Dysregulated Provision of Oxidisable Substrates to the Mitochondria in ME/CFS Lymphoblasts.

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 ß-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 ß-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.

Relationships between Mitochondrial Function, AMPK, and TORC1 Signaling in Lymphoblasts with Premutation Alleles of the FMR1 Gene.

The X-linked FMR1 gene contains a non-coding trinucleotide repeat in its 5' region that, in normal, healthy individuals contains 20-44 copies. Large expansions of this region (>200 copies) cause fragile X syndrome (FXS), but expansions of 55-199 copies (referred to as premutation alleles) predispose carriers to a neurodegenerative disease called fragile X-associated tremor/ataxia syndrome (FXTAS). The cytopathological mechanisms underlying FXTAS are poorly understood, but abnormalities in mitochondrial function are believed to play a role. We previously reported that lymphoblastoid cell lines (LCLs, or lymphoblasts) of premutation carriers have elevated mitochondrial respiratory activities. In the carriers, especially those not clinically affected with FXTAS, AMP-activated protein kinase (AMPK) activity was shown to be elevated. In the FXTAS patients, however, it was negatively correlated with brain white matter lesions, suggesting a protective role in the molecular mechanisms. Here, we report an enlarged and extended study of mitochondrial function and associated cellular stress-signaling pathways in lymphoblasts isolated from male and female premutation carriers, regardless of their clinical status, and healthy controls. The results confirmed the elevation of AMPK and mitochondrial respiratory activities and reduction in reactive O2 species (ROS) levels in premutation cells and revealed for the first time that target of rapamycin complex I (TORC1) activities are reduced. Extensive correlation, multiple regression, and principal components analysis revealed the best fitting statistical explanations of these changes in terms of the other variables measured. These suggested which variables might be the most "proximal" regulators of the others in the extensive network of known causal interactions amongst the measured parameters of mitochondrial function and cellular stress signaling. In the resulting model, the premutation alleles activate AMPK and inhibit both TORC1 and ROS production, the reduced TORC1 activity contributes to activation of AMPK and of nonmitochondrial metabolism, and the higher AMPK activity results in elevated catabolic metabolism, mitochondrial respiration, and ATP steady state levels. In addition, the results suggest a separate CGG repeat number-dependent elevation of TORC1 activity that is insufficient to overcome the inhibition of TORC1 in premutation cells but may presage the previously reported activation of TORC1 in FXS cells.

Cell-Based Blood Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

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-lymphocyte death rate, mitochondrial respiratory function and TORC1 activity-could each individually serve as a biomarker with better than 90% sensitivity but only modest specificity vís a vís 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.

An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients.

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 metabolizing 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 signaling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters, and enzymes of the ß-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 ß-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.

An ancestral non-proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum.

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

Polycystin-2 Mediated Calcium Signalling in the Dictyostelium Model for Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

Changes in the microbial community structure of bacteria, archaea and fungi in response to elevated CO(2) and warming in an Australian native grassland soil.

The microbial community structure of bacteria, archaea and fungi is described in an Australian native grassland soil after more than 5 years exposure to different atmospheric CO2 concentrations ([CO2]) (ambient, +550 ppm) and temperatures (ambient, + 2°C) under different plant functional types (C3 and C4 grasses) and at two soil depths (0-5 cm and 5-10 cm). Archaeal community diversity was influenced by elevated [CO2], while under warming archaeal 16S rRNA gene copy numbers increased for C4 plant Themeda triandra and decreased for the C3 plant community (P < 0.05). Fungal community diversity resulted in three groups based upon elevated [CO2], elevated [CO2] plus warming and ambient [CO2]. Overall bacterial community diversity was influenced primarily by depth. Specific bacterial taxa changed in richness and relative abundance in response to climate change factors when assessed by a high-resolution 16S rRNA microarray (PhyloChip). Operational taxonomic unit signal intensities increased under elevated [CO2] for both Firmicutes and Bacteroidetes, and increased under warming for Actinobacteria and Alphaproteobacteria. For the interaction of elevated [CO2] and warming there were 103 significant operational taxonomic units (P < 0.01) representing 15 phyla and 30 classes. The majority of these operational taxonomic units increased in abundance for elevated [CO2] plus warming plots, while abundance declined in warmed or elevated [CO2] plots. Bacterial abundance (16S rRNA gene copy number) was significantly different for the interaction of elevated [CO2] and depth (P < 0.05) with decreased abundance under elevated [CO2] at 5-10 cm, and for Firmicutes under elevated [CO2] (P < 0.05). Bacteria, archaea and fungi in soil responded differently to elevated [CO2], warming and their interaction. Taxa identified as significantly climate-responsive could show differing trends in the direction of response ('+' or '-') under elevated CO2 or warming, which could then not be used to predict their interactive effects supporting the need to investigate interactive effects for climate change. The approach of focusing on specific taxonomic groups provides greater potential for understanding complex microbial community changes in ecosystems under climate change.

The Dictyostelium Model for Mucolipidosis Type IV.

Mucolipidosis type IV, a devastating neurological lysosomal disease linked to mutations in the transient receptor potential channel mucolipin 1, TRPML1, a calcium permeable channel in the membranes of vesicles in endolysosomal system. TRPML1 function is still being elucidated and a better understanding of the molecular pathogenesis of Mucolipidosis type IV, may facilitate development of potential treatments. We have created a model to study mucolipin function in the eukaryotic slime mould Dictyostelium discoideum by altering expression of its single mucolipin homologue, mcln. We show that in Dictyostelium mucolipin overexpression contributes significantly to global chemotactic calcium responses in vegetative and differentiated cells. Knockdown of mucolipin also enhances calcium responses in vegetative cells but does not affect responses in 6-7 h developed cells, suggesting that in developed cells mucolipin may help regulate local calcium signals rather than global calcium waves. We found that both knocking down and overexpressing mucolipin often, but not always, presented the same phenotypes. Altering mucolipin expression levels caused an accumulation or increased acidification of Lysosensor Blue stained vesicles in vegetative cells. Nutrient uptake by phagocytosis and macropinocytosis were increased but growth rates were not, suggesting defects in catabolism. Both increasing and decreasing mucolipin expression caused the formation of smaller slugs and larger numbers of fruiting bodies during multicellular development, suggesting that mucolipin is involved in initiation of aggregation centers. The fruiting bodies that formed from these smaller aggregates had proportionately larger basal discs and thickened stalks, consistent with a regulatory role for mucolipin-dependent Ca2+ signalling in the autophagic cell death pathways involved in stalk and basal disk differentiation in Dictyostelium. Thus, we have provided evidence that mucolipin contributes to chemotactic calcium signalling and that Dictyostelium is a useful model to study the molecular mechanisms involved in the cytopathogenesis of Mucolipidosis type IV.

Cellular Bioenergetics and AMPK and TORC1 Signalling in Blood Lymphoblasts Are Biomarkers of Clinical Status in FMR1 Premutation Carriers.

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.

Cytotoxicity and Mitochondrial Dysregulation Caused by α-Synuclein in Dictyostelium discoideum.

Alpha synuclein has been linked to both sporadic and familial forms of Parkinson's disease (PD) and is the most abundant protein in Lewy bodies a hallmark of Parkinson'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 α-synuclein toxicity involves alterations to mitochondrial function. Here we expressed human α-synuclein and two PD-causing α-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 α-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 α-synuclein is required and sufficient for the localisation of the protein to the cell cortex in D. discoideum.

The Spectrum of Neurological and White Matter Changes and Premutation Status Categories of Older Male Carriers of the FMR1 Alleles Are Linked to Genetic (CGG and FMR1 mRNA) and Cellular Stress (AMPK) Markers.

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.

Immortalized Parkinson's disease lymphocytes have enhanced mitochondrial respiratory activity.

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.

In vivo measurements of cytosolic calcium in Dictyostelium discoideum.

The involvement of calcium signalling during chemotaxis in Dictyostelium discoideum is well documented. Spatiotemporal increases of intracellular calcium ([Ca(2+)](i)) have been observed within seconds of stimulation with the chemoattractants folic acid and cAMP. This rise in [Ca(2+)](i) localises to the rear cortex of the cell (J. Cell Sci. 109:2673-2678, 1996) and has been found to be not essential for chemotaxis, but likely to be involved in fine tuning of chemotactic responses (EMBO J. 19:4846-4854, 2000). Measurements of cytosolic Ca(2+) ([Ca(2+)](c)) responses have involved the use of different Ca(2+) probes including ectopically expressed aequorin (a Ca(2+)-sensitive photoprotein), the fluorescent dye fura-2-dextran and the radioisotope (45)Ca(2+). The aequorin method (J. Cell Sci. 110:2845-2853, 1997) offers nonperturbing, real-time measurement of cytosolic free Ca(2+) in suspensions of cells, but the low levels of light emission preclude measurements on individual cells. Fura-2 imaging (Cell Calcium 22:65-74, 1997; Eur. J. Cell Biol. 58:172-181, 1992; Biochem. J. 332:541-548, 1998; BMC Cell Biol. 6:13, 2005) has the advantage of allowing Ca(2+) responses to be observed in individual cells so that the subcellular localisation of the response and differences amongst individual cells can be observed. However data collection is more labour intensive, much smaller numbers of cells are sampled, the cells are unavoidably damaged physically during loading and the time resolution (s) is much less than that provided by the aequorin method (ms). (45)Ca(2+) uptake assays (Cell Biol. Int. Rep. 2:71-79, 1978; J. Cell Biol. 112:103-110, 1991) allow measurement of Ca(2+) influx from the medium by cell suspensions with a time resolution of the order of seconds. Radioactive Ca(2+) uptake measurements are unsullied by but equally do not provide information about Ca(2+) efflux, intracellular Ca(2+) release or sequestration or changes in cytosolic free Ca(2+) levels.