Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons

Brain cell metabolism is intimately associated with intracellular oxidation–reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 μM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca²⁺ abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate.     

Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences

Neurons are critically dependent on mitochondrial integrity based on specific morphological, biochemical, and physiological features. They are characterized by high rates of metabolic activity and need to respond promptly to activity-dependent fluctuations in bioenergetic demand. The dimensions and polarity of neurons require efficient transport of mitochondria to hot spots of energy consumption, such as presynaptic and postsynaptic sites. Moreover, the postmitotic state of neurons in combination with their exposure to intrinsic and extrinsic neuronal stress factors call for a high fidelity of mitochondrial quality control systems. Consequently, it is not surprising that mitochondrial alterations can promote neuronal dysfunction and degeneration. In particular, mitochondrial dysfunction has long been implicated in the etiopathogenesis of Parkinson's disease (PD), based on the observation that mitochondrial toxins can cause parkinsonism in humans and animal models. Substantial progress towards understanding the role of mitochondria in the disease process has been made by the identification and characterization of genes causing familial variants of PD. Studies on the function and dysfunction of these genes revealed that various aspects of mitochondrial biology appear to be affected in PD, comprising mitochondrial biogenesis, bioenergetics, dynamics, transport, and quality control.     

AMPK Signalling and Defective Energy Metabolism in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is caused by selective loss of upper and lower motor neurons by complex mechanisms that are incompletely understood. Motor neurons are large, highly polarised and excitable cells with unusually high energetic demands to maintain resting membrane potential and propagate action potentials. This leads to higher ATP consumption and mitochondrial metabolism in motor neurons relative to other cells. Here, we review increasing evidence that defective energy metabolism and homeostasis contributes to selective vulnerability and degeneration of motor neurons in ALS. Firstly, we provide a brief overview of major energetic pathways in the CNS, including glycolysis, oxidative phosphorylation and the AMP-activated protein kinase (AMPK) signalling pathway, while highlighting critical metabolic interactions between neurons and astrocytes. Next, we review evidence from ALS patients and transgenic mutant SOD1 mice for weight loss, hypermetabolism, hyperlipidemia and mitochondrial dysfunction in disease onset and progression. Genetic and therapeutic modifiers of energy metabolism in mutant SOD1 mice will also be summarised. We also present evidence that additional ALS-linked proteins, TDP-43 and FUS, lead to energy disruption and mitochondrial defects in motor neurons. Lastly, we review emerging evidence including our own that dysregulation of the AMPK signalling cascade in motor neurons is an early and common event in ALS pathogenesis. We suggest that an imbalance in energy metabolism should be considered an important factor in both progression and potential treatment of ALS.     

Inhibition of mitochondrial function in astrocytes: implications for neuroprotection

Much evidence suggests that astrocytes protect neurons against ischemic injury. Although astrocytes are more resistant to some insults than neurons, few studies offer insight into the real time changes of astrocytic protective functions with stress. Mitochondria are one of the primary targets of ischemic injury in astrocytes. We investigated the time course of changes in astrocytic ATP levels, plasma membrane potential, and glutamate uptake, a key protective function, induced by mitochondrial inhibition. Our results show that significant functional change precedes reduction in astrocytic viability with mitochondrial inhibition. Using the mitochondrial inhibitor fluorocitrate (FC, 0.25 mmol/L) that is preferentially taken by astrocytes we found that inhibition of astrocyte mitochondria increased vulnerability of co-cultured neurons to glutamate toxicity. In our studies, the rates of FC-induced astrocytic mitochondrial depolarization were accelerated in mixed astrocyte/neuron cultures. We hypothesized that the more rapid mitochondrial depolarization was promoted by an additional energetic demand imposed be the co-cultured neurons. To test this hypothesis, we exposed pure astrocytic cultures to 0.01-1 mmol/L aspartate as a metabolic load. Aspartate application accelerated the rates of FC-induced mitochondrial depolarization, and, at 1 mmol/L, induced astrocytic death, suggesting that strong energetic demands during ischemia can compromise astrocytic function and viability.     

ALS motor neurons exhibit hallmark metabolic defects that are rescued by SIRT3 activation

Motor neurons (MNs) are highly energetic cells and recent studies suggest that altered energy metabolism precede MN loss in amyotrophic lateral sclerosis (ALS), an age-onset neurodegenerative disease. However, clear mechanistic insights linking altered metabolism and MN death are still missing. In this study, induced pluripotent stem cells from healthy controls, familial ALS, and sporadic ALS patients were differentiated toward spinal MNs, cortical neurons, and cardiomyocytes. Metabolic flux analyses reveal an MN-specific deficiency in mitochondrial respiration in ALS. Intriguingly, all forms of familial and sporadic ALS MNs tested in our study exhibited similar defective metabolic profiles, which were attributed to hyper-acetylation of mitochondrial proteins. In the mitochondria, Sirtuin-3 (SIRT3) functions as a mitochondrial deacetylase to maintain mitochondrial function and integrity. We found that activating SIRT3 using nicotinamide or a small molecule activator reversed the defective metabolic profiles in all our ALS MNs, as well as correct a constellation of ALS-associated phenotypes.Subject terms: Neuroscience, Pathogenesis     

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.     

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1''s role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.     

Mechanisms of mitochondrial response to variations in energy demand in eukaryotic cells

This review focuses on the different mechanisms involved in the adjustment of mitochondrial ATP production to cellular energy demand. The oxidative phosphorylation steady state at constant mitochondrial enzyme content can vary in response to energy demand. However, such an adaptation is tightly linked to a modification in both oxidative phosphorylation yield and phosphate potential and is obviously very limited in eukaryotic cells. We describe the three main mechanisms involved in mitochondrial response to energy demand. In heart cells, a short-term adjustment can be reached mainly through metabolic signaling via phosphotransfer networks by the compartmentalized energy transfer and signal transmission. In such a complex regulatory mechanism, Ca²⁺ signaling participates in activation of matricial dehydrogenases as well as mitochondrial ATP synthase. These processes allow a large increase in ATP production rate without an important modification in thermodynamic forces. For a long-term adaptation, two main mechanisms are involved: modulation of the mitochondrial enzyme content as a function of energy demand and/or kinetic regulation by covalent modifications (phosphorylations) of some respiratory chain complex subunits. Regardless of the mechanism involved (kinetic regulation by covalent modification or adjustment of mitochondrial enzyme content), the cAMP signaling pathway plays a major role in molecular signaling, leading to the mitochondrial response. We discuss the energetic advantages of these mechanisms. yeast; C6 glioma cells; muscle; kinetic regulation     

Mitochondrial potassium channels and uncoupling proteins in synaptic plasticity and neuronal cell death

The function of the nervous system relies upon synaptic transmission, a process in which a neurotransmitter released from pre-synaptic terminals of one neuron (in response to membrane depolarization and calcium influx) activates post-synaptic receptors on dendrites of another neuron. Synapses are subjected to repeated bouts of oxidative and metabolic stress as the result of changing ion gradients and ATP usage. Mitochondria play central roles in meeting the demands of synapses for ATP and in regulating calcium homeostasis, and mitochondrial dysfunction can cause dysfunction and degeneration of synapses, and can trigger cell death. We have identified two types of mitochondrial proteins that serve the function of protecting synapses and neurons against dysfunction and death. Mitochondrial ATP-sensitive potassium (MitoKATP) channels modulate inner membrane potential and oxyradical production; mitochondrial potassium fluxes can affect cytochrome c release and caspase activation and may determine whether neurons live or die in experimental models of stroke and Alzheimer's disease. Uncoupling proteins (UCPs) are a family of mitochondrial membrane proteins that uncouple electron transport from ATP production by transporting protons across the inner membrane. Neurons express at least three UCPs including the widely expressed UCP-2 and the neuron-specific UCP-4 and UCP-5 (BMCP-1). We have found that UCP-4 protects neurons against apoptosis by a mechanism involving suppression of oxyradical production and stabilization of cellular calcium homeostasis. The expression of UCP-4 is itself regulated by changes in energy metabolism. In addition to their roles in neuronal cell survival and death, MitoKATP channels and UCPs may play roles in regulating neuronal differentiation during development and synaptic plasticity in the adult.     

Energetic and Dynamic: How Mitochondria Meet Neuronal Energy Demands

Mitochondria are the power houses of the cell, but unlike the static structures portrayed in textbooks, they are dynamic organelles that move about the cell to deliver energy to locations in need. These organelles fuse with each other then split apart; some appear anchored and others more free to move around, and when damaged they are engulfed by autophagosomes. Together, these processes—mitochondrial trafficking, fusion and fission, and mitophagy—are best described by the term “mitochondrial dynamics”. The molecular machineries behind these events are relatively well known yet the precise dynamics in neurons remains under debate. Neurons pose a peculiar logistical challenge to mitochondria; how do these energy suppliers manage to traffic down long axons to deliver the requisite energy supply to distant parts of the cell? To date, the majority of neuronal mitochondrial dynamics studies have used cultured neurons, Drosophila larvae, zebrafish embryos, with occasional experiments in resting mouse nerves. However, a new study in this issue of PLOS Biology from Marija Sajic and colleagues provides an in vivo look at mitochondrial dynamics along resting and electrically active neurons of live anaesthetized mice.     

Optimizing mitochondrial maintenance in extended neuronal projections

Neurons rely on localized mitochondria to fulfill spatially heterogeneous metabolic demands. Mitochondrial aging occurs on timescales shorter than the neuronal lifespan, necessitating transport of fresh material from the soma. Maintaining an optimal distribution of healthy mitochondria requires an interplay between a stationary pool localized to sites of high metabolic demand and a motile pool capable of delivering new material. Interchange between these pools can occur via transient fusion / fission events or by halting and restarting entire mitochondria. Our quantitative model of neuronal mitostasis identifies key parameters that govern steady-state mitochondrial health at discrete locations. Very infrequent exchange between stationary and motile pools optimizes this system. Exchange via transient fusion allows for robust maintenance, which can be further improved by selective recycling through mitophagy. These results provide a framework for quantifying how perturbations in organelle transport and interactions affect mitochondrial homeostasis in neurons, a key aspect underlying many neurodegenerative disorders.     

The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery

Mitochondria typically form a reticular network radiating from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites. These mitochondrial networks are regulated through the complex coordination of fission, fusion and distribution events. While a number of key mitochondrial morphology proteins have been identified, the precise mechanisms which govern their activity remain elusive. Moreover, post translational modifications including ubiquitination, phosphorylation and sumoylation of the core machinery are thought to regulate both fusion and division of the network. These proteins can undergo several different modifications depending on cellular signals, environment and energetic demands of the cell. Proteins involved in mitochondrial morphology may also have dual roles in both dynamics and apoptosis, with regulation of these proteins under tight control of the cell to ensure correct function. The absolute reliance of the cell on a functional mitochondrial network is highlighted in neurons, which are particularly vulnerable to any changes in organelle dynamics due to their unique biochemical requirements. Recent evidence suggests that defects in the shape or distribution of mitochondria correlate with the progression of neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's disease. This review focuses on our current understanding of the mitochondrial morphology machinery in cell homeostasis, apoptosis and neurodegeneration, and the post translational modifications that regulate these processes.     

Increasing Fatty Acid Oxidation Remodels the Hypothalamic Neurometabolome to Mitigate Stress and Inflammation

Modification of hypothalamic fatty acid (FA) metabolism can improve energy homeostasis and prevent hyperphagia and excessive weight gain in diet-induced obesity (DIO) from a diet high in saturated fatty acids. We have shown previously that C75, a stimulator of carnitine palmitoyl transferase-1 (CPT-1) and fatty acid oxidation (FAOx), exerts at least some of its hypophagic effects via neuronal mechanisms in the hypothalamus. In the present work, we characterized the effects of C75 and another anorexigenic compound, the glycerol-3-phosphate acyltransferase (GPAT) inhibitor FSG67, on FA metabolism, metabolomics profiles, and metabolic stress responses in cultured hypothalamic neurons and hypothalamic neuronal cell lines during lipid excess with palmitate. Both compounds enhanced palmitate oxidation, increased ATP, and inactivated AMP-activated protein kinase (AMPK) in hypothalamic neurons in vitro. Lipidomics and untargeted metabolomics revealed that enhanced catabolism of FA decreased palmitate availability and prevented the production of fatty acylglycerols, ceramides, and cholesterol esters, lipids that are associated with lipotoxicity-provoked metabolic stress. This improved metabolic signature was accompanied by increased levels of reactive oxygen species (ROS), and yet favorable changes in oxidative stress, overt ER stress, and inflammation. We propose that enhancing FAOx in hypothalamic neurons exposed to excess lipids promotes metabolic remodeling that reduces local inflammatory and cell stress responses. This shift would restore mitochondrial function such that increased FAOx can produce hypothalamic neuronal ATP and lead to decreased food intake and body weight to improve systemic metabolism.     

Metabolic stress suppresses humoral immune function in long-day,but not short-day,Siberian hamsters (<Emphasis Type="Italic">Phodopus sungorus</Emphasis>)

Individuals of many species experience marked seasonal variation in environmental conditions and must adapt to potentially large fluctuations in energy availability and expenditure. Seasonal changes in immunity have likely evolved as an adaptive mechanism to cope with seasonal stressors. In addition, these changes may be constrained by seasonal fluctuations in energy availability. The goal of this study was to assess the role of energetic trade-offs associated with seasonal variation in immunity. In addition to body fat stores, metabolic fuels (e.g., glucose) may affect immune function in seasonally breeding rodents. In this study we experimentally reduced energy availability via injections of the metabolic inhibitor 2-deoxy-d-glucose (2-DG) in long- and short-day housed Siberian hamsters (Phodopus sungorus) and then examined antigen-specific antibody production. Metabolic stress decreased antibody response compared with control animals in long days. In contrast, no difference was observed between treatment groups in short days. These data suggest that reductions in energy availability suppress immunity and short days buffer organisms against glucoprivation-induced immunosuppression.     

Mitochondrial Trafficking in Neurons

Neurons, perhaps more than any other cell type, depend on mitochondrial trafficking for their survival. Recent studies have elucidated a motor/adaptor complex on the mitochondrial surface that is shared between neurons and other animal cells. In addition to kinesin and dynein, this complex contains the proteins Miro (also called RhoT1/2) and milton (also called TRAK1/2) and is responsible for much, although not necessarily all, mitochondrial movement. Elucidation of the complex has permitted inroads for understanding how this movement is regulated by a variety of intracellular signals, although many mysteries remain. Regulating mitochondrial movement can match energy demand to energy supply throughout the extraordinary architecture of these cells and can control the clearance and replenishing of mitochondria in the periphery. Because the extended axons of neurons contain uniformly polarized microtubules, they have been useful for studying mitochondrial motility in conjunction with biochemical assays in many cell types.     

Mitochondrial Function in Human Neuroblastoma Cells Is Up-Regulated and Protected by NQO1, a Plasma Membrane Redox Enzyme

Background

Recent findings suggest that NADH-dependent enzymes of the plasma membrane redox system (PMRS) play roles in the maintenance of cell bioenergetics and oxidative state. Neurons and tumor cells exhibit differential vulnerability to oxidative and metabolic stress, with important implications for the development of therapeutic interventions that promote either cell survival (neurons) or death (cancer cells).

Methods and Findings

Here we used human neuroblastoma cells with low or high levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS modulates mitochondrial functions and cell survival. Cells with elevated NQO1 levels exhibited higher levels of oxygen consumption and ATP production, and lower production of reactive oxygen species. Cells overexpressing NQO1 were more resistant to being damaged by the mitochondrial toxins rotenone and antimycin A, and exhibited less oxidative/nitrative damage and less apoptotic cell death. Cells with basal levels of NQO1 resulted in increased oxidative damage to proteins and cellular vulnerability to mitochondrial toxins. Thus, mitochondrial functions are enhanced and oxidative stress is reduced as a result of elevated PMRS activity, enabling cells to maintain redox homeostasis under conditions of metabolic and energetic stress.

Conclusion

These findings suggest that NQO1 is a potential target for the development of therapeutic agents for either preventing neuronal degeneration or promoting the death of neural tumor cells.     

Mitochondrial therapy promotes regeneration of injured hippocampal neurons

Due to the inhibitory microenvironment and reduced intrinsic growth capacity of neurons, neuronal regeneration of central nervous system remains challenging. Neurons are highly energy demanding and require sufficient mitochondria to support cellular activities. In response to stimuli, mitochondria undergo fusion/fission cycles to adapt to environment. It is thus logical to hypothesize that the plasticity of mitochondrial dynamics is required for neuronal regeneration. In this study, we examined the role of mitochondrial dynamics during regeneration of rat hippocampal neurons. Quantitative analysis showed that injury induced mitochondrial fission. As mitochondrial dysfunction has been implicated in neurodegenerative diseases, we tested the possibility that the mitochondrial therapy may promote neuronal regeneration. Supplying freshly isolated mitochondria to the injured hippocampal neurons not only significantly increased neurite re-growth but also restored membrane potential of injured hippocampal neurons. Together, our findings support the importance of mitochondrial dynamics during regeneration of injured hippocampal neurons and highlight the therapeutic prospect of mitochondria to the injured central nervous system.     

31° South: The physiology of adaptation to arid conditions in a passerine bird

Arid environments provide ideal ground for investigating the mechanisms of adaptive evolution. High temperatures and low water availability are relentless stressors for many endotherms, including birds; yet birds persist in deserts. While physiological adaptation probably involves metabolic phenotypes, the underlying mechanisms (plasticity, genetics) are largely uncharacterized. To explore this, we took an intraspecific approach that focused on a species that is resident over a mesic to arid gradient, the Karoo scrub‐robin (Cercotrichas coryphaeus). Specifically, we integrated environmental (climatic and primary productivity), physiological (metabolic rates: a measure of energy expenditure), genotypic (genetic variation underlying the machinery of energy production) and microbiome (involved in processing food from where energy is retrieved) data, to infer the mechanism of physiological adaptation. We that found the variation in energetic physiology phenotypes and gut microbiome composition are associated with environmental features as well as with variation in genes underlying energy metabolic pathways. Specifically, we identified a small list of candidate adaptive genes, some of them with known ties to relevant physiology phenotypes. Together our results suggest that selective pressures on energetic physiology mediated by genes related to energy homeostasis and possibly microbiota composition may facilitate adaptation to local conditions and provide an explanation to the high avian intraspecific divergence observed in harsh environments.