Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Retrovirus reactivation in CHMP2BIntron5 models of frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most prevalent form of pre-senile dementia after Alzheimer's disease. Amyotrophic lateral sclerosis (ALS) can overlap genetically, pathologically and clinically with FTD indicating the two conditions are ends of a spectrum and may share common pathological mechanisms. FTD-ALS causing mutations are known to be involved in endosomal trafficking and RNA regulation. Using an unbiased genome-wide genetic screen to identify mutations affecting an FTD-ALS-related phenotype in Drosophila caused by CHMP2BIntron5 expression, we have uncovered repressors of retrovirus (RV) activity as modifiers of CHMP2BIntron5 toxicity. We report that neuronal expression of CHMP2BIntron5 causes an increase in the activity of the endogenous Drosophila RV, gypsy, in the nervous system. Genetically blocking Drosophila gypsy activation and pharmacologically inhibiting viral reverse transcriptase activity prevents degenerative phenotypes observed in fly and rat neurons. These findings directly link endosomal dysfunction to RV de-repression in an FTD-ALS model without TDP-43 pathology. These observations may contribute an understanding to previous discoveries of RV activation in ALS affected patients.

Syntaxin 13, a genetic modifier of mutant CHMP2B in frontotemporal dementia, is required for autophagosome maturation.

Phagophore maturation is a key step in the macroautophagy pathway, which is critical in many important physiological and pathological processes. Here we identified Drosophila N-ethylmaleimide-sensitive fusion protein 2 (dNSF2) and soluble NSF attachment protein (Snap) as strong genetic modifiers of mutant CHMP2B, an ESCRT-III component that causes frontotemporal dementia and autophagosome accumulation. Among several SNAP receptor (SNARE) genes, Drosophila syntaxin 13 (syx13) exhibited a strong genetic interaction with mutant CHMP2B. Knockdown of syntaxin 13 (STX13) or its binding partner Vti1a in mammalian cells caused LC3-positive puncta to accumulate and blocks autophagic flux. STX13 was present on LC3-positive phagophores induced by rapamycin and was highly enriched on multilamellar structures induced by dysfunctional ESCRT-III. Loss of STX13 also caused the accumulation of Atg5-positive puncta and the formation of multilamellar structures. These results suggest that STX13 is a genetic modifier of ESCRT-III dysfunction and participates in the maturation of phagophores into closed autophagosomes.

Leaky endosomes push tau over the seed limit.

The inter- and intracellular propagation of aggregated proteins like tau is emerging as a central mechanism behind progression of various neurodegenerative diseases. The steps by which tau aggregates and propagates is currently unclear. Chen et al. now combine a cell-based model of tau aggregation with a CRISPR interference (CRISPRi) genetic screen to identify components of the endosomal sorting complex required for transport (ESCRT) machinery as mediators of intracellular propagation of tau aggregates. These findings reveal a role for endolysosomal integrity in blocking tau propagation.

Neuroprotective activity of ursodeoxycholic acid in CHMP2BIntron5 models of frontotemporal dementia.

Frontotemporal dementia (FTD) is one of the most prevalent forms of early-onset dementia. It represents part of the FTD-Amyotrophic Lateral Sclerosis (ALS) spectrum, a continuum of genetically and pathologically overlapping disorders. FTD-causing mutations in CHMP2B, a gene encoding a core component of the heteromeric ESCRT-III Complex, lead to perturbed endosomal-lysosomal and autophagic trafficking with impaired proteostasis. While CHMP2B mutations are rare, dysfunctional endosomal-lysosomal signalling is common across the FTD-ALS spectrum. Using our established Drosophila and mammalian models of CHMP2BIntron5 induced FTD we demonstrate that the FDA-approved compound Ursodeoxycholic Acid (UDCA) conveys neuroprotection, downstream of endosomal-lysosomal dysfunction in both Drosophila and primary mammalian neurons. UDCA exhibited a dose dependent rescue of neuronal structure and function in Drosophila pan-neuronally expressing CHMP2BIntron5. Rescue of CHMP2BIntron5 dependent dendritic collapse and apoptosis with UDCA in rat primary neurons was also observed. UDCA failed to ameliorate aberrant accumulation of endosomal and autophagic organelles or ubiquitinated neuronal inclusions in both models. We demonstrate the neuroprotective activity of UDCA downstream of endosomal-lysosomal and autophagic dysfunction, delineating the molecular mode of action of UDCA and highlighting its potential as a therapeutic for the treatment of FTD-ALS spectrum disorders.

The pro-apoptotic JNK scaffold POSH/SH3RF1 mediates CHMP2BIntron5-associated toxicity in animal models of frontotemporal dementia.

Frontotemporal dementia (FTD) is one of the most prevalent forms of early-onset dementia. However, the pathological mechanisms driving neuronal atrophy in FTD remain poorly understood. Here we identify a conserved role for the novel pro-apoptotic protein plenty of SH3s (POSH)/SH3 domain containing ring finger 1 in mediating neuropathology in Drosophila and mammalian models of charged multivesicular body protein 2B (CHMP2BIntron5) associated FTD. Aberrant, AKT dependent, accumulation of POSH was observed throughout the nervous system of both Drosophila and mice expressing CHMP2BIntron5. Knockdown of POSH was shown to be neuroprotective and sufficient to alleviate aberrant neuronal morphology, behavioral deficits and premature-lethality in Drosophila models, as well as dendritic collapse and cell death in CHMP2BIntron5expressing rat primary neurons. POSH knockdown also ameliorated elevated markers of Jun N-terminal kinase and apoptotic cascades in both Drosophila and mammalian models. This study provides the first characterization of POSH as a potential component of an FTD neuropathology, identifying a novel apoptotic pathway with relevance to the FTD spectrum.

dOCRL maintains immune cell quiescence by regulating endosomal traffic.

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

Mitochondrial retrograde signaling regulates neuronal function.

Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, we induced mitochondrial dysfunction in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. We find that neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. We show that the Drosophila hypoxia inducible factor alpha (HIFα) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment.

A saposin deficiency model in Drosophila: Lysosomal storage, progressive neurodegeneration and sensory physiological decline.

Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs.

Identification of dietary alanine toxicity and trafficking dysfunction in a Drosophila model of hereditary sensory and autonomic neuropathy type 1.

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is characterized by a loss of distal peripheral sensory and motorneuronal function, neuropathic pain and tissue necrosis. The most common cause of HSAN1 is due to dominant mutations in serine palmitoyl-transferase subunit 1 (SPT1). SPT catalyses the condensation of serine with palmitoyl-CoA, the initial step in sphingolipid biogenesis. Identified mutations in SPT1 are known to both reduce sphingolipid synthesis and generate catalytic promiscuity, incorporating alanine or glycine into the precursor sphingolipid to generate a deoxysphingoid base (DSB). Why either loss of function in SPT1, or generation of DSBs should generate deficits in distal sensory function remains unclear. To address these questions, we generated a Drosophila model of HSAN1. Expression of dSpt1 bearing a disease-related mutation induced morphological deficits in synapse growth at the larval neuromuscular junction consistent with a dominant-negative action. Expression of mutant dSpt1 globally was found to be mildly toxic, but was completely toxic when the diet was supplemented with alanine, when DSBs were observed in abundance. Expression of mutant dSpt1 in sensory neurons generated developmental deficits in dendritic arborization with concomitant sensory deficits. A membrane trafficking defect was observed in soma of sensory neurons expressing mutant dSpt1, consistent with endoplasmic reticulum (ER) to Golgi block. We found that we could rescue sensory function in neurons expressing mutant dSpt1 by co-expressing an effector of ER-Golgi function, Rab1 suggesting compromised ER function in HSAN1 affected dendritic neurons. Our Drosophila model identifies a novel strategy to explore the pathological mechanisms of HSAN1.

Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle.

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Bällchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle.

Tree-mycorrhizal associations detected remotely from canopy spectral properties.

A central challenge in global ecology is the identification of key functional processes in ecosystems that scale, but do not require, data for individual species across landscapes. Given that nearly all tree species form symbiotic relationships with one of two types of mycorrhizal fungi - arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi - and that AM- and ECM-dominated forests often have distinct nutrient economies, the detection and mapping of mycorrhizae over large areas could provide valuable insights about fundamental ecosystem processes such as nutrient cycling, species interactions, and overall forest productivity. We explored remotely sensed tree canopy spectral properties to detect underlying mycorrhizal association across a gradient of AM- and ECM-dominated forest plots. Statistical mining of reflectance and reflectance derivatives across moderate/high-resolution Landsat data revealed distinctly unique phenological signals that differentiated AM and ECM associations. This approach was trained and validated against measurements of tree species and mycorrhizal association across ~130 000 trees throughout the temperate United States. We were able to predict 77% of the variation in mycorrhizal association distribution within the forest plots (P < 0.001). The implications for this work move us toward mapping mycorrhizal association globally and advancing our understanding of biogeochemical cycling and other ecosystem processes.

Class IIa histone deacetylases are conserved regulators of circadian function.

Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca(2+) and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery.

Dopaminergic expression of the Parkinsonian gene LRRK2-G2019S leads to non-autonomous visual neurodegeneration, accelerated by increased neural demands for energy.

Parkinson's disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration.

parkin-induced defects in neurophysiology and locomotion are generated by metabolic dysfunction and not oxidative stress.

Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.

Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling.

Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle.

Oxidative stress induces overgrowth of the Drosophila neuromuscular junction.

Synaptic terminals are known to expand and contract throughout an animal's life. The physiological constraints and demands that regulate appropriate synaptic growth and connectivity are currently poorly understood. In previous work, we identified a Drosophila model of lysosomal storage disease (LSD), spinster (spin), with larval neuromuscular synapse overgrowth. Here we identify a reactive oxygen species (ROS) burden in spin that may be attributable to previously identified lipofuscin deposition and lysosomal dysfunction, a cellular hallmark of LSD. Reducing ROS in spin mutants rescues synaptic overgrowth and electrophysiological deficits. Synapse overgrowth was also observed in mutants defective for protection from ROS and animals subjected to excessive ROS. ROS are known to stimulate JNK and fos signaling. Furthermore, JNK and fos in turn are known potent activators of synapse growth and function. Inhibiting JNK and fos activity in spin rescues synapse overgrowth and electrophysiological deficits. Similarly, inhibiting JNK, fos, and jun activity in animals with excessive oxidative stress rescues the overgrowth phenotype. These data suggest that ROS, via activation of the JNK signaling pathway, are a major regulator of synapse overgrowth. In LSD, increased autophagy contributes to lysosomal storage and, presumably, elevated levels of oxidative stress. In support of this suggestion, we report here that impaired autophagy function reverses synaptic overgrowth in spin. Our data describe a previously unexplored link between oxidative stress and synapse overgrowth via the JNK signaling pathway.

How neurons maintain their axons long-term: an integrated view of axon biology and pathology.

Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell bodies and up to a century in humans. This requires self-sufficient cell biology including structural proteins, organelles, and membrane trafficking, metabolic, signalling, translational, chaperone, and degradation machinery-all maintaining the homeostasis of energy, lipids, proteins, and signalling networks including reactive oxygen species and calcium. Axon maintenance also involves specialised cytoskeleton including the cortical actin-spectrin corset, and bundles of microtubules that provide the highways for motor-driven transport of components and organelles for virtually all the above-mentioned processes. Here, we aim to provide a conceptual overview of key aspects of axon biology and physiology, and the homeostatic networks they form. This homeostasis can be derailed, causing axonopathies through processes of ageing, trauma, poisoning, inflammation or genetic mutations. To illustrate which malfunctions of organelles or cell biological processes can lead to axonopathies, we focus on axonopathy-linked subcellular defects caused by genetic mutations. Based on these descriptions and backed up by our comprehensive data mining of genes linked to neural disorders, we describe the 'dependency cycle of local axon homeostasis' as an integrative model to explain why very different causes can trigger very similar axonopathies, providing new ideas that can drive the quest for strategies able to battle these devastating diseases.

GC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line.

Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 h. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeted and untargeted gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells.