17. Iworima, DG; Pasqualotto, BA; Rintoul, GL. (2016) Kif5 regulates mitochondrial movement, morphology, function and neuronal survival.Molecular and Cellular Neuroscience 72: 22-33 Kif5 regulates mitochondrial movement, morphology, function and neuronal survival
kif5; Mitochondrial dynamics; Mitochondrial remodelling excitotoxicity; Mitochondrial trafficking; ro-GFP; ATeam
Due to the unique architecture of neurons, trafficking of mitochondria throughout processes to regions of high energetic demand is critical to sustain neuronal health. It has been suggested that compromised mitochondrial trafficking may play a role in neurodegenerative diseases. We evaluated the consequences of disrupted kif5c-mediated mitochondrial trafficking on mitochondrial form and function in primary rat cortical neurons. Morphological changes in mitochondria appeared to be due to remodelling, a phenomenon distinct from mitochondrial fission, which resulted in punctate-shaped mitochondria. We also demonstrated that neurons displaying punctate mitochondria exhibited relatively decreased ROS and increased cellular ATP levels using ROS-sensitive GFP and ATP FRET probes, respectively. Somewhat unexpectedly, neurons overexpressing the dominant negative form of kif5c exhibited enhanced survival following excitotoxicity, suggesting that the impairment of mitochondrial trafficking conferred some form of neuroprotection. However, when neurons were exposed to H2O2, disruption of kif5c exacerbated cell death indicating that the effect on cell viability was dependent on the mode of toxicity. Our results suggest a novel role of kif5c. In addition to mediating mitochondrial transport, kif5c plays a role in the mechanism of regulating mitochondrial morphology. Our results also suggest that kif5c mediated mitochondrial dynamics may play an important role in regulating mitochondrial function and in turn cellular health. Moreover, our studies demonstrate an interesting interplay between the regulation of mitochondrial motility and morphology. (C) 2016 Elsevier Inc. All rights reserved. DOI
15. Deheshi, S; Dabiri, B; Fan, S; Tsang, M; Rintoul, GL. (2015) Changes in mitochondrial morphology induced by calcium or rotenone in primary astrocytes occur predominantly through ros-mediated remodeling.Journal of Neurochemistry 133: 684-699 Changes in mitochondrial morphology induced by calcium or rotenone in primary astrocytes occur predominantly through ros-mediated remodeling
antioxidants; intracellular calcium; mitochondria; mitochondrial dynamics; mitochondrial morphology; reactive oxygen species
Morphological changes in mitochondria have been primarily attributed to fission and fusion, while the more pliable transformations of mitochondria (remodeling, rounding, or stretching) have been largely overlooked. In this study, we quantify the contributions of fission and remodeling to changes in mitochondrial morphology induced by the Ca2+ ionophore 4Br-A23187 and the metabolic toxin rotenone. We also examine the role of reactive oxygen species (ROS) in the regulation of mitochondrial remodeling. In agreement with our previous studies, mitochondrial remodeling, not fission, is the primary contributor to Ca2+-mediated changes in mitochondrial morphology induced by 4Br-A23187 in rat cortical astrocytes. Treatment with rotenone produced similar results. In both paradigms, remodeling was selectively blocked by antioxidants whereas fission was not, suggesting a ROS-mediated mechanism for mitochondrial remodeling. In support of this hypothesis, inhibition of endogenous ROS by overnight incubation in antioxidants resulted in elongated reticular networks of mitochondria. Examination of inner and outer mitochondrial membranes revealed that they largely acted in concert during the remodeling process. While mitochondrial morphology is traditionally ascribed to a net output of fission and fusion processes, in this study we provide evidence that the acute pliability of mitochondria can be a dominant factor in determining their morphology. More importantly, our results suggest that the remodeling process is independently regulated through a ROS-signaling mechanism. DOI PubMed
14. Deheshi, S; Pasqualotto, BA; Rintoul, GL. (2013) Mitochondrial trafficking in neuropsychiatric diseases.Neurobiology of Disease 51: 66-71 Mitochondrial trafficking in neuropsychiatric diseases
CULTURED HIPPOCAMPAL-NEURONS; CHILDHOOD-ONSET SCHIZOPHRENIA; IMPAIRED AXONAL-TRANSPORT; ALZHEIMERS-DISEASE; NEURODEGENERATIVE DISEASES; AMYLOID-BETA; CALCIUM UNIPORTER; MONOAMINE OXIDASE; FOREBRAIN NEURONS; CORTICAL-NEURONS
Mitochondria have numerous roles in healthy neuronal functioning and in neuronal injury mechanisms. They are quite dynamic organelles in that they fuse, divide and move throughout axons and dendrites. The mechanisms of mitochondrial motility have received much attention, however the significance of the dynamic nature of mitochondria in neurons is unclear. Nonetheless, deficits in mitochondrial trafficking have been implicated in numerous neurodegenerative disorders. The role of aberrant mitochondrial trafficking in neuropsychiatric disorders is not as well understood, but may involve similar mechanisms. In this review we examine the evidence which implicates changes in mitochondrial trafficking in the pathogenesis of neuropsychiatric disorders and hypothesize how defective mitochondrial transport may contribute to disease mechanisms. (C) 2012 Elsevier Inc. All rights reserved. DOI
13. Tan, AR; Cai, AY; Deheshi, S; Rintoul, GL. (2011) Elevated intracellular calcium causes distinct mitochondrial remodelling and calcineurin-dependent fission in astrocytes.Cell Calcium 49 Elevated intracellular calcium causes distinct mitochondrial remodelling and calcineurin-dependent fission in astrocytes
Mitochondria; Astrocyte; Fission; Remodelling; Intracellular calcium
Disruptions of mitochondrial dynamics have been implicated in the pathogenesis of neurodegenerative diseases. The regulation mechanisms of mitochondrial dynamics have not been fully elucidated; however, calcium has been suggested to play a role. In the present study, we examined the role of intracellular calcium in regulating mitochondrial morphology and motility in cortical astrocytes employing different concentrations of a calcium ionophore. High levels of calcium caused a dramatic reduction in mitochondrial length, the result of two distinct phenomena: mitochondrial remodelling (or "rounding") and fission. Quantitative analysis revealed that mitochondrial remodelling/rounding was the predominant process. In addition, mitochondrial motility was reduced, as reported previously in neurons. By contrast, prolonged, more modest levels of intracellular calcium resulted in a reduction in mitochondrial length without significant effects upon mitochondrial motility. This calcium-induced reduction in mitochondrial length was not affected by the presence of calcineurin inhibitors; however, when mitochondrial fission events were specifically examined, calcineurin inhibitors had a significant inhibitory effect. This suggests that changes in mitochondrial length were primarily due to mitochondrial remodelling as opposed to fission. In the present study, we have therefore dissected the effects of calcium on mitochondrial motility, remodelling and fission. Our results suggest independent mechanisms for regulating these processes. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved. DOI
12.Rintoul, GL; Reynolds, IJ. (2010) Mitochondrial trafficking and morphology in neuronal injury.Biochimica et Biophysica Acta-Molecular Basis of Disease 1802: 143-150 Mitochondrial trafficking and morphology in neuronal injury
Mitochondria; Neurodegeneration; Fission; Fusion
Alterations in mitochondrial function may have a central role in the pathogenesis of many neurodegenerative diseases. The study of mitochondrial dysfunction has typically focused on ATP generation, calcium homeostasis and the production of reactive oxygen species. However, there is a growing appreciation of the dynamic nature of mitochondria within cells. Mitochondria are highly motile organelles, and also constantly undergo fission and fusion. This raises the possibility that impairment of mitochondrial dynamics could contribute to the pathogenesis of neuronal injury. In this review we describe the mechanisms that govern mitochondrial movement, fission and fusion. The key proteins that are involved in mitochondrial fission and fusion have also been linked to some inherited neurological diseases, including autosomal dominant optic atrophy and Charcot-Marie-Tooth disease 2A. We will discuss the evidence that altered movement, fission and fusion are associated with impaired neuronal viability. There is a growing collection of literature that links impaired mitochondrial dynamics to a number of disease models. Additionally, the concept that the failure to deliver a functional mitochondrion to the appropriate site within a neuron could contribute to neuronal dysfunction provides an attractive framework for understanding the mechanisms underlying neurologic disease. However, it remains difficult to clearly establish that altered mitochondrial dynamics clearly represent a cause of neuronal dysfunction. (C) 2009 Elsevier B.V. All rights reserved. DOI
11. Dineley, KE; Devinney, MJ; Zeak, JA; Rintoul, GL; Reynolds, IJ. (2008) Glutamate mobilizes [Zn2+](i) through Ca2+-dependent reactive oxygen species accumulation.Journal of Neurochemistry 106: 2184-2193 Glutamate mobilizes [Zn2+](i) through Ca2+-dependent reactive oxygen species accumulation
excitotoxicity; FluoZin-3; intracellular calcium; intracellular zinc; mitochondria; reactive oxygen species
Liberation of zinc from intracellular stores contributes to oxidant-induced neuronal injury. However, little is known regarding how endogenous oxidant systems regulate intracellular free zinc ([Zn2+](i)). Here we simultaneously imaged [Ca2+](i) and [Zn2+](i) to study acute [Zn2+](i) changes in cultured rat forebrain neurons after glutamate receptor activation. Neurons were loaded with fura-2FF and FluoZin-3 to follow [Ca2+](i) and [Zn2+](i), respectively. Neurons treated with glutamate (100 mu M) for 10 min gave large Ca2+ responses that did not recover after termination of the glutamate stimulus. Glutamate also increased [Zn2+](i), however glutamate-induced [Zn2+](i) changes were completely dependent on Ca2+ entry, appeared to arise entirely from internal stores, and were substantially reduced by co-application of the membrane-permeant chelator TPEN during the glutamate treatment. Pharmacological maneuvers revealed that a number of endogenous oxidant producing systems, including nitric oxide synthase, phospholipase A(2), and mitochondria all contributed to glutamate-induced [Zn2+](i) changes. We found no evidence that mitochondria buffered [Zn2+](i) during acute glutamate receptor activation. We conclude that glutamate-induced [Zn2+](i) transients are caused in part by [Ca2+](i)-induced reactive oxygen species that arises from both cytosolic and mitochondrial sources. DOI
10. Chang, D.T.W.; Rintoul, G.L.; Pandipati, S.; Reynolds, I.J. (2006) Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons.Neurobiology of Disease 22:388-400 Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons
Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine repeat in the huntingtin gene (Htt). Mitochondrial defects and protein aggregates are characteristic of affected neurons. Recent studies suggest that these aggregates impair cellular transport mechanisms by interacting with cytoskeletal components and molecular motors. Here, we investigated whether mutant Htt alters mitochondrial trafficking and morphology in primary cortical neurons. We demonstrate that full-length mutant Htt was more effective than N-terminal mutant Htt in blocking mitochondrial movement, an effect that correlated with its heightened expression in the cytosolic compartment. Aggregates impaired the passage of mitochondria along neuronal processes, causing mitochondria to accumulate adjacent to aggregates and become immobilized. Furthermore, mitochondrial trafficking was reduced specifically at sites of aggregates while remaining unaltered in regions lacking aggregates. We conclude that in cortical neurons, an early event in HD pathophysiology is the aberrant mobility and trafficking of mitochondria caused by cytosolic Htt aggregates. DOI
9.Rintoul, GL; Bennett, VJ; Papaconstandinou, NA; Reynolds, IJ. (2006) Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potential.J Neurochem 97: 800-806 Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potential
enhanced yellow fluorescent protein; mitochondrial membrane potential; mitochondrial trafficking; nitric oxide
Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3',5'-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function.
8. Malaiyandi, LM; Honick, AS; Rintoul, GL; Wang, QMJ; Reynolds, IJ. (2005) Zn2+ inhibits mitochondrial movement in neurons by phosphatidylinositol 3-kinase activation.Journal of Neuroscience 25 Zn2+ inhibits mitochondrial movement in neurons by phosphatidylinositol 3-kinase activation
green fluorescent protein; organelle transport; signal transduction; wortmannin; mitochondrial membrane potential; oxidative stress
DOI
7. Reynolds, IJ and GL Rintoul. (2004) Mitochondrial Stop and Go: Signals That Regulate Organelle Movement.Sci. Stke 2004 (251): pe46 Mitochondrial Stop and Go: Signals That Regulate Organelle Movement
Summary: In order to satisfy the metabolic and ion homeostasis demands of neurons, mitochondria must be transported to appropriate locations within cells. Although it is well established that much of this trafficking occurs on microtubules and, to a lesser extent, actin, the mechanisms by which the trafficking of mitochondria is controlled are poorly understood. A recent study by Chada and Hollenbeck shows that nerve growth factor halts the movement of mitochondria in axons by means of a mechanism that depends on activation of phosphatidylinositol 3-kinase. These studies provide important new insights into the mechanisms that regulate mitochondrial movement and control mitochondrial docking. These insights are critical to the understanding of the factors that control the distribution, location, and function of mitochondria in both healthy and injured neurons. DOI
6. Reynolds, IJ; Malaiyandi, LM; Coash, M; Rintoul, GL. (2004) Mitochondrial trafficking in neurons: A key variable in neurodegeneration?Journal of Bioenergetics and Biomembranes 36 Mitochondrial trafficking in neurons: A key variable in neurodegeneration?
mitochondrial movement; excitotoxicity; zinc; fluorescence imaging; nitric oxide
3.Rintoul, GL; Filiano, AJ; Brocard, JB; Kress, GJ; Reynolds, IJ. (2003) Glutamate decreases mitochondrial size and movement in primary forebrain neurons.Journal of Neuroscience 23 Glutamate decreases mitochondrial size and movement in primary forebrain neurons
green fluorescent protein; cytoskeleton; NMDA receptor; intracellular calcium; excitotoxicity; organelle transport
