibiting the presenilin 1 (PSEN 1), phosphatase and tensin homolog (PTEN), and tumor necrosis factor (TNF) pathways to reduce AD pathology. Collectively, these data indicate that Allo functions as a systems biology regulator of bioenergetics, cholesterol homeostasis, and β-amyloid reduction in the brain. These systems are critical to neurological health, thus providing a plausible mechanistic rationale for Allo as a therapeutic to promote neural cell function and reduce the burden of AD pathology.

Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury

Traumatic brain injury (TBI) activates multiple neuronal cell death mechanisms, leading to post-traumatic neuronal loss and neurological deficits. TBI-induced cell cycle activation (CCA) in post-mitotic neurons causes regulated cell death involving cyclin-dependent kinase (CDK) activation and initiation of an E2F transcription factor-mediated pro-apoptotic program. Here we examine the mechanisms of CCA-dependent neuronal apoptosis in primary neurons in vitro and in mice exposed to controlled cortical impact (CCI). In contrast to our prior work demonstrating robust neuroprotective effects by CDK inhibitors after TBI, examination of neuronal apoptotic mechanisms in E2F1−/−/E2F2−/− or E2F2−/− transgenic mice following CCI suggests that E2F1 and/or E2F2 likely play only a modest role in neuronal cell loss after brain trauma. To elucidate more critical CCA molecular pathways involved in post-traumatic neuronal cell death, we investigated the neuroprotective effects and mechanisms of the potent CDK inhibitor CR8 in a DNA damage model of cell death in primary cortical neurons. CR8 treatment significantly reduced caspase activation and cleavage of caspase substrates, attenuating neuronal cell death. CR8 neuroprotective effects appeared to reflect inhibition of multiple pathways converging on the mitochondrion, including injury-induced elevation of pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins Puma and Noxa, thereby attenuating mitochondrial permeabilization and release of cytochrome c and AIF, with reduction of both caspase-dependent and -independent apoptosis. CR8 administration also limited injury-induced deficits in mitochondrial respiration. These neuroprotective effects may be explained by CR8-mediated inhibition of key upstream injury responses, including attenuation of c-Jun phosphorylation/activation as well as inhibition of p53 transactivation of BH3-only targets.

Oxygen availability and skeletal muscle oxidative capacity in patients with peripheral artery disease: implications from in vivo and in vitro assessments.

Evidence suggests that the peak skeletal muscle mitochondrial ATP synthesis rate ( Vmax) in patients with peripheral artery disease (PAD) may be attenuated due to disease-related impairments in O2 supply. However, in vitro assessments suggest intrinsic deficits in mitochondrial respiration despite ample O2 availability. To address this conundrum, Doppler ultrasound, near-infrared spectroscopy, phosphorus magnetic resonance spectroscopy, and high-resolution respirometry were combined to assess convective O2 delivery, tissue oxygenation, Vmax, and skeletal muscle mitochondrial capacity (complex I + II, state 3 respiration), respectively, in the gastrocnemius muscle of 10 patients with early stage PAD and 11 physical activity-matched healthy control (HC) subjects. All participants were studied in free-flow control conditions (FF) and with reactive hyperemia (RH) induced by a period of brief ischemia during the last 30 s of submaximal plantar flexion exercise. Patients with PAD repeated the FF and RH trials under hyperoxic conditions (FF + 100% O2 and RH + 100% O2). Compared with HC subjects, patients with PAD exhibited attenuated O2 delivery at the same absolute work rate and attenuated tissue reoxygenation and Vmax after relative intensity-matched exercise. Compared with the FF condition, only RH + 100% O2 significantly increased convective O2 delivery (~44%), tissue reoxygenation (~54%), and Vmax (~60%) in patients with PAD ( P < 0.05), such that Vmax was now not different from HC subjects. Furthermore, there was no evidence of an intrinsic mitochondrial deficit in PAD, as assessed in vitro with adequate O2. Thus, in combination, this comprehensive in vivo and in vitro investigation implicates O2 supply as the predominant factor limiting mitochondrial oxidative capacity in early stage PAD. NEW & NOTEWORTHY Currently, there is little accord as to the role of O2 availability and mitochondrial function in the skeletal muscle dysfunction associated with peripheral artery disease. This is the first study to comprehensively use both in vivo and in vitro approaches to document that the skeletal muscle dysfunction associated with early stage peripheral artery disease is predominantly a consequence of limited O2 supply and not the impact of an intrinsic mitochondrial defect in this pathology.

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.

SMG-1 kinase attenuates mitochondrial ROS production but not cell respiration deficits during hyperoxia

ABSTRACTPurpose: Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia. Materials and Methods: Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes. Results: Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53. Conclusions: Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.

Sab mediates mitochondrial dysfunction involved in imatinib mesylate-induced cardiotoxicity.

Imatinib mesylate is an effective treatment for chronic myelogenous leukemia and gastrointestinal stromal tumors. Although imatinib mesylate is highly tolerable, it has been implicated in severe congestive heart failure in mouse models and patients. A hallmark of imatinib mesylate-induced cardiotoxicity is mitochondrial dysfunction. The mitochondrial scaffold Sab has been implicated in facilitating signaling responsible for mitochondrial dysfunction in a c-Jun N-terminal Kinase (JNK)-dependent manner. We examined the impact of Sab-mediated signaling on imatinib mesylate cardiotoxicity in H9c2 rat cardiomyocyte-like cells. Silencing Sab increased the LD50 of imatinib mesylate 4-fold in H9c2 cells. Disrupting Sab-mediated signaling prevented imatinib mesylate-induced apoptosis as well. Knockdown of Sab or inhibition with a small peptide prevented oxidative stress, which was indicated by decreased reactive oxygen species production, lipid peroxidation, and protein carbonylation. Further, inhibition of Sab-related signaling partially rescued deficits in mitochondrial respiration, ATP production, and membrane potential in imatinib mesylate-treated H9c2 cells. Conversely, over-expression of Sab in H9c2 cells increased the cardiotoxicity of imatinib mesylate in vitro decreasing the LD50 over 4-fold. Sab expression was induced in H9c2 cells following cardiovascular-like stress in an AP-1 dependent manner. These data demonstrate that imatinib mesylate influences mitochondrial signaling leading to mitochondrial dysfunction and cardiotoxicity.

Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent StemCells.

SummaryHuntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.

Lipopolysaccharide-induced functional and structural injury of the mitochondria in the nigrostriatal pathway

Accumulating evidence suggests that chronic inflammation plays a role in the progressive dopaminergic neurodegeneration that occurs in Parkinson’s disease. It has been hypothesized that inflammation mediates neuronal damage via exacerbation of a vicious cycle of oxidative stress and mitochondrial dysfunction. The bacterial endotoxin, lipopolysaccharide (LPS), induces microglial activation and inflammation driven dopaminergic neurodegeneration. In order to test the hypothesis that LPS-induced inflammatory response might damage mitochondrial structure and function leading to nigral dopaminergic neuron loss, we injected LPS or saline into the striatum of rats. Here, we found that intrastriatal LPS induced deficit in mitochondrial respiration, damage to mitochondrial cristae, mitochondrial oxidation and nitration. Finally, we found significant loss of dopaminergic neurons in the substantia nigra one week after LPS injection. This study indicates that LPS-induced dopaminergic neurodegeneration might be exerted by mitochondrial injury.