The role of abnormal mitochondrial dynamics in the pathogenesis of Alzheimer's disease

Mitochondria play critical roles in neuronal function and almost all aspects of mitochondrial function are altered in Alzheimer neurons. Emerging evidence shows that mitochondria are dynamic organelles that undergo continuous fission and fusion, the balance of which not only controls mitochondrial morphology and number, but also regulates mitochondrial function and distribution. In this review, after a brief overview of the basic mechanisms involved in the regulation of mitochondrial fission and fusion and how mitochondrial dynamics affects mitochondrial function, we will discuss in detail our and others' recent work demonstrating abnormal mitochondrial morphology and distribution in Alzheimer's disease (AD) models and how these abnormalities may contribute to mitochondrial and synaptic dysfunction in AD. We propose that abnormal mitochondrial dynamics plays a key role in causing the dysfunction of mitochondria that ultimately damage AD neurons.     

A new method for quantifying mitochondrial axonal transport

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.     

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO-mediated pathways. Fluorescently labeled mitochondria were studied using time-lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re-oxygenation or to diethyleneamine/nitric oxide adduct, DETA-NO (100-500 microm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 +/- 1.3% at 0 h and 7.4 +/- 1.7% at 1 h of re-oxygenation, versus 25.6 +/- 1.4% in controls (p < 0.05). Mean mitochondrial velocity (microm s(-1)) decreased from 0.374 +/- 0.01 in controls to 0.146 +/- 0.01 at 0 h and 0.177 +/- 0.02 at 1 h of re-oxygenation (p < 0.001). Exposure to DETA-NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG-nitro-L-arginine methyl ester (L-NAME) prevented the hypoxia-induced decrease in mitochondrial movement at 0 h (30.1 +/- 1.6%) and at 1 h (26.1 +/- 9%) of re-oxygenation. Exposure to either hypoxia/re-oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO-mediated pathway is at least partially involved.     

Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases

The pathological processes of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases engender synaptic and neuronal cell damage. While mild oxidative and nitrosative (nitric oxide (NO)-related) stress mediates normal neuronal signaling, excessive accumulation of these free radicals is linked to neuronal cell injury or death. In neurons, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation and subsequent Ca(2+) influx can induce the generation of NO via neuronal NO synthase. Emerging evidence has demonstrated that S-nitrosylation, representing covalent reaction of an NO group with a critical protein thiol, mediates the vast majority of NO signaling. Analogous to phosphorylation and other posttranslational modifications, S-nitrosylation can regulate the biological activity of many proteins. Here, we discuss recent studies that implicate neuropathogenic roles of S-nitrosylation in protein misfolding, mitochondrial dysfunction, synaptic injury, and eventual neuronal loss. Among a growing number of S-nitrosylated proteins that contribute to disease pathogenesis, in this review we focus on S-nitrosylated protein-disulfide isomerase (forming SNO-PDI) and dynamin-related protein 1 (forming SNO-Drp1). Furthermore, we describe drugs, such as memantine and newer derivatives of this compound that can prevent both hyperactivation of extrasynaptic NMDARs as well as downstream pathways that lead to nitrosative stress, synaptic damage, and neuronal loss.     

Docosahexaenoic acid accumulation in the prenatal brain: a purported role in combating oxidative stress

Mitochondria may be both the source and the target of oxidative stress in neurodegenerative disease. In models of excitotoxicity, neuronal injury is triggered by the influx of calcium into neurons and then into mitochondria. Our studies suggest that an important consequence of this calcium movement is the generation of ROS by mitochondria. Studies with isolated mitochondria suggest that calcium may enhance ROS generation by mitochondria, especially when complex I is impaired. However, these studies are complicated by a lack of specificity of detection methods like Amplex Red. One feature of mitochondria is their movement within neurons. We used fluorescent proteins targeted to mitochondria to follow trafficking in neurons. Neurotoxins like glutamate, zinc and peroxide, which feature oxidative stress in their mechanism of action, affect mitochondrial movement, morphology or both. We speculate that restricting the delivery of mitochondria to their targets within neurons could impair neuronal viability.     

Interaction between mitochondria and oxidative stress in neuronal injury

Mitochondria may be both the source and the target of oxidative stress in neurodegenerative disease. In models of excitotoxicity, neuronal injury is triggered by the influx of calcium into neurons and then into mitochondria. Our studies suggest that an important consequence of this calcium movement is the generation of ROS by mitochondria. Studies with isolated mitochondria suggest that calcium may enhance ROS generation by mitochondria, especially when complex I is impaired. However, these studies are complicated by a lack of specificity of detection methods like Amplex Red. One feature of mitochondria is their movement within neurons. We used fluorescent proteins targeted to mitochondria to follow trafficking in neurons. Neurotoxins like glutamate, zinc and peroxide, which feature oxidative stress in their mechanism of action, affect mitochondrial movement, morphology or both. We speculate that restricting the delivery of mitochondria to their targets within neurons could impair neuronal viability.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

The Armc10/SVH gene: genome context,regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1–6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents Aβ-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against Aβ-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.     

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.     

Pre-B-cell colony-enhancing factor exerts a neuronal protection through its enzymatic activity and the reduction of mitochondrial dysfunction in in vitro ischemic models

Pre-B-cell colony-enhancing factor (PBEF) is known as a rate-limiting enzyme that converts nicotinamide (NAM) to NMN in the salvage pathway of mammalian NAD? biosynthesis. Previously we found PBEF is exclusively expressed in neurons in the mouse brain; heterozygous PBEF knockout (Pbef?/?) mice have larger ischemic lesion than wild type mice in photothrombosis-induced ischemia. For the mechanistic study of neuronal protective role of PBEF, we used in vitro oxygen-glucose deprivation (OGD) and glutamate excitotoxicity models of primary cultured neurons in current study. Our results showed that the treatments of neurons with NAM and NAD?, the substrate and downstream product of PBEF, respectively, significantly reduced neuronal death after OGD and glutamate excitotoxicity, while treatment of neurons treated with FK866, a PBEF inhibitor, increased neuronal death after OGD. Furthermore, over-expression of human PBEF reduced glutamate excitotoxicity, while over-expression of human PBEF mutants (i.e. H247A and H247E) without enzymatic activity had no effect on neuronal death. We further tested the effect of PBEF on mitochondrial function and biogenesis. Our results show that addition of NAD? and NAM increased mitochondrial biogenesis in neurons after OGD. Over-expression of PBEF in neurons reduced mitochondrial membrane potential depolarization following glutamate stimulation, while over-expression of H247A and H247E did not affect mitochondrial membrane potential depolarization. We conclude that PBEF has a neuroprotective effect in ischemia through its enzymatic activity for NAD? production that can ameliorate mitochondrial dysfunction.     

The mitochondrial uncoupler 2,4-dinitrophenol attenuates tissue damage and improves mitochondrial homeostasis following transient focal cerebral ischemia

Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable 'upstream' targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia-reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function.     

Mitochondrial ERK plays a key role in delta-opioid receptor neuroprotection against acute mitochondrial dysfunction

It is well established that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effects of DOR activation on mitochondrial dysfunction in neurons are poorly elucidated. In this investigation, we studied the effects of [d-Ala2, d-Leu5] enkephalin (DADLE), a potent DOR agonist, on acute mitochondrial dysfunction and ensuing cell damage induced by sodium azide in primary rat cortical neuronal cultures, and explored possible mechanisms underlying. Here, we show that DADLE reverses NaN₃-induced acute mitochondrial dysfunction by selectively activating DOR, mainly including mitochondrial membrane depolarization, mitochondrial Ca²⁺ overload and reactive oxygen species generation. DOR stimulation also inhibits cytochrome c release and caspase-3 activation, and attenuates neuronal death caused by acute NaN₃ insults. Furthermore, DOR activation with DADLE protects neurons from acute NaN₃ insults mainly through PKC-ERK pathway, and mitochondrial ERK activation is especially required for DOR neuroprotection against acute mitochondrial dysfunction.     

Roles of mitochondrial dynamics modulators in cardiac ischaemia/reperfusion injury

The current therapeutic strategy for the management of acute myocardial infarction (AMI) is to return blood flow into the occluded coronary artery of the heart, a process defined as reperfusion. However, reperfusion itself can increase mortality rates in AMI patients because of cardiac tissue damage and dysfunction, which is termed ‘ischaemia/reperfusion (I/R) injury’. Mitochondria play an important role in myocardial I/R injury as disturbance of mitochondrial dynamics, especially excessive mitochondrial fission, is a predominant cause of cardiac dysfunction. Therefore, pharmacological intervention and therapeutic strategies which modulate the mitochondrial dynamics balance during I/R injury could exert great beneficial effects to the I/R heart. This review comprehensively summarizes and discusses the effects of mitochondrial fission inhibitors as well as mitochondrial fusion promoters on cardiac and mitochondrial function during myocardial I/R injury. The comparison of the effects of both compounds given at different time‐points during the course of I/R injury (i.e. prior to ischaemia, during ischaemia and at the reperfusion period) are also summarized and discussed. Finally, this review also details important information which may contribute to clinical practices using these drugs to improve the quality of life in AMI patients.     

Regulation of neuronal growth cone filopodia by nitric oxide

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.     

Neurodegeneration or Neuroprotection: The Pivotal Role of Astrocytes

Formation of nitric oxide (NO), by astrocytes, has been suggested to contribute, via impairment of mitochondrial function, to the neurodegnerative process. Thus co-culture of neuronal cells with NO-generating astrocytes leads to a loss of mitochondrial function, as reflected by diminished activities of complexes IV and II+III. However, such damage may in the first instance be limited due to upregulation of neuronal glutathione metabolism as a result of metabolic trafficking of glutathione from the astrocyte to neurone. Furthermore, exposure of astrocytes to NO leads to increased glutathione metabolism resulting in the preservation of glutathione precursors for neuronal utilization. Failure of glutathione trafficking could render neuronal cells particularly susceptible to NO, leading to cell death. In addition, depletion with time of the nitric oxide synthase cofactor, tetrahydrobiopterin, may result in the astrocytic generation of more potent oxidizing species, which could contribute to the neurodegenerative process.     

Glycogen synthase kinase-3 inhibition reduces ischemic cerebral damage, restores impaired mitochondrial biogenesis and prevents ROS production

This study was designed to test the hypothesis that improved mitochondrial biogenesis could help reducing ischemic cerebral injury. We found that levels of proliferator-activated receptor γ coactivator 1α and nuclear respiratory factor-1, mitochondrial DNA content and other markers of mitochondrial biogenesis and function were reduced in primary mouse cortical neurons under oxygen-glucose deprivation (OGD). The glycogen synthase kinase-3 (GSK-3) inhibitor SB216763 activated an efficient mitochondrial biogenesis program in control cortical neurons and counteracted the OGD-mediated mitochondrial biogenesis impairment. This was accompanied by the activation of an antioxidant response that reduced mitochondrial reactive oxygen species generation and ischemic neuronal damage. The in vitro effects of SB216763 were mimicked by two other structurally unrelated GSK-3 inhibitors. The protective effects of SB216763 on OGD-mediated neuronal damage were abolished in the presence of diverse mitochondrial inhibitors. Finally, when systemically administered in vivo, SB216763 reduced the infarct size and recovered the loss of mitochondrial DNA in mice subjected to permanent middle cerebral artery occlusion. We conclude that GSK-3 inhibition by SB216763 might pave the way of novel promising therapies aimed at stimulating the renewal of functional mitochondria and reducing reactive oxygen species-mediated damage in ischemic stroke.     

Refractoriness of urethral striated muscle contractility to nitric oxide-dependent cyclic GMP production

The purpose of this study was to investigate the role of cyclic GMP (cGMP) in the effects of nitric oxide (NO) on urethral striated muscle and its involvement in contractile function. The localization of cGMP, neuronal NO synthase (nNOS), vimentin, and neuronal markers was assessed by immunofluorescence in the sheep and rat urethra and the expression of nNOS was determined in Western blots. Nerve-mediated contractile responses to electrical field stimulation (EFS) were recorded in the sheep urethra. The scant nitrergic innervation of the striated muscle layer suggests that autonomic control of its activity is unlikely. The striated fiber itself may be the source of high levels NO produced by sarcolemmal and/or cytosolic μ or α variant of nNOS. This endogenous NO may provoke high basal production of soluble guanylate cyclase (GC) dependent cGMP, mainly in non-NO producing muscle fibers, which is not further enhanced by NO donors. cGMP co-localizes with neurofilament and PGP 9.5 at muscle endplates. Modulators of the cGMP pathway did not affect nerve-mediated contractile activity induced by EFS, suggesting that cGMP is not a significant mediator of neuromuscular transmission. In addition, NO donors did increase the accumulation of cGMP in dense networks of vimentin immunoreactive interstitial cells of Cajal (ICC), whose function is not yet known. These data suggest that there is a strong but non-regulated production of cGMP under resting conditions, which does not seem to affect contractile function. Modulation of cholinergic neurotransmission by NO through cGMP-independent mechanisms cannot be discarded.     

Lack of mitochondrial nitric oxide production in the mouse brain

Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF-fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark-type NO-sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5-ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS-dependent mechanism.