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Institute
Manganese (Mn) is an essential micronutrient for development and function of the nervous system. Deficiencies in Mn transport have been implicated in the pathogenesis of Huntington's disease (HD), an autosomal dominant neurodegenerative disorder characterized by loss of medium spiny neurons of the striatum. Brain Mn levels are highest in striatum and other basal ganglia structures, the most sensitive brain regions to Mn neurotoxicity. Mouse models of HD exhibit decreased striatal Mn accumulation and HD striatal neuron models are resistant to Mn cytotoxicity. We hypothesized that the observed modulation of Mn cellular transport is associated with compensatory metabolic responses to HD pathology. Here we use an untargeted metabolomics approach by performing ultraperformance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS) on control and HD immortalized mouse striatal neurons to identify metabolic disruptions under three Mn exposure conditions, low (vehicle), moderate (non-cytotoxic) and high (cytotoxic). Our analysis revealed lower metabolite levels of pantothenic acid, and glutathione (GSH) in HD striatal cells relative to control cells. HD striatal cells also exhibited lower abundance and impaired induction of isobutyryl carnitine in response to increasing Mn exposure. In addition, we observed induction of metabolites in the pentose shunt pathway in HD striatal cells after high Mn exposure. These findings provide metabolic evidence of an interaction between the HD genotype and biologically relevant levels of Mn in a striatal cell model with known HD by Mn exposure interactions. The metabolic phenotypes detected support existing hypotheses that changes in energetic processes underlie the pathobiology of both HD and Mn neurotoxicity.
Aims
Vitellogenesis is the yolk production process which provides the essential nutrients for the developing embryos. Yolk is a lipoprotein particle that presents lipids and lipid-binding proteins, referred to as vitellogenins (VIT). The Caenorhabditis elegans nematode has six genes encoding VIT lipoproteins. Several pathways are known to regulate vitellogenesis, including the DAF-16 transcription factor. Some reports have shown that heavy metals, such as manganese (Mn), impair brood size in C. elegans; however the mechanisms associated with this effect have yet to be identified. Our aim was to evaluate Mn′s effects on C. elegans reproduction and better understand the pathways related to these effects.
Main methods.
Young adult larval stage worms were treated for 4 h with Mn in 85 mM NaCl and Escherichia coli OP50 medium.
Key findings.
Mn reduced egg-production and egg-laying during the first 24 h after the treatment, although the total number of progenies were indistinguishable from the control group levels. This delay may have occurred due to DAF-16 activation, which was noted only after the treatment and was not apparent 24 h later. Moreover, the expression, protein levels and green fluorescent protein (GFP) fluorescence associated with VIT were decreased soon after Mn treatment and recovered after 24 h.
Significance
Combined, these data suggest that the delay in egg-production is likely regulated by DAF-16 and followed by the inhibition of VIT transport activity. Further studies are needed to clarify the mechanisms associated with Mn-induced DAF-16 activation.
Manganese (Mn) is an essential trace element for physiological functions since it acts as an enzymatic co-factor. Nevertheless, overexposure to Mn has been associated with a pathologic condition called manganism. Furthermore, Mn has been reported to affect lipid metabolism by mechanisms which have yet to be established. Herein, we used the nematode Caenorhabditis elegans to examine Mn’s effects on the dopaminergic (DAergic) system and determine which transcription factors that regulate with lipid metabolism are affected by it. Worms were exposed to Mn for four hours in the presence of bacteria and in a liquid medium (85 mM NaCl). Mn increased fat storage as evidenced both by Oil Red O accumulation and triglyceride levels. In addition, metabolic activity was reduced as a reflection of decreased oxygen consumption caused by Mn. Mn also affected feeding behavior as evidenced by decreased pharyngeal pumping rate. DAergic neurons viability were not altered by Mn, however the dopamine levels were significantly reduced following Mn exposure. Furthermore, the expression of sbp-1 transcription factor and let-363 protein kinase responsible for lipid accumulation control was increased and decreased, respectively, by Mn. Altogether, our data suggest that Mn increases the fat storage in C. elegans, secondary to DAergic system alterations, under the control of SBP-1 and LET-363 proteins.
Methylmercury (MeHg), an abundant environmental pollutant, has long been known to adversely affect neurodevelopment in both animals and humans. Several reports from epidemiological studies, as well as experimental data indicate sex-specific susceptibility to this neurotoxicant; however, the molecular bases of this process are still not clear. In the present study, we used Caenorhabditis elegans (C. elegans), to investigate sex differences in response to MeHg toxicity during development. Worms at different developmental stage (L1, L4, and adult) were treated with MeHg for 1h. Lethality assays revealed that male worms exhibited significantly higher resistance to MeHg than hermaphrodites, when at L4 stage or adults. However, the number of worms with degenerated neurons was unaffected by MeHg, both in males and hermaphrodites. Lower susceptibility of males was not related to changes in mercury (Hg) accumulation, which was analogous for both wild-type (wt) and male-rich him-8 strain. Total glutathione (GSH) levels decreased upon MeHg in him-8, but not in wt. Moreover, the sex-dependent response of the cytoplasmic thioredoxin system was observedmales exhibited significantly higher expression of thioredoxin TRX-1, and thioredoxin reductase TRXR-1 expression was downregulated upon MeHg treatment only in hermaphrodites. These outcomes indicate that the redox status is an important contributor to sex-specific sensitivity to MeHg in C. elegans.
cis-Diamminedichloroplatinum(II) (Cisplatin) is one of the most important and frequently used cytostatic drugs for the treatment of various solid tumors. Herein, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method incorporating a fast and simple sample preparation protocol was developed for the elemental mapping of Cisplatin in the model organism Caenorhabditis elegans (C. elegans). The method allows imaging of the spatially-resolved elemental distribution of platinum in the whole organism with respect to the anatomic structure in L4 stage worms at a lateral resolution of 5 μm. In addition, a dose- and time-dependent Cisplatin uptake was corroborated quantitatively by a total reflection X-ray fluorescence spectroscopy (TXRF) method, and the elemental mapping indicated that Cisplatin is located in the intestine and in the head of the worms. Better understanding of the distribution of Cisplatin in this well-established model organism will be instrumental in deciphering Cisplatin toxicity and pharmacokinetics. Since the cytostatic effect of Cisplatin is based on binding the DNA by forming intra- and interstrand crosslinks, the response of poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutants to Cisplatin was also examined. Loss of pme-1, which is the C. elegans ortholog of human poly(ADP-ribose) polymerase 1 (PARP-1) led to disturbed DNA damage response. With respect to survival and brood size, pme-1 deletion mutants were more sensitive to Cisplatin as compared to wildtype worms, while Cisplatin uptake was indistinguishable.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
Exposure to high manganese (Mn) levels may damage the basal ganglia, leading to a syndrome analogous to Parkinson's disease, with motor and cognitive impairments. The molecular mechanisms underlying Mn neurotoxicity, particularly during development, still deserve further investigation. Herein, we addressed whether early-life Mn exposure affects motor coordination and cognitive function in adulthood and potential underlying mechanisms. Male Wistar rats were exposed intraperitoneally to saline (control) or MnCl2 (5, 10 or 20 mg/kg/day) from post-natal day (PND) 8-12. Behavioral tests were performed on PND 60-65 and biochemical analysis in the striatum and hippocampus were performed on PND14 or PND70. Rats exposed to Mn (10 and 20 mg/kg) performed significantly worse on the rotarod test than controls indicating motor coordination and balance impairments. The object and social recognition tasks were used to evaluate short-term memory. Rats exposed to the highest Mn dose failed to recognize a familiar object when replaced by a novel object as well as to recognize a familiar juvenile rat after a short period of time. However, Mn did not alter olfactory discrimination ability. In addition, Mn-treated rats displayed decreased levels of non-protein thiols (e.g. glutathione) and increased levels of glial fibrillary acidic protein (GFAP) in the striatum. Moreover, Mn significantly increased hippocampal glutathione peroxidase (GPx) activity. These findings demonstrate that acute low-level exposure to Mn during a critical neurodevelopmental period causes cognitive and motor dysfunctions that last into adulthood, that are accompanied by alterations in antioxidant defense system in both the hippocampus and striatum. (C) 2015 Elsevier Inc. All rights reserved.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time-and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.