Acute manganese exposure impairs glutamatergic function in a young mouse model of Alzheimer's disease.

Manganese (Mn) is an essential metal that serves as a cofactor for metalloenzymes important in moderating oxidative stress and the glutamate/glutamine cycle. Mn is typically obtained through the diet, but toxic overexposure can occur through other environmental or occupational exposure routes such as inhalation. Mn is known to accumulate in the brain following exposure and may contribute to the etiology of neurodegenerative disorders such as Alzheimer's disease (AD) even in the absence of acute neurotoxicity. In the present study, we used in vitro primary cell culture, ex vivo slice electrophysiology and in vivo behavioral approaches to determine if Mn-induced changes in glutamatergic signaling may be altered by genetic risk factors for AD neuropathology. Primary cortical astrocytes incubated with Mn exhibited early rapid clearance of glutamate compared to saline treated astrocytes but decreased clearance over longer time periods, with no effect of the AD genotype. Further, we found that in vivo exposure to a subcutaneous subacute, high dose of Mn as manganese chloride tetrahydrate (3 ×50 mg/kg MnCl2·4(H2O) over 7 days) resulted in increased expression of cortical GLAST protein regardless of genotype, with no changes in GLT-1. Hippocampal long-term potentiation was not altered in APP/PSEN1 mice at this age and neither was it disrupted following Mn exposure. Mn exposure did increase sensitivity to seizure onset following treatment with the excitatory agonist kainic acid, with differing responses between APP/PSEN1 and control mice. These results highlight the sensitivity of the glutamatergic system to Mn exposure. Experiments were performed in young adult APP/PSEN1 mice, prior to cognitive decline or accumulation of hallmark amyloid plaque pathology and following subacute exposure to Mn. The data support a role of Mn in pathophysiology of AD in early stages of the disease and support the need to better understand neurological consequences of Mn exposure in vulnerable populations.

Ascorbate deficiency decreases dopamine release in gulo-/- and APP/PSEN1 mice.

Dopamine (DA) has important roles in learning, memory, and motivational processes and is highly susceptible to oxidation. In addition to dementia, Alzheimer's disease (AD) patients frequently exhibit decreased motivation, anhedonia, and sleep disorders, suggesting deficits in dopaminergic neurotransmission. Vitamin C (ascorbate, ASC) is a critical antioxidant in the brain and is often depleted in AD patients as a result of disease-related oxidative stress and dietary deficiencies. To probe the effects of ASC deficiency and AD pathology on the DAergic system, gulo-/- mice, which like humans depend on dietary ASC to maintain adequate tissue levels, were crossed with APP/PSEN1 mice and provided sufficient or depleted ASC supplementation from weaning until 12 months of age. Ex vivo fast-scan cyclic voltammetry showed that chronic ASC depletion and APP/PSEN1 genotype both independently decreased dopamine release in the nucleus accumbens, a hub for motivational behavior and reward, while DA clearance was similar across all groups. In striatal tissue containing nucleus accumbens, low ASC treatment led to decreased levels of DA and its metabolites 3,4-dihydroxyohenyl-acetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA). Decreased enzyme activity observed through lower pTH/TH ratio was driven by a cumulative effect of ASC depletion and APP/PSEN1 genotype. Together the data show that deficits in dopaminergic neurotransmission resulting from age and disease status are magnified in conditions of low ASC which decrease DA availability during synaptic transmission. Such deficits may contribute to the non-cognitive behavioral changes observed in AD including decreased motivation, anhedonia, and sleep disorders.

A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens.

Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess metal uptake can be toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. aureus through disruption of metal homeostasis. VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship, reactive oxygen species (ROS) generation assays, metal binding assays, and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of enzymes, generation of reactive oxygen species, or disruption of other cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple Gram-positive bacteria, leading to increased reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates VU0026921 as a probe to further decipher mechanisms required to maintain metal homeostasis in Gram-positive bacteria.IMPORTANCEStaphylococcus aureus is a leading agent of antibiotic-resistant bacterial infections in the world. S. aureus tightly controls metal homeostasis during infection, and disruption of metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of VU0026921 is modulated by metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in metal detoxification. This work defines a small-molecule probe for further defining the role of metal toxicity in S. aureus and validates future antibiotic development targeting metal toxicity pathways.

Manganese-induced neurodegenerative diseases and possible therapeutic approaches.

INTRODUCTION: Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and prion disease represent important public health concerns. Exposure to high levels of  heavy metals such as manganese (Mn) may contribute to their development. AREAS COVERED: In this critical review, we address the role of Mn in the etiology of neurodegenerative diseases and discuss emerging treatments of Mn overload, such as chelation therapy. In addition, we discuss natural and synthetic compounds under development as prospective therapeutics. Moreover, bioinformatic approaches to identify new potential targets and therapeutic substances to reverse the neurodegenerative diseases are discussed. EXPERT OPINION: Here, the authors highlight the importance of better understanding the molecular mechanisms of toxicity associated with neurodegenerative diseases, and the role of Mn in these diseases. Additional emphasis should be directed to the discovery of new agents to treat Mn-induced diseases, since present day chelator therapies have limited bioavailability. Furthermore, the authors encourage the scientific community to develop research using libraries of compounds to screen those compounds that show efficacy in regulating brain Mn levels. In addition, bioinformatics may provide novel insight for pathways and clinical treatments associated with Mn-induced neurodegeneration, leading to a new direction in Mn toxicological research.

Manganese Acts upon Insulin/IGF Receptors to Phosphorylate AKT and Increase Glucose Uptake in Huntington's Disease Cells.

Perturbations in insulin/IGF signaling and manganese (Mn2+) uptake and signaling have been separately reported in Huntington's disease (HD) models. Insulin/IGF supplementation ameliorates HD phenotypes via upregulation of AKT, a known Mn2+-responsive kinase. Limited evidence both in vivo and in purified biochemical systems suggest Mn2+ enhances insulin/IGF receptor (IR/IGFR), an upstream tyrosine kinase of AKT. Conversely, Mn2+ deficiency impairs insulin release and associated glucose tolerance in vivo. Here, we test the hypothesis that Mn2+-dependent AKT signaling is predominantly mediated by direct Mn2+ activation of the insulin/IGF receptors, and HD-related impairments in insulin/IGF signaling are due to HD genotype-associated deficits in Mn2+ bioavailability. We examined the combined effects of IGF-1 and/or Mn2+ treatments on AKT signaling in multiple HD cellular models. Mn2+ treatment potentiates p-IGFR/IR-dependent AKT phosphorylation under physiological (1 nM) or saturating (10 nM) concentrations of IGF-1 directly at the level of intracellular activation of IGFR/IR. Using a multi-pharmacological approach, we find that > 70-80% of Mn2+-associated AKT signaling across rodent and human neuronal cell models is specifically dependent on IR/IGFR, versus other signaling pathways upstream of AKT activation. Mn2+-induced p-IGFR and p-AKT were diminished in HD cell models, and, consistent with our hypothesis, were rescued by co-treatment of Mn2+ and IGF-1. Lastly, Mn2+-induced IGF signaling can modulate HD-relevant biological processes, as the reduced glucose uptake in HD STHdh cells was partially reversed by Mn2+ supplementation. Our data demonstrate that Mn2+ supplementation increases peak IGFR/IR-induced p-AKT likely via direct effects on IGFR/IR, consistent with its role as a cofactor, and suggests reduced Mn2+ bioavailability contributes to impaired IGF signaling and glucose uptake in HD models.

A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs.

Human induced pluripotent stem cell (iPSC)-derived developmental lineages are key tools for in vitro mechanistic interrogations, drug discovery, and disease modeling. iPSCs have previously been differentiated to endothelial cells with blood-brain barrier (BBB) properties, as defined by high transendothelial electrical resistance (TEER), low passive permeability, and active transporter functions. Typical protocols use undefined components, which impart unacceptable variability on the differentiation process. We demonstrate that replacement of serum with fully defined components, from common medium supplements to a simple mixture of insulin, transferrin, and selenium, yields BBB endothelium with TEER in the range of 2,000-8,000 Ω × cm2 across multiple iPSC lines, with appropriate marker expression and active transporters. The use of a fully defined medium vastly improves the consistency of differentiation, and co-culture of BBB endothelium with iPSC-derived astrocytes produces a robust in vitro neurovascular model. This defined differentiation scheme should broadly enable the use of human BBB endothelium for diverse applications.

Iron and manganese-related CNS toxicity: mechanisms, diagnosis and treatment.

INTRODUCTION: Iron (Fe) and manganese (Mn) are essential nutrients for humans. They act as cofactors for a variety of enzymes. In the central nervous system (CNS), these two metals are involved in diverse neurological activities. Dyshomeostasis may interfere with the critical enzymatic activities, hence altering the neurophysiological status and resulting in neurological diseases. Areas covered: In this review, the authors cover the molecular mechanisms of Fe/Mn-induced toxicity and neurological diseases, as well as the diagnosis and potential treatment. Given that both Fe and Mn are abundant in the earth crust, nutritional deficiency is rare. In this review the authors focus on the neurological disorders associated with Mn and Fe overload. Expert commentary: Oxidative stress and mitochondrial dysfunction are the primary molecular mechanism that mediates Fe/Mn-induced neurotoxicity. Although increased Fe or Mn concentrations have been found in brain of patients, it remains controversial whether the elevated metal amounts are the primary cause or secondary consequence of neurological diseases. Currently, treatments are far from satisfactory, although chelation therapy can significantly decrease brain Fe and Mn levels. Studies to determine the primary cause and establish the molecular mechanism of toxicity may help to adapt more comprehensive and satisfactory treatments in the future.

Reduced bioavailable manganese causes striatal urea cycle pathology in Huntington's disease mouse model.

Huntington's disease (HD) is caused by a mutation in the huntingtin gene (HTT), resulting in profound striatal neurodegeneration through an unknown mechanism. Perturbations in the urea cycle have been reported in HD models and in HD patient blood and brain. In neurons, arginase is a central urea cycle enzyme, and the metal manganese (Mn) is an essential cofactor. Deficient biological responses to Mn, and reduced Mn accumulation have been observed in HD striatal mouse and cell models. Here we report in vivo and ex vivo evidence of a urea cycle metabolic phenotype in a prodromal HD mouse model. Further, either in vivo or in vitro Mn supplementation reverses the urea-cycle pathology by restoring arginase activity. We show that Arginase 2 (ARG2) is the arginase enzyme present in these mouse brain models, with ARG2 protein levels directly increased by Mn exposure. ARG2 protein is not reduced in the prodromal stage, though enzyme activity is reduced, indicating that altered Mn bioavailability as a cofactor leads to the deficient enzymatic activity. These data support a hypothesis that mutant HTT leads to a selective deficiency of neuronal Mn at an early disease stage, contributing to HD striatal urea-cycle pathophysiology through an effect on arginase activity.

"Manganese-induced neurotoxicity: a review of its behavioral consequences and neuroprotective strategies".

Manganese (Mn) is an essential heavy metal. However, Mn's nutritional aspects are paralleled by its role as a neurotoxicant upon excessive exposure. In this review, we covered recent advances in identifying mechanisms of Mn uptake and its molecular actions in the brain as well as promising neuroprotective strategies. The authors focused on reporting findings regarding Mn transport mechanisms, Mn effects on cholinergic system, behavioral alterations induced by Mn exposure and studies of neuroprotective strategies against Mn intoxication. We report that exposure to Mn may arise from environmental sources, occupational settings, food, total parenteral nutrition (TPN), methcathinone drug abuse or even genetic factors, such as mutation in the transporter SLC30A10. Accumulation of Mn occurs mainly in the basal ganglia and leads to a syndrome called manganism, whose symptoms of cognitive dysfunction and motor impairment resemble Parkinson's disease (PD). Various neurotransmitter systems may be impaired due to Mn, especially dopaminergic, but also cholinergic and GABAergic. Several proteins have been identified to transport Mn, including divalent metal tranporter-1 (DMT-1), SLC30A10, transferrin and ferroportin and allow its accumulation in the central nervous system. Parallel to identification of Mn neurotoxic properties, neuroprotective strategies have been reported, and these include endogenous antioxidants (for instance, vitamin E), plant extracts (complex mixtures containing polyphenols and non-characterized components), iron chelating agents, precursors of glutathione (GSH), and synthetic compounds that can experimentally afford protection against Mn-induced neurotoxicity.

Novel high-throughput assay to assess cellular manganese levels in a striatal cell line model of Huntington's disease confirms a deficit in manganese accumulation.

In spite of the essentiality of manganese (Mn) as a trace element necessary for a variety of physiological processes, Mn in excess accumulates in the brain and has been associated with dysfunction and degeneration of the basal ganglia. Despite the high sensitivity, limited chemical interference, and multi-elemental advantages of traditional methods for measuring Mn levels, they lack the feasibility to assess Mn transport dynamics in a high-throughput manner. Our lab has previously reported decreased net Mn accumulation in a mutant striatal cell line model of Huntington's disease (STHdh(Q111/Q111)) relative to wild-type following Mn exposure. To evaluate Mn transport dynamics in these striatal cell lines, we have developed a high-throughput fluorescence-quenching extraction assay (Cellular Fura-2 Manganese Extraction Assay - CFMEA). CFMEA utilizes changes in fura-2 fluorescence upon excitation at 360 nm (Ca(2+) isosbestic point) and emission at 535 nm, as an indirect measurement of total cellular Mn content. Here, we report the establishment, development, and application of CFMEA. Specifically, we evaluate critical extraction and assay conditions (e.g. extraction buffer, temperature, and fura-2 concentration) required for efficient extraction and quantitative detection of cellular Mn from cultured cells. Mn concentrations can be derived from quenching of fura-2 fluorescence with standard curves based on saturation one-site specific binding kinetics. Importantly, we show that extracted calcium and magnesium concentrations below 10 µM have negligible influence on measurements of Mn by fura-2. CFMEA is able to accurately measure extracted Mn levels from cultured striatal cells over a range of at least 0.1-10 µM. We have used two independent Mn supplementation approaches to validate the quantitative accuracy of CFMEA over a 0-200 µM cellular Mn-exposure range. Finally, we have utilized CFMEA to experimentally confirm a deficit in net Mn accumulation in the mutant HD striatal cell line versus wild-type cells. To conclude, we have developed and applied a novel assay to assess Mn transport dynamics in cultured striatal cell lines. CFMEA provides a rapid means of evaluating Mn transport kinetics in cellular toxicity and disease models.

Disease-toxicant screen reveals a neuroprotective interaction between Huntington's disease and manganese exposure.

Recognizing the similarities between Huntington's disease (HD) pathophysiology and the neurotoxicology of various metals, we hypothesized that they may exhibit disease-toxicant interactions revealing cellular pathways underlying neurodegeneration. Here, we utilize metals and the STHdh mouse striatal cell line model of HD to perform a gene-environment interaction screen. We report that striatal cells expressing mutant Huntingtin exhibit elevated sensitivity to cadmium toxicity and resistance to manganese toxicity. This neuroprotective gene-environment interaction with manganese is highly specific, as it does not occur with iron, copper, zinc, cobalt, cadmium, lead, or nickel ions. Analysis of the Akt cell stress signaling pathway showed diminished activation with manganese exposure and elevated activation after cadmium exposure in the mutant cells. Direct examination of intracellular manganese levels found that mutant cells have a significant impairment in manganese accumulation. Furthermore, YAC128Q mice, a HD model, showed decreased total striatal manganese levels following manganese exposure relative to wild-type mice. Thus, this disease-toxicant interaction screen has revealed that expression of mutant Huntingtin results in heightened sensitivity to cadmium neurotoxicity and a selective impairment of manganese accumulation.