Gordon L. Rintoul

Glutamate Decreases Mitochondrial Size and Movement in Primary Forebrain Neurons

Mitochondria are essential to maintain neuronal viability. In addition to the generation of ATP and maintenance of calcium homeostasis, the effective delivery of mitochondria to the appropriate location within neurons is also likely to influence their function. In this study we examined mitochondrial movement and morphology in primary cultures of rat forebrain using a mitochondrially targeted enhanced yellow fluorescent protein (mt-eYFP). Mt-eYFP-labeled mitochondria display a characteristic elongated phenotype and also move extensively. Application of glutamate to cultures results in a rapid diminution of movement and also an alteration from elongated to rounded morphology. This effect required the entry of calcium and was mediated by activation of the NMDA subtype of glutamate receptor. Treatment of cultures with an uncoupler or blocking ATP synthesis with oligomycin also stopped movement but did not alter morphology. Interestingly, application of glutamate together with the uncoupler did not prevent the changes in movement or shape but facilitated recovery after washout of the stimuli. This suggests that the critical target for calcium in this paradigm is cytosolic. These studies demonstrate that in addition to altering the bioenergetic properties of mitochondria, neurotoxins can also alter mitochondrial movement and morphology. We speculate that neurotoxin-mediated impairment of mitochondrial delivery may contribute to the injurious effects of neurotoxins.

Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons.

Huntingtons 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.

Zn2+ Inhibits Mitochondrial Movement in Neurons by Phosphatidylinositol 3-Kinase Activation

Mitochondria have been identified as targets of the neurotoxic actions of zinc, possibly through decreased mitochondrial energy production and increased reactive oxygen species accumulation. It has been hypothesized that impairment of mitochondrial trafficking may be a mechanism of neuronal injury. Here, we report that elevated intraneuronal zinc impairs mitochondrial trafficking. At concentrations just sufficient to cause injury, zinc rapidly inhibited mitochondrial movement without altering morphology. Zinc chelation initially restored movement, but the actions of zinc became insensitive to chelator in <10 min. A search for downstream signaling events revealed that inhibitors of phosphatidylinositol (PI) 3-kinase prevented this zinc effect on movement. Moreover, transient inhibition of PI 3-kinase afforded neuroprotection against zinc-mediated toxicity. These data illustrate a novel mechanism that regulates mitochondrial trafficking in neurons and also suggest that mitochondrial trafficking may be closely coupled to neuronal viability.

Mitochondrial trafficking and morphology in neuronal injury

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.

Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potential

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.

Glutamate mobilizes [Zn2+]i through Ca2+-dependent reactive oxygen species accumulation

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 μM) 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 A2, 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.

New perspectives on mitochondrial morphology in cell function.

Mitochondria are a node of integration for intracellular signaling pathways and their morphology changes seem to be tightly associated with their function. New data show that morphology is one of the parameters involved in mitochondrias choice between promoting cell death and protecting cells against general metabolic jeopardy.

Mitochondrial Stop and Go: Signals That Regulate Organelle Movement

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.

Inhibition of Calcium‐Dependent NMDA Receptor Current Rundown by Calbindin‐D28k

Abstract : NMDA receptors are regulated by several different calcium‐dependent processes. To determine if the presence of the intracellular calcium‐binding protein calbindin‐D28k can influence the calcium regulation of NMDA receptor activity,human embryonic kidney 293 cells were co‐transfected with cDNAs for NMDA receptor subunits and cablinding. Recordings were made using the nystatin perforated patch technique to preserve intracellular contents. When compared with control cells (transfected with cDNA enconding β‐galactosidase in place of calbindin), the presence of calbindin had no effect on either calcium‐dependent inactivation or the calciumsensitive, time‐dependent increase in glycine‐independent desensitization of NMDA receptor‐mediated currents. However, the development of calcium‐dependent rundown of peak glutamate‐evoked current was slowed significantly in calbindin versus β‐galactosidase cotransfected cells. This result was true for cells transfected with either NR1/NR2A or NR1/NR2B subunits, although calbindin was relatively less effective at inhibiting rundown in NR1/NR2B‐expressing cells. NMDA peak current rundown has been attributed to calcium‐induced depolymerization of the actin cytoskeleton. Therefore, our results indicate that although calbindin may not influence calcium‐dependent regulatory processes occurring very near the NMDA receptor channel, it appears to be more effective at buffering local elevations in intracellular calcium at the actin cytoskeleton.

Changes in mitochondrial morphology induced by calcium or rotenone in primary astrocytes occur predominantly through ros-mediated remodeling

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. Mitochondrial morphology is traditionally ascribed to a balance of fission and fusion processes. We have shown that mitochondria can undergo more pliable transformations; remodeling, rounding, or stretching. We demonstrate that remodeling, not fission, is the primary contributor to calcium mediated changes in mitochondrial morphology in primary astrocytes. Others have shown fission is mediated by calcineurin. Our results suggest the remodeling process distinct from fission and is independently regulated through a ROS‐signaling mechanism (CsA: Cyclosporine A; NAC: N‐acetyl‐l‐cysteine; GSH: Reduced‐L‐Glutathione).