Diverse cytopathologies in mitochondrial disease are caused by AMP-activated protein kinase signaling

The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK alpha subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKalpha expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKalpha expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial "mass" and ATP levels were reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKalpha antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.     

Dictyostelium, a microbial model for brain disease

Background

Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum.

Scope of review

This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders.

Major conclusions

Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se. Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein.

General significance

Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research     

Protective effects of AMP‐activated protein kinase in the cardiovascular system

Cardiovascular diseases remain the leading cause of mortality worldwide. Recent studies of AMP-activated protein kinase (AMPK), a highly conserved sensor of cellular energy status, suggest that there might be therapeutic value in targeting the AMPK signaling pathway. AMPK is found in most mammalian tissues, including those of the cardiovascular system. As cardiovascular diseases are typically associated with blood flow occlusion and blood occlusion may induce rapid energy deficit, AMPK activation may occur during the early phase upon nutrient deprivation in cardiovascular organs. Therefore, investigation of AMPK in cardiovascular organs may help us to understand the pathophysiology of defence mechanisms in these organs. Recent studies have provided proof of concept for the idea that AMPK is protective in heart as well as in vascular endothelial and smooth muscle cells. Moreover, dysfunction of the AMPK signalling pathway is involved in the genesis and development of various cardiovascular diseases, including atherosclerosis, hypertension and stroke. The roles of AMPK in the cardiovascular system, as they are currently understood, will be presented in this review. The interaction between AMPK and other cardiovascular signalling pathways such as nitric oxide signalling is also discussed.     

mTOR integrates amino acid- and energy-sensing pathways

The AMP-activated protein kinase (AMPK) exists as a heterotrimetric complex comprising a catalytic alpha subunit and non-catalytic beta and gamma subunits. Under conditions of hypoxia, exercise, ischemia, heat shock, and low glucose, AMPK is activated allosterically by rising cellular AMP and by phosphorylation of the catalytic alpha subunit. The mammalian target of rapamycin (mTOR) controls cellular functions in response to amino acids and growth factors. Recent reports including our study have demonstrated the possible interplay between mTOR and AMPK signaling pathways, supporting a model in which mitochondrial dysfunction caused by the mitochondrial inhibitors or ATP depletion inhibits activation of p70 S6 kinase alpha (p70alpha), a downstream effector of mTOR, by activating AMPK. Leucine may stimulate p70alpha phosphorylation via mTOR pathway, in part, by serving both as a mitochondrial fuel through oxidative carboxylation and an allosteric activation of glutamate dehydrogenase. This hypothesis may support an idea in which leucine modulates mTOR function, in part by regulating mitochondrial function and AMPK. Further understanding of the role of mTOR in coordinating amino acid- and energy-sensing pathways would provide new insights into relationship between nutrients and cellular functions.     

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.     

AMP‐activated protein kinase deficiency exacerbates aging‐induced myocardial contractile dysfunction

Aging is associated with myocardial dysfunction although the underlying mechanism is unclear. AMPK, a key cellular fuel sensor for energy metabolism, is compromised with aging. This study examined the role of AMPK deficiency in aging‐associated myocardial dysfunction. Young or old wild‐type (WT) and transgenic mice with overexpression of a mutant AMPK α₂ subunit (kinase dead, KD) were used. AMPK α isoform activity, myocardial function and morphology were examined. DCF and JC‐1 fluorescence probes were employed to quantify reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), respectively. KD mice displayed significantly reduced α₂ but not α₁ AMPK isoform activity at both ages with a greater effect at old age. Aging itself decreased α₁ isoform activity. Cardiomyocyte contractile function, intracellular Ca²⁺ handling, and SERCA2a levels were compromised with aging, the effects of which were exacerbated by AMPK deficiency. H&E staining revealed cardiomyocyte hypertrophy with aging, which was more pronounced in KD mice. TEM micrographs displayed severe disruption of mitochondrial ultrastructure characterized by swollen, irregular shape and disrupted cristae in aged KD compared with WT mice. Aging enhanced ROS production and reduced ΔΨm, the effects of which were accentuated by AMPK deficiency. Immunoblotting data depicted unchanged Akt phosphorylation and a significant decrease in mitochondrial biogenesis cofactor PGC‐1α in aged groups. AMPK deficiency but not aging decreased the phosphorylation of ACC and eNOS. Expression of membrane Glut4 and HSP90 was decreased in aged KD mice. Moreover, treatment of the AMPK activator metformin attenuated aging‐induced cardiomyocyte contractile defects. Collectively, our data suggest a role for AMPK deficiency in aging‐induced cardiac dysfunction possibly through disrupted mitochondrial function and ROS production.     

AMP-activated protein kinase--the key role in metabolic regulation

AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that acts as an energy sensor maintaining the energy balance at the cellular as well as at the whole body level. Within the healthy cell, metabolic stress leading to an increase in AMP concentration results in AMPK activation. Once activated, AMPK "switches off" many anabolic pathways e.g. fatty acid and protein synthesis while "switches on" catabolic pathways such as fatty acid oxidation or glycolysis which serve to restore intracellular ATP level. Adipocyte derived hormones leptin and adiponectin activate AMPK in peripheral tissues increasing energy expenditure. AMPK also regulates food intake due to response to hormonal and nutrient signals in hypothalamus. Antidiabetic drugs that mimic the action of insulin activate the AMPK signaling pathways. Further studies are needed to clarify the importance of the AMPK activation for therapeutic effects of this drugs.     

Regulation of nutrient uptake by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that is a key regulator of energy balance at both the cellular and whole-body level. AMPK acts to stimulate ATP production and reduce ATP consumption when cellular ATP levels fall, thereby normalizing energy balance. Given the central role of AMPK in cellular carbohydrate and lipid metabolism, AMPK activation has been proposed to be a therapeutic target for conditions associated with dysfunctional nutrient metabolism including obesity, type 2 diabetes, hepatic steatosis, cardiovascular diseases and cancer. One way by which increased ATP production can be achieved is by increasing the supply of nutrient substrates. In the 1990s, AMPK activation was demonstrated to stimulate glucose uptake in striated muscle, thereby improving substrate supply for ATP production. Subsequently AMPK activation was postulated to underlie the increase in glucose uptake that occurs during muscle contraction. More recently, however, several lines of evidence have demonstrated that AMPK activation is unlikely to be required for contraction-mediated glucose uptake. Furthermore, despite the importance of AMPK in cellular and whole-body metabolism, far fewer studies have investigated either the role of AMPK in glucose uptake by non-muscle tissues or whether AMPK regulates the uptake of fatty acids. In the present review, we discuss the role of AMPK in nutrient uptake by tissues, focusing on glucose uptake out with muscle and fatty acid uptake.     

Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury

The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose.     

Methionine supplementation stimulates mitochondrial respiration

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases.By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular metabolism, considering its role in the regulation of key cellular processes. Methionine supplementation induces an up-regulation of proteins related to mitochondrial functions such as TCA cycle, electron transport chain and respiration, combined with an enhancement of mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, snf1Δ cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is detrimental for this mutant in methionine condition, indicating that respiration mostly relies on pyruvate flux into mitochondrial pathways.These data provide new insights into the regulation of mitochondrial metabolism and extends our understanding on the role of methionine in regulating energy signalling pathways.     

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψ_m) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψ_m in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.     

AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser2?2 and Ser3??, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1-42) leads to increased phosphorylation of tau at Ser2?2/Ser3?? and Ser33??. Activation of AMPK by Aβ-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1-42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.     

A single β adaptin contributes to AP1 and AP2 complexes and clathrin function in Dictyostelium

The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a β subunit. We identified a single β subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding β1/2 resulted in severe defects in growth, cytokinesis and development. Additionally, cells lacking β1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of β1/2 null cells were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of β1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the β subunit in the stability of the medium subunits. Dictyostelium β1/2 could resemble a common ancestor of the more specialized β1 and β2 subunits of the vertebrate AP complexes. Our results support the essential contribution of a single β subunit to the stability and function of AP1 and AP2 in a simple eukaryote.     

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.     

Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase

Strategies to prevent and treat obesity aim to decrease energy intake and/or increase energy expenditure. Regarding the increase of energy expenditure, two key intracellular targets may be considered (1) mitochondrial oxidative phosphorylation, the major site of ATP production, and (2) AMP-activated protein kinase (AMPK), the master regulator of cellular energy homeostasis. Experiments performed mainly in transgenic mice revealed a possibility to ameliorate obesity and associated disorders by mitochondrial uncoupling in metabolically relevant tissues, especially in white adipose tissue (WAT), skeletal muscle (SM), and liver. Thus, ectopic expression of brown fat-specific mitochondrial uncoupling protein 1 (UCP1) elicited major metabolic effects both at the cellular/tissue level and at the whole-body level. In addition to expected increases in energy expenditure, surprisingly complex phenotypic effects were detected. The consequences of mitochondrial uncoupling in WAT and SM are not identical, showing robust and stable obesity resistance accompanied by improvement of lipid metabolism in the case of ectopic UCP1 in WAT, while preservation of insulin sensitivity in the context of high-fat feeding represents the major outcome of muscle UCP1 expression. These complex responses could be largely explained by tissue-specific activation of AMPK, triggered by a depression of cellular energy charge. Experimental data support the idea that (1) while being always activated in response to mitochondrial uncoupling and compromised intracellular energy status in general, AMPK could augment energy expenditure and mediate local as well as whole-body effects; and (2) activation of AMPK alone does not lead to induction of energy expenditure and weight reduction.     

Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function.     

AMPK Activation through Mitochondrial Regulation Results in Increased Substrate Oxidation and Improved Metabolic Parameters in Models of Diabetes

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5''-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC₅₀ 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both ¹³C-palmitate and ¹³C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO₂ in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.