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Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex and debilitating disease with a substantial social and economic impact on individuals and their community. Despite its importance and deteriorating impact, progresses in diagnosis and treatment of ME/CFS is limited. This is due to the unclear pathophysiology of the disease and consequently lack of prognostic biomarkers. To investigate pathophysiology of ME/CFS, several potential pathologic hallmarks have been investigated; however, these studies have failed to report a consistent result. These failures in introducing the underlying reason for ME/CFS have stimulated considering other possible contributing mechanisms such as tryptophan (TRP) metabolism and in particular kynurenine pathway (KP). KP plays a central role in cellular energy production through the production of nicotinamide adenine dinucleotide (NADH). In addition, this pathway has been shown to mediate immune response and neuroinflammation through its metabolites. This review, we will discuss the pathology and management of ME/CFS and provide evidence pertaining KP abnormalities and symptoms that are classic characteristics of ME/CFS. Targeting the KP regulation may provide innovative approaches to the management of ME/CFS.

Fragile X Associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder affecting carriers of premutation alleles (PM) of the X-linked FMR1 gene, which contain CGG repeat expansions of 55–200 range in a non-coding region. This late-onset disorder is characterised by the presence of tremor/ataxia and cognitive decline, associated with the white matter lesions throughout the brain, especially involving the middle cerebellar peduncles. Nearly half of older male and ~ 20% of female PM carriers develop FXTAS. While there is evidence for mitochondrial dysfunction in neural and some peripheral tissues from FXTAS patients (though less obvious in the non-FXTAS PM carriers), the results from peripheral blood mononuclear cells (PBMC) are still controversial. Motor, cognitive, and neuropsychiatric impairments were correlated with measures of mitochondrial and non-mitochondrial respiratory activity, AMPK, and TORC1 cellular stress-sensing protein kinases, and CGG repeat size, in a sample of adult FXTAS male and female carriers. Moreover, the levels of these cellular measures, all derived from Epstein- Barr virus (EBV)- transformed and easily accessible blood lymphoblasts, were compared between the FXTAS (N = 23) and non-FXTAS (n = 30) subgroups, and with baseline data from 33 healthy non-carriers. A significant hyperactivity of cellular bioenergetics components as compared with the baseline data, more marked in the non-FXTAS PMs, was negatively correlated with repeat numbers at the lower end of the CGG-PM distribution. Significant associations of these components with motor impairment measures, including tremor-ataxia and parkinsonism, and neuropsychiatric changes, were prevalent in the FXTAS subgroup. Moreover, a striking elevation of AMPK activity, and a decrease in TORC1 levels, especially in the non-FXTAS carriers, were related to the size of CGG expansion. The bioenergetics changes in blood lymphoblasts are biomarkers of the clinical status of FMR1 carriers. The relationship between these changes and neurological involvement in the affected carriers suggests that brain bioenergetic alterations are reflected in this peripheral tissue. A possible neuroprotective role of stress sensing kinase, AMPK, in PM carriers, should be addressed in future longitudinal studies. A decreased level of TORC1—the mechanistic target of the rapamycin complex, suggests a possible future approach to therapy in FXTAS.

The abnormal accumulation of the tau protein into aggregates is a hallmark in neurodegenerative diseases collectively known as tauopathies. In normal conditions, tau binds off and on microtubules aiding in their assembly and stability dependent on the phosphorylation state of the protein. In disease-affected neurons, hyperphosphorylation leads to the accumulation of the tau protein into aggregates, mainly neurofibrillary tangles (NFT) which have been seen to colocalise with other protein aggregates in neurodegeneration. One such protein is α-synuclein, the main constituent of Lewy bodies (LB), a hallmark of Parkinson’s disease (PD). In many neurodegenerative diseases, including PD, the colocalisation of tau and α-synuclein has been observed, suggesting possible interactions between the two proteins. To explore the cytotoxicity and interactions between these two proteins, we expressed full length human tau and α-synuclein inDictyostelium discoideumalone, and in combination. We show that tau is phosphorylated inD. discoideumand colocalises closely (within 40 nm) with tubulin throughout the cytoplasm of the cell as well as with α-synuclein at the cortex. Expressing wild type α-synuclein alone caused inhibited growth on bacterial lawns, phagocytosis and intracellularLegionellaproliferation rates, but activated mitochondrial respiration and non-mitochondrial oxygen consumption. The expression of tau alone impaired multicellular morphogenesis, axenic growth and phototaxis, while enhancing intracellularLegionellaproliferation. Direct respirometric assays showed that tau impairs mitochondrial ATP synthesis and increased the “proton leak,” while having no impact on respiratory complex I or II function. In most cases depending on the phenotype, the coexpression of tau and α-synuclein exacerbated (phototaxis, fruiting body morphology), or reversed (phagocytosis, growth on plates, mitochondrial respiratory function,Legionellaproliferation) the defects caused by either tau or α-synuclein expressed individually. Proteomics data revealed distinct patterns of dysregulation in strains ectopically expressing tau or α-synuclein or both, but down regulation of expression of cytoskeletal proteins was apparent in all three groups and most evident in the strain expressing both proteins. These results indicate that tau and α-synuclein exhibit different but overlapping patterns of intracellular localisation, that they individually exert distinct but overlapping patterns of cytotoxic effects and that they interact, probably physically in the cell cortex as well as directly or indirectly in affecting some phenotypes. The results show the efficacy of usingD. discoideumas a model to study the interaction of proteins involved in neurodegeneration.

Mitochondrial dysfunction has been implicated in the pathology of Parkinson’s disease (PD). In Dictyostelium discoideum, strains with mitochondrial dysfunction present consistent, AMPK-dependent phenotypes. This provides an opportunity to investigate if the loss of function of specific PD-associated genes produces cellular pathology by causing mitochondrial dysfunction with AMPK-mediated consequences. DJ-1 is a PD-associated, cytosolic protein with a conserved oxidizable cysteine residue that is important for the protein’s ability to protect cells from the pathological consequences of oxidative stress. Dictyostelium DJ-1 (encoded by the gene deeJ) is located in the cytosol from where it indirectly inhibits mitochondrial respiration and also exerts a positive, nonmitochondrial role in endocytosis (particularly phagocytosis). Its loss in unstressed cells impairs endocytosis and causes correspondingly slower growth, while also stimulating mitochondrial respiration. We report here that oxidative stress in Dictyostelium cells inhibits mitochondrial respiration and impairs phagocytosis in an AMPK-dependent manner. This adds to the separate impairment of phagocytosis caused by DJ-1 knockdown. Oxidative stress also combines with DJ-1 loss in an AMPK-dependent manner to impair or exacerbate defects in phototaxis, morphogenesis and growth. It thereby phenocopies mitochondrial dysfunction. These results support a model in which the oxidized but not the reduced form of DJ-1 inhibits AMPK in the cytosol, thereby protecting cells from the adverse consequences of oxidative stress, mitochondrial dysfunction and the resulting AMPK hyperactivity.

The unicellular slime mould Dictyostelium discoideum is a valuable eukaryotic model organism in the study of mitochondrial biology and disease. As a member of the Amoebozoa, a sister clade to the animals and fungi, Dictyostelium mitochondrial biology shares commonalities with these organisms, but also exhibits some features of plants. As such it has made significant contributions to the study of eukaryotic mitochondrial biology. This review provides an overview of the advances in mitochondrial biology made by the study of Dictyostelium and examines several examples where Dictyostelium has and will contribute to the understanding of mitochondrial disease. The study of Dictyostelium’s mitochondrial biology has contributed to the understanding of mitochondrial genetics, transcription, protein import, respiration, morphology and trafficking, and the role of mitochondria in cellular differentiation. Dictyostelium is also proving to be a versatile model organism in the study both of classical mitochondrial disease e.g. Leigh syndrome, and in mitochondria-associated neurodegenerative diseases like Parkinson’s disease. The study of mitochondrial diseases presents a unique challenge due to the cryptic nature of their genotype-phenotype relationship. The use of Dictyostelium can contribute to resolving this problem by providing a genetically tractable, haploid eukaryotic organism with a suite of readily characterised phenotype readouts of cellular signalling pathways. Dictyostelium has provided insight into the signalling pathways involved in multiple neurodegenerative diseases and will continue to provide a significant contribution to the understanding of mitochondrial biology and disease.

Neurological disorders, including neurodegenerative diseases, are collectively a major cause of death and disability worldwide. Whilst the underlying disease mechanisms remain elusive, altered mitochondrial function has been clearly implicated and is a key area of study in these disorders. Studying mitochondrial function in these disorders is difficult due to the inaccessibility of brain tissue, which is the key tissue affected in these diseases. To overcome this issue, numerous cell models have been used, each providing unique benefits and limitations. Here, we focussed on the use of lymphoblastoid cell lines (LCLs) to study mitochondrial function in neurological disorders. LCLs have long been used as tools for genomic analyses, but here we described their use in functional studies specifically in regard to mitochondrial function. These models have enabled characterisation of the underlying mitochondrial defect, identification of altered signalling pathways and proteins, differences in mitochondrial function between subsets of particular disorders and identification of biomarkers of the disease. The examples provided here suggest that these cells will be useful for development of diagnostic tests (which in most cases do not exist), identification of drug targets and testing of pharmacological agents, and are a worthwhile model for studying mitochondrial function in neurological disorders.

Although understanding of the biomedical basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is growing, the underlying pathological mechanisms remain uncertain. We recently reported a reduction in the proportion of basal oxygen consumption due to ATP synthesis by Complex V in ME/CFS patient-derived lymphoblast cell lines, suggesting mitochondrial respiratory inefficiency. This was accompanied by elevated respiratory capacity, elevated mammalian target of rapamycin complex 1 (mTORC1) signaling activity and elevated expression of enzymes involved in the TCA cycle, fatty acid β-oxidation and mitochondrial transport. These and other observations led us to hypothesise the dysregulation of pathways providing the mitochondria with oxidisable substrates. In our current study, we aimed to revisit this hypothesis by applying a combination of whole-cell transcriptomics, proteomics and energy stress signaling activity measures using subsets of up to 34 ME/CFS and 31 healthy control lymphoblast cell lines from our growing library. While levels of glycolytic enzymes were unchanged in accordance with our previous observations of unaltered glycolytic rates, the whole-cell proteomes of ME/CFS lymphoblasts contained elevated levels of enzymes involved in the TCA cycle (p = 1.03 × 10−4), the pentose phosphate pathway (p = 0.034, G6PD p = 5.5 × 10−4), mitochondrial fatty acid β-oxidation (p = 9.2 × 10−3), and degradation of amino acids including glutamine/glutamate (GLS p = 0.034, GLUD1 p = 0.048, GOT2 p = 0.026), branched-chain amino acids (BCKDHA p = 0.028, BCKDHB p = 0.031) and essential amino acids (FAH p = 0.036, GCDH p = 0.006). The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance. The results suggest that ME/CFS metabolism is dysregulated such that alternatives to glycolysis are more heavily utilised than in controls to provide the mitochondria with oxidisable substrates.

Alpha synuclein has been linked to both sporadic and familial forms of Parkinson&rsquo, s disease (PD) and is the most abundant protein in Lewy bodies a hallmark of Parkinson&rsquo, s disease. The function of this protein and the molecular mechanisms underlying its toxicity are still unclear, but many studies have suggested that the mechanism of &alpha, synuclein toxicity involves alterations to mitochondrial function. Here we expressed human &alpha, synuclein and two PD-causing &alpha, synuclein mutant proteins (with a point mutation, A53T, and a C-terminal 20 amino acid truncation) in the eukaryotic model Dictyostelium discoideum. Mitochondrial disease has been well studied in D. discoideum and, unlike in mammals, mitochondrial dysfunction results in a clear set of defective phenotypes. These defective phenotypes are caused by the chronic hyperactivation of the cellular energy sensor, AMP-activated protein kinase (AMPK). Expression of &alpha, synuclein wild type and mutant forms was toxic to the cells and mitochondrial function was dysregulated. Some but not all of the defective phenotypes could be rescued by down regulation of AMPK revealing both AMPK-dependent and -independent mechanisms. Importantly, we also show that the C-terminus of &alpha, synuclein is required and sufficient for the localisation of the protein to the cell cortex in D. discoideum.

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function., Highlights • Live-cell visualization of mitochondria dynamics is currently difficult in Dictyostelium discoideum. • We provide an easy and rapid procedure for visualizing mitochondria. • This technology employs a plasmid, REMIT-mRFPmars, expressing a mitochondrial targeting sequence linked to mRFPmars. • Expression of REMIT-mRFPmars does not alter mitochondrial load or function, providing a suitable tag for live-cell studies.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a devastating illness whose biomedical basis is now beginning to be elucidated. We reported previously that, after recovery from frozen storage, lymphocytes (peripheral blood mononuclear cells, PBMCs) from ME/CFS patients die faster in culture medium than those from healthy controls. We also found that lymphoblastoid cell lines (lymphoblasts) derived from these PBMCs exhibit multiple abnormalities in mitochondrial respiratory function and signalling activity by the cellular stress-sensing kinase Target Of Rapamycin Complex 1 (TORC1). These differences were correlated with disease severity, as measured by the Richardson and Lidbury weighted standing test. The clarity of the differences between these cells derived from ME/CFS patient blood and those from healthy controls suggested that they may provide useful biomarkers for ME/CFS. Here, we report a preliminary investigation into that possibility using a variety of analytical classification tools, including linear discriminant analysis, logistic regression and receiver operating characteristic (ROC) curve analysis. We found that results from three different tests&mdash, lymphocyte death rate, mitochondrial respiratory function and TORC1 activity&mdash, could each individually serve as a biomarker with better than 90% sensitivity but only modest specificity v&iacute, s a v&iacute, s healthy controls. However, in combination, they provided a cell-based biomarker with sensitivity and specificity approaching 100% in our sample. This level of sensitivity and specificity was almost equalled by a suggested protocol in which the frozen lymphocyte death rate was used as a highly sensitive test to triage positive samples to the more time consuming and expensive tests measuring lymphoblast respiratory function and TORC1 activity. This protocol provides a promising biomarker that could assist in more rapid and accurate diagnosis of ME/CFS.

Parkinson's disease (PD) was initially thought to be a solely sporadic disorder; however, many PD-associated genes have been identified with mutations that increase the likelihood of disease. PARK7 is one of these genes. Mutant alleles of PARK7 that result in loss of function of the encoded protein DJ-1 have been reported to cause early-onset, familial Parkinson's disease. DJ-1 is thought to be involved in many cellular pathways in multiple biochemical capacities as a chaperone, antioxidant, transcriptional regulator, deglycase, and protease. Despite extensive work supporting these roles, the mechanisms by which mutant DJ-1 causes PD remain unclear. Loss of DJ-1 function has been reported to cause elevated levels of reactive oxygen species and increased aggregation of other Parkinson's associated proteins, ultimately leading to neurodegeneration. DJ-1 overoxidation reportedly results in loss of function and increased buildup of nonfunctional protein within cells. Most studies report that wild-type DJ-1 in unstressed cells is located in the cytoplasm, but its location under conditions of oxidative stress is more controversial; localization in the cytoplasm, mitochondria, and nucleus has been reported. It has also been suggested that mutant, but not wild-type DJ-1 can localize to the mitochondria, leaving lower levels of the protein in the cytoplasm and other subcellular locations that may result in cytopathology.

Tau is a microtubule-associated protein that binds dynamically to microtubules, dependent on the protein's phosphorylation state. In disease-affected neurons, hyperphosphorylation causes the accumulation of tau protein into aggregates. Neurodegenerative diseases with prominent tau pathology are collectively known as tauopathies. The most common of these and the most prevalent neurodegenerative disease is Alzheimer's disease, in which tau is associated with amyloid-beta (the other hallmark in this disease) and α-synuclein. Tau has also been associated with α-synuclein in Parkinson's disease and with other proteins found in the brain such as prion protein, DJ-1, and filamin. Since the evidence of protein accumulations is usually determined from postmortem brains, many models have been created to study the possible interactions of these proteins. Cellular and animal models expressing tau in combination with other pathological proteins have assisted in gaining insight into these relationships.

In combination with studies of post-mortem Parkinson's disease (PD) brains, pharmacological and genetic models of PD have suggested that two fundamental interacting cellular processes are impaired – proteostasis and mitochondrial respiration. We have re-examined the role of mitochondrial dysfunction in lymphoblasts isolated from individuals with idiopathic PD and an age-matched control group. As previously reported for various PD cell types, the production of reactive oxygen species (ROS) by PD lymphoblasts was significantly elevated. However, this was not due to an impairment of mitochondrial respiration, as is often assumed. Instead, basal mitochondrial respiration and ATP synthesis are dramatically elevated in PD lymphoblasts. The mitochondrial mass, genome copy number and membrane potential were unaltered, but the expression of indicative respiratory complex proteins was also elevated. This explains the increased oxygen consumption rates by each of the respiratory complexes in experimentally uncoupled mitochondria of iPD cells. However, it was not attributable to increased activity of the stress- and energy-sensing protein kinase AMPK, a regulator of mitochondrial biogenesis and activity. The respiratory differences between iPD and control cells were sufficiently dramatic as to provide a potentially sensitive and reliable biomarker of the disease state, unaffected by disease duration (time since diagnosis) or clinical severity. Lymphoblasts from control and PD individuals thus occupy two distinct, quasi-stable steady states; a ‘normal’ and a ‘hyperactive’ state characterized by two different metabolic rates. The apparent stability of the ‘hyperactive’ state in patient-derived lymphoblasts in the face of patient ageing, ongoing disease and mounting disease severity suggests an early, permanent switch to an alternative metabolic steady state. With its associated, elevated ROS production, the ‘hyperactive’ state might not cause pathology to cells that are rapidly turned over, but brain cells might accumulate long-term damage leading ultimately to neurodegeneration and the loss of mitochondrial function observed post-mortem. Whether the ‘hyperactive’ state in lymphoblasts is a biomarker specifically of PD or more generally of neurodegenerative disease remains to be determined., Editors' choice: Cultured Parkinson's disease lymphocytes are metabolically hyperactive, suggesting a new understanding of the underlying cytopathology and biomarkers for this and potentially other neurodegenerative diseases.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”), and by fatigue whose severity and associated requirement for rest are excessive and disproportionate to the fatigue-inducing activity. There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by a reduction of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in the resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

The underlying molecular basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is not well understood. Characterized by chronic, unexplained fatigue, a disabling payback following exertion (“post-exertional malaise”) and variably presenting, multi-system symptoms, ME/CFS is a complex disease which demands concerted biomedical investigation from disparate fields of expertise. ME/CFS research and patient treatment have been challenged by the lack of diagnostic biomarkers and finding these is a prominent direction of current work. Despite these challenges, modern research demonstrates a tangible biomedical basis for the disorder across many body systems. This evidence is largely comprised of disturbances to immunological and inflammatory pathways, autonomic and neurologic systems, abnormalities in muscle and mitochondrial function, shifts in metabolism, and gut physiology or gut microbiome disturbances. It is possible that these threads are together entangled as parts of an underlying molecular pathology reflecting a far-reaching homeostatic shift affecting each of these systems. Due to the variability of non-overlapping symptom presentation or precipitating events such as infection or other bodily stresses, the initiation of body-wide pathological cascades with similar outcomes stemming from different causes may be implicated in the condition. Patient stratification to account for this heterogeneity is therefore one important consideration during exploration of potential diagnostic developments.

The misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies, a group of neurodegenerative disorders that includes Parkinson's disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the protein misfolding cyclic amplification (PMCA) assay. To do this, previous published methods were adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate whether such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show that CA both accelerates the rate of α-synuclein fibrillization and produces species that harbour enhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live non-transgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed that misfolded α-synuclein causes hyperactive mitochondrial respiration without causing any functional deficit. These data give strong support for the mitochondrion as a target for misfolded α-synuclein and reveal persistent, hyperactive respiration as a potential upstream pathogenic event associated with the synucleinopathies. This article has an associated First Person interview with the first author of the paper., Summary: Misfolded α-synuclein produced using the protein misfolding cyclic amplification assay associates with cardiolipin and causes hyperactive respiration in neuronal cells.

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative disorders with similar clinical manifestations whose precise mechanisms of disease are presently unknown. We created multiple cell lines each with different levels of reduction of expression of the gene coding for the type 2 variant of the disease, Tripeptidyl peptidase (Tpp1), in the cellular slime mould Dictyostelium discoideum. Knocking down Tpp1 in Dictyostelium resulted in the accumulation of autofluorescent material, a characteristic trait of Batten disease. Phenotypic characterisation of the mutants revealed phenotypic deficiencies in growth and development, whilst endocytic uptake of nutrients was enhanced. Furthermore, the severity of the phenotypes correlated with the expression levels of Tpp1. We propose that the phenotypic defects are due to altered Target of Rapamycin (TOR) signalling. We show that treatment of wild type Dictyostelium cells with rapamycin (a specific TOR complex inhibitor) or antisense inhibition of expression of Rheb (Ras homologue enriched in the brain) (an upstream TOR complex activator) phenocopied the Tpp1 mutants. We also show that overexpression of Rheb rescued the defects caused by antisense inhibition of Tpp1. These results suggest that the TOR signalling pathway is responsible for the cytopathological outcomes in the Dictyostelium Tpp1 model of Batten disease.

The fragile X premutation (PM) allele contains a CGG expansion of 55-200 repeats in the FMR1 gene's promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39-81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers - CGG repeat number and the levels of the FMR1 mRNA - were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85-90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings - suggestive of a role of this enzyme in the risk of FXTAS - need to be verified and expanded in future studies using larger samples and longitudinal assessment.

HTRA2 is a mitochondrial protein, mutations in which are associated with autosomal dominant late-onset Parkinson’s disease (PD). The mechanisms by which HTRA2 mutations result in PD are poorly understood. HTRA2 is proposed to play a proteolytic role in protein quality control and homeostasis in the mitochondrial intermembrane space. Its loss has been reported to result in accumulation of unfolded and misfolded proteins. However, in at least one case, PD-associated HTRA2 mutation can cause its hyperphosphorylation, possibly resulting in protease hyperactivity. The consequences of overactive mitochondrial HTRA2 are not clear. Dictyostelium discoideum provides a well-established model for studying mitochondrial dysfunction, such as has been implicated in the pathology of PD. We identified a single homologue of human HTRA2 encoded in the Dictyostelium discoideum genome and showed that it is localized to the mitochondria where it plays a cytoprotective role. Knockdown of HTRA2 expression caused defective morphogenesis in the multicellular phases of the Dictyostelium life cycle. In vegetative cells, it did not impair mitochondrial respiration but nonetheless caused slow growth (particularly when the cells were utilizing a bacterial food source), unaccompanied by significant defects in the requisite endocytic pathways. Despite its protective roles, we could not ectopically overexpress wild type HTRA2, suggesting that mitochondrial HTRA2 hyperactivity is lethal. This toxicity was abolished by replacing the essential catalytic serine S300 with alanine to ablate serine protease activity. Overexpression of protease-dead HTRA2 phenocopied the effects of knockdown, suggesting that the mutant protein competitively inhibits interactions between wild type HTRA2 and its binding partners. Our results show that cytopathological dysfunction can be caused either by too little or too much HTRA2 activity in the mitochondria and suggest that either could be a cause of PD.

Summary The human protein arginine methyltransferase NDUFAF7 controls the assembly of the ∼1-MDa mitochondrial complex I (CI; the NADH ubiquinone oxidoreductase) by methylating its subunit NDUFS2. We determined crystal structures of MidA, the Dictyostelium ortholog of NDUFAF7. The MidA catalytic core domain resembles other eukaryotic methyltransferases. However, three large core loops assemble into a regulatory domain that is likely to control ligand selection. Binding of MidA to NDUFS2 is weakened by methylation, suggesting a mechanism for methylation-controlled substrate release. Structural and bioinformatic analyses support that MidA and NDUFAF7 and their role in CI assembly are conserved from bacteria to humans, implying that protein methylation already existed in proteobacteria. In vivo studies confirmed the critical role of the MidA methyltransferase activity for CI assembly, growth, and phototaxis of Dictyostelium . Collectively, our data elucidate the origin of protein arginine methylation and its use by MidA/NDUFAF7 to regulate CI assembly.

The loss of function of DJ-1 caused by mutations in DJ1 causes a form of familial Parkinson's disease (PD). However, the role of DJ-1 in healthy and in PD cells is poorly understood. Even its subcellular localization in mammalian cells is uncertain, with both cytosolic and mitochondrial locations having been reported. We show here that DJ-1 is normally located in the cytoplasm in healthy Dictyostelium discoideum cells. With its unique life cycle, straightforward genotype-phenotype relationships, experimental accessibility and genetic tractability, D. discoideum offers an attractive model to investigate the roles of PD-associated genes. Furthermore, the study of mitochondrial biology, mitochondrial genome transcription and AMP-activated protein kinase-mediated cytopathologies in mitochondrial dysfunction have been well developed in this organism. Unlike mammalian systems, Dictyostelium mitochondrial dysfunction causes a reproducible and readily assayed array of aberrant phenotypes: defective phototaxis, impaired growth, normal rates of endocytosis and characteristic defects in multicellular morphogenesis. This makes it possible to study whether the underlying cytopathological mechanisms of familial PD involve mitochondrial dysfunction. DJ-1 has a single homologue in the Dictyostelium genome. By regulating the expression level of DJ-1 in D. discoideum, we show here that in unstressed cells, DJ-1 is required for normal rates of endocytic nutrient uptake (phagocytosis and, to a lesser extent, pinocytosis) and thus growth. Reduced expression of DJ-1 had no effect on phototaxis in the multicellular migratory ‘slug’ stage of the life cycle, but resulted in thickened stalks in the final fruiting bodies. This pattern of phenotypes is distinct from that observed in Dictyostelium to result from mitochondrial dyfunction. Direct measurement of mitochondrial respiratory function in intact cells revealed that DJ-1 knockdown stimulates whereas DJ-1 overexpression inhibits mitochondrial activity. Together, our results suggest positive roles for DJ-1 in endocytic pathways and loss-of-function cytopathologies that are not associated with impaired mitochondrial function., Editor's choice: The Dictyostelium homologue of the Parkinson's disease-associated protein DJ-1 is located in the cytosol, and its loss causes cytopathological defects in endocytic and autophagic cell death pathways, but stimulates respiration by functionally normal mitochondrial respiratory complexes.

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License., Ndufaf5 (also known as C20orf7) is a mitochondrial complex I (CI) assembly factor whose mutations lead to human mitochondrial disease. Little is known about the function of the protein and the cytopathological consequences of the mutations. Disruption of Dictyostelium Ndufaf5 leads to CI deficiency and defects in growth and development. The predicted sequence of Ndufaf5 contains a putative methyltransferase domain. Site-directed mutagenesis indicates that the methyltransferase motif is essential for its function. Pathological mutations were recreated in the Dictyostelium protein and expressed in the mutant background. These proteins were unable to complement the phenotypes, which further validates Dictyostelium as a model of the disease. Chronic activation of AMP-activated protein kinase (AMPK) has been proposed to play a role in Dictyostelium and human cytopathology in mitochondrial diseases. However, inhibition of the expression of AMPK gene in the Ndufaf5-null mutant does not rescue the phenotypes associated with the lack of Ndufaf5, suggesting that novel AMPK-independent pathways are responsible for Ndufaf5 cytopathology. Of interest, the Ndufaf5-deficient strain shows an increase in autophagy. This phenomenon was also observed in a Dictyostelium mutant lacking MidA (C2orf56/PRO1853/Ndufaf7), another CI assembly factor, suggesting that autophagy activation might be a common feature in mitochondrial CI dysfunction., This work was supported by Grants BFU2009-09050 and BFU2012-32536 from the Spanish Ministerio de Ciencia e Innovación.

Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells. Here, we describe a method to successfully use the Seahorse XF(e)24 Extracellular Flux Analyzer to assess the mitochondrial respirometry of the cellular slime mold Dictyostelium discoideum.

Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells. Here, we describe a method to successfully use the Seahorse XF(e)24 Extracellular Flux Analyzer to assess the mitochondrial respirometry of the cellular slime mold Dictyostelium discoideum.

A new pathway that controls pseudopod extension is identified. Results support a model in which Rab1A is required for chemoattractant-mediated Roco2 activation that functions through the F-actin cross-linker filamin to promote pseudopod extension., We identify a new pathway that is required for proper pseudopod formation. We show that Roco2, a leucine-rich repeat kinase 2 (LRRK2)-related Roco kinase, is activated in response to chemoattractant stimulation and helps mediate cell polarization and chemotaxis by regulating cortical F-actin polymerization and pseudopod extension in a pathway that requires Rab1A. We found that Roco2 binds the small GTPase Rab1A as well as the F-actin cross-linking protein filamin (actin-binding protein 120, abp120) in vivo. We show that active Rab1A (Rab1A-GTP) is required for and regulates Roco2 kinase activity in vivo and that filamin lies downstream from Roco2 and controls pseudopod extension during chemotaxis and random cell motility. Therefore our study uncovered a new signaling pathway that involves Rab1A and controls the actin cytoskeleton and pseudopod extension, and thereby, cell polarity and motility. These findings also may have implications in the regulation of other Roco kinases, including possibly LRRK2, in metazoans.

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and ‘salt and pepper’ patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA− pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA− pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA− background rescues pstO cell localisation. hfnA− cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on ‘salt and pepper’ differentiation and sorting out.

Many of the expanding roles of nucleoside diphosphate kinase have been attributed to its ability to interact with other proteins. One proposal is an interaction with the cellular energy sensor AMP-activated protein kinase, and here, we apply the simple eukaryotic organism, Dictyostelium discoideum as a test model. Stable cotransformants were created in which NDPK expression was knocked down by antisense inhibition, and AMPK activity was chronically elevated either by constitutive overexpression of its active, catalytic domain (AMPKαT) or as a result of mitochondrial dysfunction (created by antisense inhibition of expression of a mitochondrial chaperone protein, chaperonin 60). To investigate a biochemical interaction, transformants were created which contained constructs expressing FLAG-NDPK and hexahistidine-tagged full-length AMPK or AMPKαT. The protein extract from these transformants was used in coimmunoprecipitations. Knock down of NDPK expression suppressed the phenotypic defects that are caused by AMPK hyperactivity resulting either from overexpression of AMPKαT or from mitochondrial dysfunction. These included rescue of defects in slug phototaxis, fruiting body morphology and growth in a liquid medium. Coimmunoprecipitation experiments failed to demonstrate a biochemical interaction between the two proteins. The results demonstrate a genetic interaction between NDPK and AMPK in Dictyostelium in that NDPK is required for the phenotypic effects of activated AMPK. Coimmunoprecipitations suggest that this interaction is not mediated by a direct interaction between the two proteins.

10 páginas, 6 figuras.-- et al., Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA- mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology., This work was supported by grants BMC2006-00394 and BMC2009-09050 to R.E. from the Spanish Ministerio de Ciencia e Innovación; to P.R.F. from the Thyne Reid Memorial Trusts and the Australian Research Council; to A.V. and O.G. from the Spanish National Bioinformatics Institute (www.inab.org), a platform of Genome Spain; to R.G. from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (PI070167) and from the Comunidad de Madrid (GEN-0269/2006). S.C. is supported by a research contract from Consejería de Educación de la Comunidad de Madrid y del Fondo Social Europeo (FSE).

The social amoeba or cellular slime mould Dictyostelium discoideum is a "professional" phagocyte that has long been recognized for its value as a biomedical model organism, particularly in studying the actomyosin cytoskeleton and chemotactic motility in non-muscle cells. The complete genome sequence of D. discoideum is known, it is genetically tractable, readily grown clonally as a eukaryotic microorganism and is highly accessible for biochemical, cell biological and physiological studies. These are the properties it shares with other microbial model organisms. However, Dictyostelium combines these with a unique life style, with motile unicellular and multicellular stages, and multiple cell types that offer for study an unparalleled variety of phenotypes and associated signalling pathways. These advantages have led to its recent emergence as a valuable model organism for studying the molecular pathogenesis and treatment of human disease, including a variety of infectious diseases caused by bacterial and fungal pathogens. Perhaps surprisingly, this organism, without neurons or brain, has begun to yield novel insights into the cytopathology of mitochondrial diseases as well as other genetic and idiopathic disorders affecting the central nervous system. Dictyostelium has also contributed significantly to our understanding of NDP kinase, as it was the Dictyostelium enzyme whose structure was first determined and related to enzymatic activity. The phenotypic richness and tractability of Dictyostelium should provide a fertile arena for future exploration of NDPK's cellular roles.

Phototaxis has been studied in a variety of organisms belonging to all three major taxonomic domains - the bacteria, the archaea and the eukarya. Dictyostelium discoideum is one of a small number of eukaryotic organisms which are amenable to studying the signalling pathways involved in phototaxis. In this study we provide evidence based on protein coimmunoprecipitation for a phototaxis signalling complex in Dictyostelium that includes the proteins RasD, filamin, ErkB, GRP125 and PKB.

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

Dictyostelium provides a well established model system for the study of mitochondrial biology and disease. Mitochondrial dysfunction in Dictyostelium has been generated by knockout of nonessential nuclear genes encoding mitochondrial proteins, by knockout of targeted mitochondrial genes in a subset of the mitochondria, and by knock down of essential nuclear-encoded mitochondrial proteins. The resulting effects on mitochondrial electron transport and membrane potential can be studied by directly measuring the activities, composition, and assembly or stability of individual mitochondrial respiratory complexes and by using fluorescent probes to assay the mitochondrial membrane potential in vivo. Assays for these are described here. The complexities of mammalian developmental biology have obscured the phenotype-genotype relationships in mitochondrial disease and this has inhibited understanding of the underlying cytopathological mechanisms. By contrast, the Dictyostelium model has revealed a characteristic constellation of downstream phenotypic outcomes that, e.g., point to/show common underlying cytopathological mechanisms in mitochondrial disease. These aberrant phenotypes arise from chronic hyperactivity of the energy-sensing protein kinase AMPK and the assay of the most prominent of them is described here.