Julia Bornhorst

Impact of Manganese on and Transfer across Blood-Brain and Blood-Cerebrospinal Fluid Barrier in Vitro

Background: Modes of neurotoxic action after Mn overexposure are not fully understood. Results: The blood-CSF barrier showed higher Mn sensitivity and active Mn transport properties. Conclusion: The blood-CSF barrier might be the major route for Mn into the brain. Significance: Deeper insight in the impact of Mn on and its transfer across brain barrier systems might help to prevent irreversible neurological damage. Manganese occupational and dietary overexposure has been shown to result in specific clinical central nervous system syndromes, which are similar to those observed in Parkinson disease. To date, modes of neurotoxic action of Mn are still to be elucidated but are thought to be strongly related to Mn accumulation in brain and oxidative stress. However, the pathway and the exact process of Mn uptake in the brain are yet not fully understood. Here, two well characterized primary porcine in vitro models of the blood-brain and the blood-cerebrospinal fluid (CSF) barrier were applied to assess the transfer of Mn in the brain while monitoring its effect on the barrier properties. Thus, for the first time effects of MnCl2 on the integrity of these two barriers as well as Mn transfer across the respective barriers are compared in one study. The data reveal a stronger Mn sensitivity of the in vitro blood-CSF barrier compared with the blood-brain barrier. Very interestingly, the negative effects of Mn on the structural and functional properties of the highly Mn-sensitive blood-CSF barrier were partly reversible after incubation with calcium. In summary, both the observed stronger Mn sensitivity of the in vitro blood-CSF barrier and the observed site-directed, most probably active, Mn transport toward the brain facing compartment, reveal that, in contrast to the general assumption in literature, after oral Mn intake the blood-CSF barrier might be the major route for Mn into the brain.

The effects of pdr1, djr1.1 and pink1 loss in manganese-induced toxicity and the role of α-synuclein in C. elegans

Parkinsons disease (PD) is a neurodegenerative brain disorder characterized by selective dopaminergic (DAergic) cell loss that results in overt motor and cognitive deficits. Current treatment options exist to combat PD symptomatology, but are unable to directly target its pathogenesis due to a lack of knowledge concerning its etiology. Several genes have been linked to PD, including three genes associated with an early-onset familial form: parkin, pink1 and dj1. All three genes are implicated in regulating oxidative stress pathways. Another hallmark of PD pathophysiology is Lewy body deposition, associated with the gain-of-function genetic risk factor α-synuclein. The function of α-synuclein is poorly understood, as it shows both neurotoxic and neuroprotective activities in PD. Using the genetically tractable invertebrate Caenorhabditis elegans (C. elegans) model system, the neurotoxic or neuroprotective role of α-synuclein upon acute Mn exposure in the background of mutated pdr1, pink1 or djr1.1 was examined. The pdr1 and djr1.1 mutants showed enhanced Mn accumulation and oxidative stress that was reduced by α-synuclein. Moreover, DAergic neurodegeneration, while unchanged with Mn exposure, returned to wild-type (WT) levels for pdr1, but not djr1.1 mutants expressing α-synuclein. Taken together, this study uncovers a novel, neuroprotective role for WT human α-synuclein in attenuating Mn-induced toxicity in the background of PD-associated genes, and further supports the role of extracellular dopamine in exacerbating Mn neurotoxicity.

Manganese inhibits poly(ADP-ribosyl)ation in human cells: a possible mechanism behind manganese-induced toxicity?

For humans manganese is both an essential trace element and, at higher doses, a toxic metal. Due to the ubiquitous occurrence of manganese in foodstuff, in industrial countries daily dietary uptake is higher as compared to the estimated daily requirement. Therefore manganese deficiency is extremely rare. In contrast chronic manganese toxicity, affecting primarily the central nervous system, is more prevalent. Thus manganese occupational and dietary overexposure has been shown to cause progressive, permanent, neurodegenerative damage, resulting in syndromes similar to idiopathic Parkinsons disease. To date modes of manganese neurotoxic action are poorly understood and in most studies oxidative stress is postulated as the underlying mechanism. The present study searched on the cellular level for a molecular mechanism behind manganese-induced neurotoxicity and investigated bioavailability, cytotoxicity and genotoxicity of MnCl(2), as well as its impact on the DNA damage response in human cells (HeLa S3) in culture. Whereas up to 10 µM MnCl(2) showed no induction of DNA strand breaks after 24 h incubation, manganese strongly inhibited H(2)O(2)-stimulated poly(ADP-ribosyl)ation at low, completely non-cytotoxic, for certain human exposure, relevant concentrations starting at 1 µM. Thereby inhibition of this essential DNA damage response signalling reaction was not due to a reduced gene expression or protein level of the responsible polymerase PARP-1. Taken together, the results indicate that manganese, under conditions of either overload due to high exposure or disturbed homeostasis, can disturb the cellular response to DNA strand breaks, which has been shown before (S. Katyal and P. J. McKinnon, Mech. Ageing Dev., 2008, 129, 483-491) to result in neurological diseases.

Oxidative stress mechanisms underlying Parkinson's disease-associated neurodegeneration in C. elegans.

Oxidative stress is thought to play a significant role in the development and progression of neurodegenerative diseases. Although it is currently considered a hallmark of such processes, the interweaving of a multitude of signaling cascades hinders complete understanding of the direct role of oxidative stress in neurodegeneration. In addition to its extensive use as an aging model, some researchers have turned to the invertebrate model Caenorhabditis elegans (C. elegans) in order to further investigate molecular mediators that either exacerbate or protect against reactive oxygen species (ROS)-mediated neurodegeneration. Due to their fully characterized genome and short life cycle, rapid generation of C. elegans genetic models can be useful to study upstream markers of oxidative stress within interconnected signaling pathways. This report will focus on the roles of C. elegans homologs for the oxidative stress-associated transcription factor Nrf2, as well as the autosomal recessive, early-onset Parkinson’s disease (PD)-associated proteins Parkin, DJ-1, and PINK1, in neurodegenerative processes.

Metal-induced neurodegeneration in C. elegans

The model species, Caenorhabditis elegans, has been used as a tool to probe for mechanisms underlying numerous neurodegenerative diseases. This use has been exploited to study neurodegeneration induced by metals. The allure of the nematode comes from the ease of genetic manipulation, the ability to fluorescently label neuronal subtypes, and the relative simplicity of the nervous system. Notably, C. elegans have approximately 60–80% of human genes and contain genes involved in metal homeostasis and transport, allowing for the study of metal-induced degeneration in the nematode. This review discusses methods to assess degeneration as well as outlines techniques for genetic manipulation and presents a comprehensive survey of the existing literature on metal-induced degeneration studies in the worm.

Real-time Imaging Reveals That P2Y2 and P2Y12 Receptor Agonists Are Not Chemoattractants and Macrophage Chemotaxis to Complement C5a Is Phosphatidylinositol 3-Kinase (PI3K)- and p38 Mitogen-activated Protein Kinase (MAPK)-independent

Background: ATP, PI3K, and p38 MAPK signaling is implicated in the recruitment of immune cells. Results: Macrophages did not migrate toward ATPγS (ATP analog) but migrated to C5a independent of PI3K and p38 MAPK. Conclusion: ATP does not recruit macrophages but locally induces lamellipodial membrane extensions. Significance: ATP can promote chemotaxis and phagocytosis via autocrine/paracrine signaling but itself is not a chemoattractant. Adenosine 5′-triphosphate (ATP) has been implicated in the recruitment of professional phagocytes (neutrophils and macrophages) to sites of infection and tissue injury in two distinct ways. First, ATP itself is thought to be a chemotactic “find me” signal released by dying cells, and second, autocrine ATP signaling is implicated as an amplifier mechanism for chemotactic navigation to end-target chemoattractants, such as complement C5a. Here we show using real-time chemotaxis assays that mouse peritoneal macrophages do not directionally migrate to stable analogs of ATP (adenosine-5′-(γ-thio)-triphosphate (ATPγS)) or its hydrolysis product ADP (adenosine-5′-(β-thio)-diphosphate (ADPβS)). HPLC revealed that these synthetic P2Y2 (ATPγS) and P2Y12 (ADPβS) receptor ligands were in fact slowly degraded. We also found that ATPγS, but not ADPβS, promoted chemokinesis (increased random migration). Furthermore, we found that photorelease of ATP or ADP induced lamellipodial membrane extensions. At the cell signaling level, C5a, but not ATPγS, activated Akt, whereas both ligands induced p38 MAPK activation. p38 MAPK and Akt activation are strongly implicated in neutrophil chemotaxis. However, we found that inhibitors of phosphatidylinositol 3-kinase (PI3K; upstream of Akt) and p38 MAPK (or conditional deletion of p38α MAPK) did not impair macrophage chemotactic efficiency or migration velocity. Our results suggest that PI3K and p38 MAPK are redundant for macrophage chemotaxis and that purinergic P2Y2 and P2Y12 receptor ligands are not chemotactic. We propose that ATP signaling is strictly autocrine or paracrine and that ATP and ADP may act as short-range “touch me” (rather than long-range find me) signals to promote phagocytic clearance via cell spreading.

Differing cytotoxicity and bioavailability of selenite, methylselenocysteine, selenomethionine, selenosugar 1 and trimethylselenonium ion and their underlying metabolic transformations in human cells

SCOPE The trace element selenium (Se) is an integral component of our diet. However, its metabolism and toxicity following elevated uptake are not fully understood. Since the either adverse or beneficial health effects strongly depend on the ingested Se species, five low molecular weight species were investigated regarding their toxicological effects, cellular bioavailability and species-specific metabolism in human cells. METHODS AND RESULTS For the first time, the urinary metabolites methyl-2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 1) and trimethylselenonium ion (TMSe) were toxicologically characterised in comparison to the food relevant species methylselenocysteine (MeSeCys), selenomethionine (SeMet) and selenite in human urothelial, astrocytoma and hepatoma cells. In all cell lines selenosugar 1 and TMSe showed no cytotoxicity. Selenite, MeSeCys and SeMet exerted substantial cytotoxicity, which was strongest in the urothelial cells. There was no correlation between the potencies of the respective toxic effects and the measured cellular Se concentrations. Se speciation indicated that metabolism of the respective species is likely to affect cellular toxicity. CONCLUSION Despite being taken up, selenosugar 1 and TMSe are non-cytotoxic to urothelial cells, most likely because they are not metabolically activated. The absent cytotoxicity of selenosugar 1 and TMSe up to supra-physiological concentrations, support their importance as metabolites for Se detoxification.

Age- and manganese-dependent modulation of dopaminergic phenotypes in a C. elegans DJ-1 genetic model of Parkinson's disease

Parkinsons disease (PD) is the second most common neurodegenerative disease, yet its etiology and pathogenesis are poorly understood. PD is characterized by selective dopaminergic (DAergic) degeneration and progressive hypokinetic motor impairment. Mutations in dj-1 cause autosomal recessive early-onset PD. DJ-1 is thought to protect DAergic neurons via an antioxidant mechanism, but the precise basis of this protection has not yet been resolved. Aging and manganese (Mn) exposure are significant non-genetic risk factors for PD. Caenorhabditis elegans (C. elegans) is an optimal model for PD and aging studies because of its simple nervous system, conserved DAergic machinery, and short 20-day lifespan. Here we tested the hypothesis that C. elegans DJ-1 homologues were protective against Mn-induced DAergic toxicity in an age-dependent manner. We showed that the deletion of C. elegans DJ-1 related (djr) genes, djr-1.2, decreased survival after Mn exposure. djr-1.2, the DJ-1 homologue was expressed in DAergic neurons and its deletion decreased lifespan and dopamine (DA)-dependent dauer movement behavior after Mn exposure. We also tested the role of DAF-16 as a regulator of dj-1.2 interaction with Mn toxicity. Lifespan defects resulting from djr-1.2 deletion could be restored to normal by overexpression of either DJR-1.2 or DAF-16. Furthermore, dauer movement alterations after djr-1.2 deletion were abolished by constitutive activation of DAF-16 through mutation of its inhibitor, DAF-2 insulin receptor. Taken together, our results reveal PD-relevant interactions between aging, the PD environmental risk factor manganese, and homologues of the established PD genetic risk factor DJ-1. Our data demonstrate a novel role for the DJ-1 homologue, djr-1.2, in mitigating Mn-dependent lifespan reduction and DA signaling alterations, involving DAF-2/DAF-16 signaling.

Developmental exposure to manganese induces lasting motor and cognitive impairment in rats

Exposure to high manganese (Mn) levels may damage the basal ganglia, leading to a syndrome analogous to Parkinsons 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.

Untargeted metabolic profiling identifies interactions between Huntington's disease and neuronal manganese status

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 Huntingtons 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.