Altered Mitochondrial Dynamics Research Articles

Techniques for Quantitative Analysis of Mitochondrial Dynamics

Alterations of mitochondrial dynamics (fusion/fission and motility) and ER-mitochondria associations have emerged as feature of a number of pathways. Analysis of these aspects has remained largely qualitative and manual, limiting their potential use as biomarkers. Here we demonstrate techniques for unbiased, quantitative and automatized image analysis of mitochondrial dynamics and ER-mitochondrial interactions. Mitochondrial fusion is evaluated in cultured cells expressing photoactivatable GFP and DsRed in the mitochondria by a simple count of green-only pixels following photoactivation. This approach is validated against a manual count of fusion events. We have employed difference image calculation to measure motility for several years; now we have developed a method to automatically calculate the optimal threshold for this assay. Finally, we have developed methods for quantitative evaluation of the interface between ER and mitochondria in electron micrographs. These scripts provide both granular data on individual interactions (distances, surface covered, etc.) and also attempt to create a simple ‘score’ that can easily be used to compare across conditions.

Physical exercise mitigates doxorubicin-induced brain cortex and cerebellum mitochondrial alterations and cellular quality control signaling

Doxorubicin (DOX) is a highly effective anti-neoplastic agent, whose clinical use is limited by a dose-dependent mitochondrial toxicity in non-target tissues, including the brain. Here we analyzed the effects of distinct exercise modalities (12-week endurance treadmill—TM or voluntary free-wheel activity—FW) performed before and during sub-chronic DOX treatment on brain cortex and cerebellum mitochondrial bioenergetics, oxidative stress, permeability transition pore (mPTP), and proteins involved in mitochondrial biogenesis, apoptosis and auto(mito)phagy.Male Sprague–Dawley rats were divided into saline-sedentary (SAL+SED), DOX-sedentary (DOX+SED; 7-week DOX (2mg·kg−1per week)), DOX+TM and DOX+FW. Animal behavior and post-sacrifice mitochondrial function were assessed. Oxidative phosphorylation (OXPHOS) subunits, oxidative stress markers or related proteins (SIRT3, p66shc, UCP2, carbonyls, MDA, –SH, aconitase, Mn-SOD), as well as proteins involved in mitochondrial biogenesis (PGC1α and TFAM) were evaluated. Apoptotic signaling was followed through caspases 3, 8 and 9-like activities, Bax, Bcl2, CypD, ANT and cofilin expression. Mitochondrial dynamics (Mfn1, Mfn2, OPA1 and DRP1) and auto(mito)phagy (LC3II, Beclin1, Pink1, Parkin and p62)-related proteins were measured by semi-quantitative Western blotting.DOX impaired behavioral performance, mitochondrial function, including lower resistance to mPTP and increased apoptotic signaling, decreased the content in OXPHOS complex subunits and increased oxidative stress in brain cortex and cerebellum. Molecular markers of mitochondrial biogenesis, dynamics and autophagy were also altered by DOX treatment in both brain subareas. Generally, TM and FW were able to mitigate DOX-related impairments in brain cortex and cerebellum mitochondrial activity, mPTP and apoptotic signaling.We conclude that the alterations in mitochondrial biogenesis, dynamics and autophagy markers induced by exercise performed before and during treatment may contribute to the observed protective brain cortex and cerebellum mitochondrial phenotype, which is more resistant to oxidative damage and apoptotic signaling in sub-chronically DOX treated animals.

Protective effect of tetrahydropalmatine against d-galactose induced memory impairment in rat

Aging is associated with Alzheimer's disease (AD), cardiovascular disease and cancer. Oxidative stress is considered as a major factor that accelerates the aging process. d-galactose (d-gal), a reducing sugar, induces oxidative stress resulting in alteration in mitochondrial dynamics and apoptosis of neurons. To understand the ability of tetrahydropalmatine (THP) to ameliorate memory impairment caused by aging, we investigated the effect of THP on d-gal induced memory impairment in rats. Subcutaneous injection of d-gal (100mg/kg/d) for 8weeks caused memory loss as detected by the Morris water maze and morphologic abnormalities of neurons in the hippocampus regions and cortex of rat brain. THP treatment ameliorated d-gal induced memory impairment associated with the decrease of malondialdehyde (MDA) and nitric oxide (NO) contents, as well as the increase of glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. THP treatment was also found to reverse the abnormality of acetylcholine (ACh) levels and acetylcholinesterase (AChE) activities. In addition, treatment with THP could decrease the expression of nuclear factor κ (NF-κB) and glial fibrillary acidic protein (GFAP) which prevented the neuroinflammation and memory impairment in the d-gal treated rats. Taken together, these results clearly demonstrated that subcutaneous injection of d-gal produced memory deficits, meanwhile THP could protect neuron from d-gal insults and improve cognition. This study provided an experimental basis for clinical application of THP in AD therapy.

Abstract 16277: Gender-dimorphic Effect of Optic Atrophy Protein (OPA)-1 on Platelet Activation and Thrombosis

Mitochondrial dysfunction is strongly associated with many disorders including metabolic and cardiovascular diseases and their complications. Preliminary studies of platelets from the Framingham offspring cohort, revealed enrichment in transcripts encoding for the mitochondrial dynamics proteins: optic atrophy-I in females, and mitofusin II in both genders that correlated with cardiovascular risk. These transcripts encode established regulators of mitochondrial fusion, electron transport complex (ETC) assembly and apoptosis, thereby suggesting that altered mitochondrial dynamics may contribute to platelet dysfunction. Thus, to determine whether the enrichment in OPA1 transcripts in females that correlates with increased CVD risk represents an adaptive or pathophysiological response, mice with platelet-specific deletion of OPA1 (pOPA-1 KO) were generated. Male pOPA-1 KO mice exhibited compromised cristae morphology and a 50% reduction in mitochondrial DNA. Additionally, mitochondrial function assessed by an extracellular flux analyzer (XF24 Seahorse biosciences) was reduced by 50%. Mitochondrial dysfunction in male pOPA-1 KO mice was associated with heightened agonist response (a hyperactive phenotype). Male pOPA-1 KO mice had a shortened time to stable occlusion of the carotid artery as assessed in vivo by rose Bengal photochemical injury, and were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava. In contrast, cristae morphology was normal and mitochondrial DNA unchanged in females. Additionally, the females revealed no evidence of heightened agonist response or increased incidence of thrombus formation. Thus OPA1 plays an essential role in regulating platelet mitochondrial function, platelet activation and thrombosis in males but not in females. These findings suggest that whereas maintaining OPA1 expression might be a protective adaptation in males, OPA1 induction in platelets of females with diabetes might not protect against platelet hyperactivation.

Activation of the NLRP3 inflammasome induces vascular dysfunction in obese OLETF rats

ObjectiveObesity-induced vascular dysfunction is related to chronic low-grade systemic inflammation. Recent studies indicate that NLRP3, a multiprotein complex formed by NOD-like receptor (NLR) family members, is a key component mediating internal sterile inflammation, but the role in obesity-related vascular dysfunction is largely unknown. In the present study, we investigate whether NLRP3 activation is involved in vascular inflammation in obese Otsuka Long-Evans Tokushima Fatty rats (OLETF). Methods and resultsMale OLETF with their control Long-Evans Tokushima Otsuka rats (LETO) were studied at 3 and 12 months of age. Aortic relaxation in response to acetylcholine decreased gradually with age in both strains, with early and persistent endothelium dysfunction in obese OLETF compared with age-matched LETO controls. These changes are associated with parallel changes of aortic endothelial nitric oxide synthase (eNOS) content, macrophage accumulation and intimal thickening. NLRP3 increased in OLETF rats compared to LETO. Consistent with inflammasome activation, the conversion of procaspase-1 to cleaved and activated forms as well as IL-1β markedly increased in OLETF rats. Additionally, we observed increased expression of dynamin-related protein-1 (Drp1) and decreased fusion-relative protein optic atropy-1(OPA1). Altered mitochondrial dynamics was associated with elevated oxidative stress level in OLETF aortas. ConclusionsThese results demonstrate that obesity seems to accelerate endothelial dysfunction in OLETFs via the activation of NLRP3 and mitochondrial dysfunction.

DRP1 inhibition rescues retinal ganglion cells and their axons by preserving mitochondrial integrity in a mouse model of glaucoma.

Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic neuropathies.

Mitochondrial dynamic abnormalities in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease characterized by progressive loss of motor neurons in the brainstem and spinal cord. Currently, there is no cure or effective treatment for ALS and the cause of disease is unknown in the majority of ALS cases. Neuronal mitochondria dysfunction is one of the earliest features of ALS. Mitochondria are highly dynamic organelles that undergo continuous fission, fusion, trafficking and turnover, all of which contribute to the maintenance of mitochondrial function. Abnormal mitochondrial dynamics have been repeatedly reported in ALS and increasing evidence suggests altered mitochondrial dynamics as possible pathomechanisms underlying mitochondrial dysfunction in ALS. Here, we provide an overview of mitochondrial dysfunction and dynamic abnormalities observed in ALS, and discuss the possibility of targeting mitochondrial dynamics as a novel therapeutic approach for ALS.

Alterations in mitochondrial dynamics induced by tebufenpyrad and pyridaben in a dopaminergic neuronal cell culture model

Tebufenpyrad and pyridaben are two agro-chemically important acaricides that function like the known mitochondrial toxicant rotenone. Although these two compounds have been commonly used to kill populations of mites and ticks in commercial greenhouses, their neurotoxic profiles remain largely unknown. Therefore, we investigated the effects of these two pesticides on mitochondrial structure and function in an in vitro cell culture model using the Seahorse bioanalyzer and confocal fluorescence imaging. The effects were compared with rotenone. Exposing rat dopaminergic neuronal cells (N27 cells) to tebufenpyrad and pyridaben for 3h induced dose-dependent cell death with an EC50 of 3.98μM and 3.77μM, respectively. Also, tebufenpyrad and pyridaben (3μM) exposure induced reactive oxygen species (ROS) generation and m-aconitase damage, suggesting that the pesticide toxicity is associated with oxidative damage. Morphometric image analysis with the MitoTracker red fluorescent probe indicated that tebufenpyrad and pyridaben, as well as rotenone, caused abnormalities in mitochondrial morphology, including reduced mitochondrial length and circularity. Functional bioenergetic experiments using the Seahorse XF96 analyzer revealed that tebufenpyrad and pyridaben very rapidly suppressed the basal mitochondrial oxygen consumption rate similar to that of rotenone. Further analysis of bioenergetic curves also revealed dose-dependent decreases in ATP-linked respiration and respiratory capacity. The luminescence-based ATP measurement further confirmed that pesticide-induced mitochondrial inhibition of respiration is accompanied by the loss of cellular ATP. Collectively, our results suggest that exposure to the pesticides tebufenpyrad and pyridaben induces neurotoxicity by rapidly initiating mitochondrial dysfunction and oxidative damage in dopaminergic neuronal cells. Our findings also reveal that monitoring the kinetics of mitochondrial respiration with Seahorse could be used as an early neurotoxicological high-throughput index for assessing the risk that pesticides pose to the dopaminergic neuronal system.

Insulin resistance by TNF-α is associated with mitochondrial dysfunction in 3T3-L1 adipocytes and is ameliorated by punicic acid, a PPARγ agonist

Punicic acid (PA), a poly unsaturated fatty acid found abundantly in pomegranate seed oil is reported to have PPARγ agonist property. TNF-α mediated insulin resistance plays an important role in the pathogenesis of diabetes and is associated with severe mitochondrial impairment. In this study, PA was evaluated for its ability to ameliorate TNF-α induced mitochondrial dysfunctions in 3T3-L1 adipocytes. For this, we examined the alterations in mitochondrial energetics, biogenesis, transmembrane potential and dynamics in TNF-α induced insulin resistant model of 3T3-L1 adipocytes. PA improved glucose uptake, ROS accumulation, mitochondrial biogenesis and energetics in TNF-α treated cells. In addition, treatment with PA was found to ameliorate TNF-α induced alterations in proteins associated with mitochondrial dynamics like FIS1 and OPA1. These findings suggest that PA can be considered as an active lead for the management of insulin resistance and associated mitochondrial dysfunctions.

Physical exercise improves brain cortex and cerebellum mitochondrial bioenergetics and alters apoptotic, dynamic and auto(mito)phagy markers

We here investigate the effects of two exercise modalities (endurance treadmill training-TM and voluntary free-wheel activity-FW) on the brain cortex and cerebellum mitochondrial bioenergetics, permeability transition pore (mPTP), oxidative stress, as well as on proteins involved in mitochondrial biogenesis, apoptosis, and quality control. Eighteen male rats were assigned to sedentary-SED, TM and FW groups. Behavioral alterations and ex vivo brain mitochondrial function endpoints were assessed. Proteins involved in oxidative phosphorylation (OXPHOS, including the adenine nucleotide translocator), oxidative stress markers and regulatory proteins (SIRT3, p66shc, UCP2, carbonyls, MDA, -SH, aconitase, Mn-SOD), as well as proteins involved in mitochondrial biogenesis (PGC1α, TFAM) were evaluated. Apoptotic signaling was measured through quantifying caspase 3, 8 and 9-like activities, Bax, Bcl2, CypD, and cofilin expression. Mitochondrial dynamics (Mfn1/2, OPA1 and DRP1) and auto(mito)phagy (LC3II, Beclin1, Pink1, Parkin, p62)-related proteins were also measured by Western blotting. Only the TM exercise group showed increased spontaneous alternation and exploratory activity. Both exercise regimens improved mitochondrial respiratory activity, increased OXPHOS complexes I, III and V subunits in both brain subareas and decreased oxidative stress markers. Increased resistance to mPTP and decreased apoptotic signaling were observed in the brain cortex from TM and in the cerebellum from TM and FW groups. Also, exercise increased the expression of proteins involved in mitochondrial biogenesis, autophagy and fusion, simultaneous with decreased expression of mitochondrial fission-related protein DRP1. In conclusion, physical exercise improves brain cortex and cerebellum mitochondrial function, decreasing oxidative stress and apoptotic related markers. It is also possible that favorable alterations in mitochondrial biogenesis, dynamics and autophagy signaling induced by exercise contributed to increased mitochondrial plasticity leading to a more robust phenotype.

Regulation of mitochondrial trafficking, function and quality control by the mitochondrial GTPases Miro1 and Miro2.

Regulated trafficking of mitochondria in neurons is essential for providing ATP at the correct spatial location to power neural function and computation, and for providing calcium buffering at sites of calcium entry or release. Indeed the regulation of mitochondrial distribution, morphology and function are proposed to play an important role in neuronal development and survival but the regulatory mechanisms remain unclear. Miro family proteins (Miro1 and Miro2 in mammals) contain a transmembrane domain locating them to the outer mitochondrial membrane, along with two GTPase domains and two calcium-sensing EF-hand domains that face into the cytosol, and play a key role in regulating mitochondrial transport. Miro proteins mediate mitochondrial trafficking in neurons by linking mitochondria to kinesin and dynein motor proteins for their transport in axons and dendrites. Miro proteins are also targets for the Parkinson’s Disease associated PINK1/Parkin mitophagy pathway and are therefore implicated in altered mitochondrial dynamics during mitophagy. Here I will present our recent results on the role played by Miro proteins in regulating mitochondrial trafficking and quality control. The role that Miro-mediated control of mitochondrial trafficking and turnover plays in regulating neuronal development, function and pathology will also be explored.

Implications of mitochondrial dynamics on neurodegeneration and on hypothalamic dysfunction

Mitochondrial dynamics is a term that encompasses the movement of mitochondria along the cytoskeleton, regulation of their architecture, and connectivity mediated by tethering and fusion/fission. The importance of these events in cell physiology and pathology has been partially unraveled with the identification of the genes responsible for the catalysis of mitochondrial fusion and fission. Mutations in two mitochondrial fusion genes (MFN2 and OPA1) cause neurodegenerative diseases, namely Charcot-Marie Tooth type 2A and autosomal dominant optic atrophy (ADOA). Alterations in mitochondrial dynamics may be involved in the pathophysiology of prevalent neurodegenerative conditions. Moreover, impairment of the activity of mitochondrial fusion proteins dysregulates the function of hypothalamic neurons, leading to alterations in food intake and in energy homeostasis. Here we review selected findings in the field of mitochondrial dynamics and their relevance for neurodegeneration and hypothalamic dysfunction.

Downregulation of dynamin-related protein 1 contributes to impaired autophagic flux and angiogenic function in senescent endothelial cells.

Recent studies have shown that altered mitochondrial dynamics impairs the function in senescent endothelial cells (ECs). However, the underlying molecular mechanism remains to be elucidated. Herein, we investigated the role and underlying mechanism of mitochondrial fission protein dynamin-related protein 1 (DRP1) in vascular aging. We found that DRP1 expression is decreased in senescent ECs, accompanied with long interconnected mitochondria and impaired angiogenic function. In addition, there was marked increase of autophagosomes but not of autolysosomes (assessed as punctate dual fluorescent mCherry-GFP (green fluorescent protein) tandem-tagged light chain 3 expression) in senescent ECs, indicating impaired autophagic flux. DRP1 knockdown or pharmacological inhibition in young ECs resulted in elongated mitochondria, suppressed autophagic flux, premature senescence, and impaired angiogenic function. In contrast, adenoviral-mediated overexpression of DRP1 in senescent ECs restored autophagic flux and improved angiogenic function. EC senescence was associated with the increase of mitochondrial reactive oxygen species and antioxidant N-acetyl-cysteine restored autophagosome clearance and improved angiogenic function. Consistently, en face staining of old rat thoracic aorta revealed a decrease of DRP1 expression and increase of autophagosomes accumulation. Furthermore, in vivo knockdown of Drp1 in common carotid arteries significantly impaired the autophagosome clearance. Importantly, downregulation of Drp1 directly abrogated microvessels outgrowth from ex vivo aortic rings. These results suggest that loss of DRP1 during senescence exacerbates ECs dysfunction by increasing mitochondrial reactive oxygen species and subsequently inhibiting autophagic flux.

Metabolic dysfunction and altered mitochondrial dynamics in the utrophin-dystrophin deficient mouse model of duchenne muscular dystrophy.

The utrophin-dystrophin deficient (DKO) mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD). However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL) muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1) and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients.

Tetracyclines Disturb Mitochondrial Function across Eukaryotic Models: A Call for Caution in Biomedical Research.

In recent years, tetracyclines, such as doxycycline, have become broadly used to control gene expression by virtue of the Tet-on/Tet-off systems. However, the wide range of direct effects of tetracycline use has not been fully appreciated. We show here that these antibiotics induce a mitonuclear protein imbalance through their effects on mitochondrial translation, an effect that likely reflects the evolutionary relationship between mitochondria and proteobacteria. Even at low concentrations, tetracyclines induce mitochondrial proteotoxic stress, leading to changes in nuclear gene expression and altered mitochondrial dynamics and function in commonly used cell types, as well as worms, flies, mice, and plants. Given that tetracyclines are so widely applied in research, scientists should be aware of their potentially confounding effects on experimental results. Furthermore, these results caution against extensive use of tetracyclines in livestock due to potential downstream impacts on the environment and human health.

Mitochondrial dynamics in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an idiopathic cardiopulmonary disease characterized by obstruction of small pulmonary arteries. Vascular obstruction is the consequence of excessive proliferation and apoptosis resistance of vascular cells, as well as inflammation, thrombosis, and vasoconstriction. Vascular obstruction increases the afterload faced by the right ventricle (RV), leading to RV failure. The proliferative, obstructive vasculopathy of PAH shares several mitochondrial abnormalities with cancer, notably a shift to aerobic glycolysis and mitochondrial fragmentation. Mitochondria in the pulmonary artery smooth muscle cell (PASMC) normally serve as oxygen sensors. In PAH, acquired mitochondrial abnormalities, including epigenetic silencing of superoxide dismutase (SOD2), disrupt oxygen sensing creating a pseudo-hypoxic environment characterized by normoxic activation of hypoxia-inducible factor-1α (HIF-1α). The resulting metabolic shift to aerobic glycolysis (the Warburg phenomenon) reflects inhibition of pyruvate dehydrogenase by pyruvate dehydrogenase kinases. In addition, altered mitochondrial dynamics result in mitochondrial fragmentation. The molecular basis of this structural change includes upregulation and activation of fission mediators, notably dynamin-related protein 1 (DRP-1), and downregulation of fusion mediators, especially mitofusin-2 (MFN2). These pathogenic mitochondrial abnormalities offer new therapeutic targets. Inhibition of mitotic fission or enhancement of fusion in PAH PASMC slows cell proliferation, causes cell cycle arrest, and induces apoptosis. DRP-1 inhibition or MFN2 gene therapy can regress PAH in experimental models of PAH. This review focuses on the etiology of mitochondrial fragmentation in PAH and explores the therapeutic implications of mitochondrial dynamics in the pulmonary vasculature and RV.

The role of alterations in mitochondrial dynamics and PGC-1α over-expression in fast muscle atrophy following hindlimb unloading.

Skeletal muscle atrophy occurs as a result of disuse. Although several studies have established that a decrease in protein synthesis and increase in protein degradation lead to muscle atrophy, little is known about the triggers underlying such processes. A growing body of evidence challenges oxidative stress as a trigger of disuse atrophy; furthermore, it is also becoming evident that mitochondrial dysfunction may play a causative role in determining muscle atrophy. Mitochondrial fusion and fission have emerged as important processes that govern mitochondrial function and PGC-1α may regulate fusion/fission events. Although most studies on mice have focused on the anti-gravitary slow soleus muscle as it is preferentially affected by disuse atrophy, several fast muscles (including gastrocnemius) go through a significant loss of mass following unloading. Here we found that in fast muscles an early down-regulation of pro-fusion proteins, through concomitant AMP-activated protein kinase (AMPK) activation, can activate catabolic systems, and ultimately cause muscle mass loss in disuse. Elevated muscle PGC-1α completely preserves muscle mass by preventing the fall in pro-fusion protein expression, AMPK and catabolic system activation, suggesting that compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy. The mechanisms triggering disuse muscle atrophy remain of debate. It is becoming evident that mitochondrial dysfunction may regulate pathways controlling muscle mass. We have recently shown that mitochondrial dysfunction plays a major role in disuse atrophy of soleus, a slow, oxidative muscle. Here we tested the hypothesis that hindlimb unloading-induced atrophy could be due to mitochondrial dysfunction in fast muscles too, notwithstanding their much lower mitochondrial content. Gastrocnemius displayed atrophy following both 3 and 7days of unloading. SOD1 and catalase up-regulation, no H2 O2 accumulation and no increase of protein carbonylation suggest the antioxidant defence system efficiently reacted to redox imbalance in the early phases of disuse. A defective mitochondrial fusion (Mfn1, Mfn2 and OPA1 down-regulation) occurred together with an impairment of OXPHOS capacity. Furthermore, at 3days of unloading higher acetyl-CoA carboxylase (ACC) phosphorylation was found, suggesting AMP-activated protein kinase (AMPK) pathway activation. To test the role of mitochondrial alterations we used Tg-mice overexpressing PGC-1α because of the known effect of PGC-1α on stimulation of Mfn2 expression. PGC-α overexpression was sufficient to prevent (i) the decrease of pro-fusion proteins (Mfn1, Mfn2 and OPA1), (ii) activation of the AMPK pathway, (iii) the inducible expression of MuRF1 and atrogin1 and of authopagic factors, and (iv) any muscle mass loss in response to disuse. As the effects of increased PGC-1α activity were sustained throughout disuse, compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy also in fast muscles.

Hypobaric Hypoxia Imbalances Mitochondrial Dynamics in Rat Brain Hippocampus.

Brain is predominantly susceptible to oxidative stress and mitochondrial dysfunction during hypobaric hypoxia, and therefore undergoes neurodegeneration due to energy crisis. Evidences illustrate a high degree of association for mitochondrial fusion/fission imbalance and mitochondrial dysfunction. Mitochondrial fusion/fission is a recently reported dynamic mechanism which frequently occurs among cellular mitochondrial network. Hence, the study investigated the temporal alteration and involvement of abnormal mitochondrial dynamics (fusion/fission) along with disturbed mitochondrial functionality during chronic exposure to hypobaric hypoxia (HH). The Sprague-Dawley rats were exposed to simulated high altitude equivalent to 25000 ft for 3, 7, 14, 21, and 28 days. Mitochondrial morphology, distribution within neurons, enzyme activity of respiratory complexes, Δψ m, ADP: ATP, and expression of fission/fusion key proteins were determined. Results demonstrated HH induced alteration in mitochondrial morphology by damaged, small mitochondria observed in neurons with disturbance of mitochondrial functionality and reduced mitochondrial density in neuronal processes manifested by excessive mitochondrial fragmentation (fission) and decreased mitochondrial fusion as compared to unexposed rat brain hippocampus. The study suggested that imbalance in mitochondrial dynamics is one of the noteworthy mechanisms occurring in hippocampal neurons during HH insult.

Hyperglycemia- and neuropathy-induced changes in mitochondria within sensory nerves.

ObjectiveThis study focused on altered mitochondrial dynamics as a potential mechanism for diabetic peripheral neuropathy (DPN). We employed both an in vitro sensory neuron model and an in situ analysis of human intraepidermal nerve fibers (IENFs) from cutaneous biopsies to measure alterations in the size distribution of mitochondria as a result of hyperglycemia and diabetes, respectively.MethodsNeurite- and nerve-specific mitochondrial signals within cultured rodent sensory neurons and human IENFs were measured by employing a three-dimensional visualization and quantification technique. Skin biopsies from distal thigh (DT) and distal leg (DL) were analyzed from three groups of patients; patients with diabetes and no DPN, patients with diabetes and confirmed DPN, and healthy controls.ResultsThis analysis demonstrated an increase in mitochondria distributed within the neurites of cultured sensory neurons exposed to hyperglycemic conditions. Similar changes were observed within IENFs of the DT in DPN patients compared to controls. This change was represented by a significant shift in the size frequency distribution of mitochondria toward larger mitochondria volumes within DT nerves of DPN patients. There was a length-dependent difference in mitochondria within IENFs. Distal leg IENFs from control patients had a significant shift toward larger volumes of mitochondrial signal compared to DT IENFs.InterpretationThe results of this study support the hypothesis that altered mitochondrial dynamics may contribute to DPN pathogenesis. Future studies will examine the potential mechanisms that are responsible for mitochondrial changes within IENFs and its effect on DPN pathogenesis.

Abstract 3510: N-Myc positively influences mitochondrial biogenesis in neuroblastoma

Abstract Background: Neuroblastoma is the most common extracranial tumor of childhood., accounting for approximately 7% of all cancer in children. The morbidity and mortality are affected by a number of factors - perhaps most significantly by amplification of the oncogene, N-MYC. Advanced stage disease with N-MYC overexpression confers the highest mortality. The N-MYC gene product (N-Myc) regulates the transcription of many genes, and when overexpressed is thought to enhance the cell cycle and dysregulate apoptosis. The function of N-Myc is virtually identical to another oncoprotein of the same family, c-Myc, yet is restricted by developmental stage and cell type. Overexpression of c-Myc has been shown to increase mitochondrial biogenesis by alterations in mitochondrial dynamics. It has been proposed that these changes may contribute to c-Myc induced tumorigenesis. We hypothesize that N-Myc amplification will have similar effects on mitochondrial biogenesis in neuroblastoma. Design/Methods: To perform these studies we utilized a cell line with endogenous levels of N-Myc (SK-N-SH). In these cells we have ectopically overexpressed N-Myc and examined the cells for changes in the mitochondria with various assays, including: Western blots, electron microscopy, flow cytometry, ATP assay and confocal microscopy. Results: We found a X-fold increase in mitochondrial mass in cells that overexpress N-Myc,, as well as a more tubular appearance with less organized cristae. Additionally, N-Myc changed the expression of various mitochondrial fusion and fission proteins. The most dramatic change was seen in the expression of the fusion protein Opa1, which changed by ten-fold. This, combined with a decrease in expression of fission proteins DRP1 and MFF, may account for the more tubular appearance of mitochondria in N-Myc overexpressing cells. Conclusions N-Myc amplification appeared to increase mitochondrial mass. This may occur as a result of increased expression of mitochondrial fusion proteins, as previously observed in c-Myc overexpressing cells. Additionally, mitochondrial function is influenced by N-Myc overexpression. Therefore, N-Myc amplification appears to positively influence mitochondrial biogenesis in neuroblastoma. Further studies are needed to determine if this increase in mitochondrial mass and function is necessary for the development and maintenance of neuroblastoma. Citation Format: Manika Sharma, James A. Graves. N-Myc positively influences mitochondrial biogenesis in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3510. doi:10.1158/1538-7445.AM2014-3510