Could the kynurenine pathway be the key missing piece of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) complex puzzle?

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.

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.

Novel Blood Biomarkers Are Associated with White Matter Lesions in Fragile X- Associated Tremor/Ataxia Syndrome.

BACKGROUND: The need for accessible cellular biomarkers of neurodegeneration in carriers of the fragile X mental retardation 1 (FMR1) premutation (PM) alleles. OBJECTIVE: To assess the mitochondrial status and respiration in blood lymphoblasts from PM carriers manifesting the fragile X-associated tremor/ataxia syndrome (FXTAS) and non-FXTAS carriers, and their relationship with the brain white matter lesions. METHODS: Oxygen consumption rates (OCR) and ATP synthesis using a Seahorse XFe24 Extracellular Flux Analyser, and steady-state parameters of mitochondrial function were assessed in cultured lymphoblasts from 16 PM males (including 11 FXTAS patients) and 9 matched controls. The regional white matter hyperintensity (WMH) scores were obtained from MRI. RESULTS: Mitochondrial respiratory activity was significantly elevated in lymphoblasts from PM carriers compared with controls, with a 2- to 3-fold increase in basal and maximum OCR attributable to complex I activity, and ATP synthesis, accompanied by unaltered mitochondrial mass and membrane potential. The changes, which were more advanced in FXTAS patients, were significantly associated with the WMH scores in the supratentorial regions. CONCLUSION: The dramatic increase in mitochondrial activity in lymphoblasts from PM carriers may represent either the early stages of disease (specific alterations in short-lived blood cells) or an activation of the lymphocytes under pathological situations. These changes may provide early, convenient blood biomarkers of clinical involvements.

Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning.

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.

Unravelling shared mechanisms: insights from recent ME/CFS research to illuminate long COVID pathologies.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic illness often triggered by an initiating acute event, mainly viral infections. The transition from acute to chronic disease remains unknown, but interest in this phenomenon has escalated since the COVID-19 pandemic and the post-COVID-19 illness, termed 'long COVID' (LC). Both ME/CFS and LC share many clinical similarities. Here, we present recent findings in ME/CFS research focussing on proposed disease pathologies shared with LC. Understanding these disease pathologies and how they influence each other is key to developing effective therapeutics and diagnostic tests. Given that ME/CFS typically has a longer disease duration compared with LC, with symptoms and pathologies evolving over time, ME/CFS may provide insights into the future progression of LC.

Oxygen consumption rate of flatworms under the influence of wake- and sleep-promoting neurotransmitters.

Flatworms are among the best studied animal models for regeneration; however, they also represent an emerging opportunity to investigate other biological processes as well. For instance, flatworms are nocturnal and sleep during the day, a state that is regulated by sleep/wake history and the action of the sleep-promoting neurotransmitter gamma-aminobutyric acid (or GABA). Sleep is widespread across the animal kingdom, where it serves many nonexclusive functions. Notably, sleep saves energy by reducing metabolic rate and by not doing something more energetically taxing. Whether the conservation of energy is apparent in sleeping flatworms is unclear. We measured the oxygen consumption rate (OCR) of flatworms dosed with either (1) GABA (n = 29) which makes flatworms inactive or (2) dopamine (n = 20) which stimulates flatworms to move, or (3) day and night neurotransmitter-free controls (n = 28 and 27, respectively). While OCR did not differ between the day and night, flatworms treated with GABA used less oxygen than those treated with dopamine, and less than the day-time control. Thus, GABA affected flatworm physiology, ostensibly by enforcing energy-conserving sleep. Evidence that dopamine increased metabolism was less strong. This work broadens our understanding of flatworm physiology and expands the phylogenetic applicability of energy conservation as a function of sleep.

The Dictyostelium model for mitochondrial disease.

Mitochondrial diseases are a diverse family of genetic disorders caused by mutations affecting mitochondrial proteins encoded in either the nuclear or the mitochondrial genome. By impairing mitochondrial oxidative phosphorylation, they compromise cellular energy production and the downstream consequences in humans are a bewilderingly complex array of signs and symptoms that can affect any of the major organ systems in unpredictable combinations. This complexity and unpredictability has limited our understanding of the cytopathological consequences of mitochondrial dysfunction. By contrast, in Dictyostelium the mitochondrial disease phenotypes are consistent, measurable "readouts" of dysregulated intracellular signalling pathways. When the underlying genetic defects would produce coordinate, generalized deficiencies in multiple mitochondrial respiratory complexes, the disease phenotypes are mediated by chronic activation of an energy-sensing protein kinase, AMP-activated protein kinase (AMPK). This chronic AMPK hyperactivity maintains mitochondrial mass and cellular ATP concentrations at normal levels, but chronically impairs growth, cell cycle progression, multicellular development, photosensory and thermosensory signal transduction. It also causes the cells to support greater proliferation of the intracellular bacterial pathogen, Legionella pneumophila. Notably however, phagocytic and macropinocytic nutrient uptake are impervious both to AMPK signalling and to these types of mitochondrial dysfunction. Surprisingly, a Complex I-specific deficiency (midA knockout) not only causes the foregoing AMPK-mediated defects, but also produces a dramatic deficit in endocytic nutrient uptake accompanied by an additional secondary defect in growth. More restricted and specific phenotypic outcomes are produced by knocking out genes for nuclear-encoded mitochondrial proteins that are not required for respiration. The Dictyostelium model for mitochondrial disease has thus revealed consistent patterns of sublethal dysregulation of intracellular signalling pathways that are produced by different types of underlying mitochondrial dysfunction.

Dysregulation of the Kynurenine Pathway, Cytokine Expression Pattern, and Proteomics Profile Link to Symptomology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Dysregulation of the kynurenine pathway (KP) is believed to play a significant role in neurodegenerative and cognitive disorders. While some evidence links the KP to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), further studies are needed to clarify the overall picture of how inflammation-driven KP disturbances may contribute to symptomology in ME/CFS. Here, we report that plasma levels of most bioactive KP metabolites differed significantly between ME/CFS patients and healthy controls in a manner consistent with their known contribution to symptomology in other neurological disorders. Importantly, we found that enhanced production of the first KP metabolite, kynurenine (KYN), correlated with symptom severity, highlighting the relationship between inflammation, KP dysregulation, and ME/CFS symptomology. Other significant changes in the KP included lower levels of the downstream KP metabolites 3-HK, 3-HAA, QUIN, and PIC that could negatively impact cellular energetics. We also rationalized KP dysregulation to changes in the expression of inflammatory cytokines and, for the first time, assessed levels of the iron (Fe)-regulating hormone hepcidin that is also inflammation-responsive. Levels of hepcidin in ME/CFS decreased nearly by half, which might reflect systemic low Fe levels or possibly ongoing hypoxia. We next performed a proteomics screen to survey for other significant differences in protein expression in ME/CFS. Interestingly, out of the seven most significantly modulated proteins in ME/CFS patient plasma, 5 proteins have roles in maintaining gut health, which considering the new appreciation of how gut microbiome and health modulates systemic KP could highlight a new explanation of symptomology in ME/CFS patients and potential new prognostic biomarker/s and/or treatment avenues.

A Dictyostelium SH2 adaptor protein required for correct DIF-1 signaling and pattern formation.

Dictyostelium is the only non-metazoan with functionally analyzed SH2 domains and studying them can give insights into their evolution and wider potential. LrrB has a novel domain configuration with leucine-rich repeat, 14-3-3 and SH2 protein-protein interaction modules. It is required for the correct expression of several specific genes in early development and here we characterize its role in later, multicellular development. During development in the light, slug formation in LrrB null (lrrB-) mutants is delayed relative to the parental strain, and the slugs are highly defective in phototaxis and thermotaxis. In the dark the mutant arrests development as an elongated mound, in a hitherto unreported process we term dark stalling. The developmental and phototaxis defects are cell autonomous and marker analysis shows that the pstO prestalk sub-region of the slug is aberrant in the lrrB- mutant. Expression profiling, by parallel micro-array and deep RNA sequence analyses, reveals many other alterations in prestalk-specific gene expression in lrrB- slugs, including reduced expression of the ecmB gene and elevated expression of ampA. During culmination ampA is ectopically expressed in the stalk, there is no expression of ampA and ecmB in the lower cup and the mutant fruiting bodies lack a basal disc. The basal disc cup derives from the pstB cells and this population is greatly reduced in the lrrB- mutant. This anatomical feature is a hallmark of mutants aberrant in signaling by DIF-1, the polyketide that induces prestalk and stalk cell differentiation. In a DIF-1 induction assay the lrrB- mutant is profoundly defective in ecmB activation but only marginally defective in ecmA induction. Thus the mutation partially uncouples these two inductive events. In early development LrrB interacts physically and functionally with CldA, another SH2 domain containing protein. However, the CldA null mutant does not phenocopy the lrrB- in its aberrant multicellular development or phototaxis defect, implying that the early and late functions of LrrB are affected in different ways. These observations, coupled with its domain structure, suggest that LrrB is an SH2 adaptor protein active in diverse developmental signaling pathways.

MidA is a putative methyltransferase that is required for mitochondrial complex I function.

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.

Interactions and Cytotoxicity of Human Neurodegeneration- Associated Proteins Tau and α-Synuclein in the Simple Model Dictyostelium discoideum.

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 in Dictyostelium discoideum alone, and in combination. We show that tau is phosphorylated in D. discoideum and 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 intracellular Legionella proliferation rates, but activated mitochondrial respiration and non-mitochondrial oxygen consumption. The expression of tau alone impaired multicellular morphogenesis, axenic growth and phototaxis, while enhancing intracellular Legionella proliferation. 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, Legionella proliferation) 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 using D. discoideum as a model to study the interaction of proteins involved in neurodegeneration.

The Parkinson's Disease-Associated Protein DJ-1 Protects Dictyostelium Cells from AMPK-Dependent Outcomes of Oxidative Stress.

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.

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.

A Conserved Role for LRRK2 and Roco Proteins in the Regulation of Mitochondrial Activity.

Parkinson's Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo. To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects.

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.

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.

Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells.

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.