Gunnar F. Kwakye

Role of manganese in neurodegenerative diseases.

Manganese (Mn) is an essential ubiquitous trace element that is required for normal growth, development and cellular homeostasis. Exposure to high Mn levels causes a clinical disease characterized by extrapyramidal symptom resembling idiopathic Parkinsons disease (IPD). The present review focuses on the role of various transporters in maintaining brain Mn homeostasis along with recent methodological advances in real-time measurements of intracellular Mn levels. We also provide an overview on the role for Mn in IPD, discussing the similarities (and differences) between manganism and IPD, and the relationship between α-synuclein and Mn-related protein aggregation, as well as mitochondrial dysfunction, Mn and PD. Additional sections of the review discuss the link between Mn and Huntingtons disease (HD), with emphasis on huntingtin function and the potential role for altered Mn homeostasis and toxicity in HD. We conclude with a brief survey on the potential role of Mn in the etiologies of Alzheimers disease (AD), amyotrophic lateral sclerosis (ALS) and prion disease. Where possible, we discuss the mechanistic commonalities inherent to Mn-induced neurotoxicity and neurodegenerative disorders.

Manganese-induced parkinsonism and Parkinson’s disease: Shared and distinguishable features

Manganese (Mn) is an essential trace element necessary for physiological processes that support development, growth and neuronal function. Secondary to elevated exposure or decreased excretion, Mn accumulates in the basal ganglia region of the brain and may cause a parkinsonian-like syndrome, referred to as manganism. The present review discusses the advances made in understanding the essentiality and neurotoxicity of Mn. We review occupational Mn-induced parkinsonism and the dynamic modes of Mn transport in biological systems, as well as the detection and pharmacokinetic modeling of Mn trafficking. In addition, we review some of the shared similarities, pathologic and clinical distinctions between Mn-induced parkinsonism and Parkinson’s disease. Where possible, we review the influence of Mn toxicity on dopamine, gamma aminobutyric acid (GABA), and glutamate neurotransmitter levels and function. We conclude with a survey of the preventive and treatment strategies for manganism and idiopathic Parkinson’s disease (PD).

Disease‐toxicant screen reveals a neuroprotective interaction between Huntington’s disease and manganese exposure

J. Neurochem. (2010) 112, 227–237.

Altered Manganese Homeostasis and Manganese Toxicity in a Huntington's Disease Striatal Cell Model Are Not Explained by Defects in the Iron Transport System

Expansion of a polyglutamine tract in Huntingtin (Htt) leads to the degeneration of medium spiny neurons in Huntingtons disease (HD). Furthermore, the HTT gene has been functionally linked to iron (Fe) metabolism, and HD patients show alterations in brain and peripheral Fe homeostasis. Recently, we discovered that expression of mutant HTT is associated with impaired manganese (Mn) uptake following overexposure in a striatal neuronal cell line and mouse model of HD. Here we test the hypothesis that the transferrin receptor (TfR)-mediated Fe uptake pathway is responsible for the HD-associated defects in Mn uptake. Western blot analysis showed that TfR levels are reduced in the mutant STHdh(Q111/Q111) striatal cell line, whereas levels of the Fe and Mn transporter, divalent metal transporter 1 (DMT1), are unchanged. To stress the Fe transport system, we exposed mutant and wild-type cells to elevated Fe(III), which revealed a subtle impairment in net Fe uptake only at the highest Fe exposures. In contrast, the HD mutant line exhibited substantial deficits in net Mn uptake, even under basal conditions. Finally, to functionally evaluate a role for Fe transporters in the Mn uptake deficit, we examined Mn toxicity in the presence of saturating Fe(III) levels. Although Fe(III) exposure decreased Mn neurotoxicity, it did so equally for wild-type and mutant cells. Therefore, although Fe transporters contribute to Mn uptake and toxicity in the striatal cell lines, functional alterations in this pathway are insufficient to explain the strong Mn resistance phenotype of this HD cell model.

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 Huntingtons 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.

Cellular fura‐2 Manganese Extraction Assay (CFMEA)

Cellular manganese (Mn) uptake and transport dynamics can be measured using a cellular fura‐2 manganese extraction assay (CFMEA). The assay described here uses immortalized murine striatal cell line and primary cortical astrocytes, but the method is equally adaptable to other cultured mammalian cells. An ultrasensitive fluorescent nucleic acid stain for quantification of double‐stranded DNA (dsDNA) in solution, Quant‐iT PicoGreen, has been utilized for normalization of Mn concentration in the cultured cells, following Mn (II) chloride (MnCl2) exposure. Depending on the cell type and density, other methods, e.g., protein determination assays or cell counts, may also be used for normalization. Methods are described for rapidly stopping Mn uptake and transport processes at specified times, extraction, and quantification of cellular Mn content, and normalization of Mn levels to dsDNA concentration. Curr. Protoc. Toxicol. 48:12.18.1‐12.18.20.

Disease-Toxicant Interactions in Parkinson’s Disease Neuropathology

Human disease commonly manifests as a result of complex genetic and environmental interactions. In the case of neurodegenerative diseases, such as Parkinson’s disease (PD), understanding how environmental exposures collude with genetic polymorphisms in the central nervous system to cause dysfunction is critical in order to develop better treatment strategies, therapies, and a more cohesive paradigm for future research. The intersection of genetics and the environment in disease etiology is particularly relevant in the context of their shared pathophysiological mechanisms. This review offers an integrated view of disease-toxicant interactions in PD. Particular attention is dedicated to how mutations in the genes SNCA, parkin, leucine-rich repeat kinase 2 (LRRK2) and DJ-1, as well as dysfunction of the ubiquitin proteasome system, may contribute to PD and how exposure to heavy metals, pesticides and illicit drugs may further the consequences of these mutations to exacerbate PD and PD-like disorders. Although the toxic effects induced by exposure to these environmental factors may not be the primary causes of PD, their mechanisms of action are critical for our current understanding of the neuropathologies driving PD. Elucidating how environment and genetics collude to cause pathogenesis of PD will facilitate the development of more effective treatments for the disease. Additionally, we discuss the neuroprotection exerted by estrogen and other compounds that may prevent PD and provide an overview of current treatment strategies and therapies.

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

Huntingtons 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.

Atropa belladonna neurotoxicity: Implications to neurological disorders

Atropa belladonna, commonly known as belladonna or deadly nightshade, ranks among one of the most poisonous plants in Europe and other parts of the world. The plant contains tropane alkaloids including atropine, scopolamine, and hyoscyamine, which are used as anticholinergics in Food and Drug Administration (FDA) approved drugs and homeopathic remedies. These alkaloids can be very toxic at high dose. The FDA has recently reported that Hylands baby teething tablets contain inconsistent amounts of Atropa belladonna that may have adverse effects on the nervous system and cause death in children, thus recalled the product in 2017. A greater understanding of the neurotoxicity of Atropa belladonna and its modification of genetic polymorphisms in the nervous system is critical in order to develop better treatment strategies, therapies, regulations, education of at-risk populations, and a more cohesive paradigm for future research. This review offers an integrated view of the homeopathy and neurotoxicity of Atropa belladonna in children, adults, and animal models as well as its implications to neurological disorders. Particular attention is dedicated to the pharmaco/toxicodynamics, pharmaco/toxicokinetics, pathophysiology, epidemiological cases, and animal studies associated with the effects of Atropa belladonna on the nervous system. Additionally, we discuss the influence of active tropane alkaloids in Atropa belladonna and other similar plants on FDA-approved therapeutic drugs for treatment of neurological disorders.

Acute exposure to chlorpyrifos caused NADPH oxidase mediated oxidative stress and neurotoxicity in a striatal cell model of Huntington’s disease

HIGHLIGHTSA disease‐toxicant interaction between mutant Huntingtin and chlorpyrifos (CPF).Mutant huntingtin enhances CPF‐induced neurotoxicity in a striatal cell model.NADPH‐oxidase (NOX) mediated ROS production is involved in the neurotoxicity.Exogenous antioxidants and NOX inhibitors ameliorate CPF‐induced neurotoxicity. ABSTRACT We hypothesized that expression of mutant Huntingtin (HTT) would modulate the neurotoxicity of the commonly used organophosphate insecticide, chlorpyrifos (CPF), revealing cellular mechanisms underlying neurodegeneration. Using a mouse striatal cell model of HD, we report that mutant HD cells are more susceptible to CPF‐induced cytotoxicity as compared to wild‐type. This CPF‐induced cytotoxicity caused increased production of reactive oxygen species, reduced glutathione levels, decreased superoxide dismutase activity, and increased malondialdehyde levels in mutant HD cells relative to wild‐type. Furthermore, we show that co‐treatment with antioxidant agents attenuated the CPF‐induced ROS levels and cytotoxicity. Co‐treatment with a NADPH oxidase (NOX) inhibitor, apocynin, also attenuated the CPF‐induced ROS production and neurotoxicity. CPF caused increased NOX activity in mutant HD lines that was ameliorated following co‐treatment with apocynin. Finally, CPF‐induced neurotoxicity significantly increased the protein expression of nuclear factor erythroid 2‐related factor (Nrf2) in mutant HD cells as compared to wild‐type. This study is the first report of CPF‐induced toxicity in HD pathophysiology and suggests that mutant HTT and CPF exhibit a disease‐toxicant interaction wherein expression of mutant HTT enhances CPF‐induced neurotoxicity via a NOX‐mediated oxidative stress mechanism to cause neuronal loss in the full length HTT expressing striatal cells.