Bioavailable Trace Metals in Neurological Diseases

Medical treatment in Wilson’s disease includes chelators (d-penicillamine and trientine) or zinc salts that have to be maintain all the lifelong. This pharmacological treatment is categorised into two phases; the first being a de-coppering phase and the second a maintenance one. The best therapeutic approach remains controversial, as only a few non-controlled trials have compared these treatments. During the initial phase, progressive increase of chelators’ doses adjusted to exchangeable copper and urinary copper might help to avoid neurological deterioration. Liver transplantation is indicated in acute fulminant liver failure and decompensated cirrhosis; in cases of neurologic deterioration, it must be individually discussed. During the maintenance phase, the most important challenge is to obtain a good adherence to lifelong medical therapy. Neurodegenerative diseases that lead to a mislocalisation of iron can be caused by a culmination of localised overload (pro-oxidant siderosis) and localised deficiency (metabolic distress). A new therapeutic concept with conservative iron chelation rescues iron-overloaded neurons by scavenging labile iron and, by delivering this chelated metal to endogenous apo-transferrin, allows iron redistribution to avoid systemic loss of iron

Repositioning: the fast track to new anti-malarial medicines?

Repositioning of existing drugs has been suggested as a fast track for developing new anti-malarial agents. The compound libraries of GlaxoSmithKline (GSK), Pfizer and AstraZeneca (AZ) comprising drugs that have undergone clinical studies in other therapeutic areas, but not achieved approval, and a set of US Food and Drug Administration (FDA)-approved drugs and other bio-actives were tested against Plasmodium falciparum blood stages. Repositioning of existing therapeutics in malaria is an attractive proposal. Compounds active in vitro at muM concentrations were identified. However, therapeutic concentrations may not be effectively achieved in mice or humans because of poor bio-availability and/or safety concerns. Stringent safety requirements for anti-malarial drugs, given their widespread use in children, make this a challenging area in which to reposition therapy.This work was supported by the Medicines Malaria Venture, St Jude Children's Research Hospital, GlaxoSmithKline Plc and Pfizer Inc

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

α-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death

Adenosine A2A receptors in Parkinson’s disease treatment

Latest results on the action of adenosine A2A receptor antagonists indicate their potential therapeutic usefulness in the treatment of Parkinson’s disease. Basal ganglia possess high levels of adenosine A2A receptors, mainly on the external surfaces of neurons located at the indirect tracts between the striatum, globus pallidus, and substantia nigra. Experiments with animal models of Parkinson’s disease indicate that adenosine A2A receptors are strongly involved in the regulation of the central nervous system. Co-localization of adenosine A2A and dopaminergic D2 receptors in striatum creates a milieu for antagonistic interaction between adenosine and dopamine. The experimental data prove that the best improvement of mobility in patients with Parkinson’s disease could be achieved with simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A2A receptors. In animal models of Parkinson’s disease, the use of selective antagonists of adenosine A2A receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve mobility during both monotherapy and co-administration with L-DOPA and dopamine receptor agonists. The use of adenosine A2A receptor antagonists in combination therapy enables the reduction of the L-DOPA doses, as well as a reduction of side effects. In combination therapy, the adenosine A2A receptor antagonists might be used in both moderate and advanced stages of Parkinson’s disease. The long-lasting administration of adenosine A2A receptor antagonists does not decrease the patient response and does not cause side effects typical of L-DOPA therapy. It was demonstrated in various animal models that inhibition of adenosine A2A receptors not only decreases the movement disturbance, but also reveals a neuroprotective activity, which might impede or stop the progression of the disease. Recently, clinical trials were completed on the use of istradefylline (KW-6002), an inhibitor of adenosine A2A receptors, as an anti-Parkinson drug

Survivin Mutant Protects Differentiated Dopaminergic SK-N-SH Cells Against Oxidative Stress

Oxidative stress is due to an imbalance of antioxidant/pro-oxidant homeostasis and is associated with the progression of several neurological diseases, including Parkinson's and Alzheimer's disease and amyotrophic lateral sclerosis. Furthermore, oxidative stress is responsible for the neuronal loss and dysfunction associated with disease pathogenesis. Survivin is a member of the inhibitors of the apoptosis (IAP) family of proteins, but its neuroprotective effects have not been studied. Here, we demonstrate that SurR9-C84A, a survivin mutant, has neuroprotective effects against H2O2-induced neurotoxicity. Our results show that H2O2 toxicity is associated with an increase in cell death, mitochondrial membrane depolarisation, and the expression of cyclin D1 and caspases 9 and 3. In addition, pre-treatment with SurR9-C84A reduces cell death by decreasing both the level of mitochondrial depolarisation and the expression of cyclin D1 and caspases 9 and 3. We further show that SurR9-C84A increases the antioxidant activity of GSH-peroxidase and catalase, and effectively counteracts oxidant activity following exposure to H2O2. These results suggest for the first time that SurR9-C84A is a promising treatment to protect neuronal cells against H2O2-induced neurotoxicity

Modulatory Communication Signal Performance Is Associated with a Distinct Neurogenomic State in Honey Bees

Studies of animal communication systems have revealed that the perception of a salient signal can cause large-scale changes in brain gene expression, but little is known about how communication affects the neurogenomic state of the sender. We explored this issue by studying honey bees that produce a vibratory modulatory signal. We chose this system because it represents an extreme case of animal communication; some bees perform this behavior intensively, effectively acting as communication specialists. We show large differences in patterns of brain gene expression between individuals producing vibratory signal as compared with carefully matched non-senders. Some of the differentially regulated genes have previously been implicated in the performance of other motor activities, including courtship behavior in Drosophila melanogaster and Parkinson's Disease in humans. Our results demonstrate for the first time a neurogenomic brain state associated with sending a communication signal and provide suggestive glimpses of molecular roots for motor control

Oxidation of DJ-1 Induced by 6-Hydroxydopamine Decreasing Intracellular Glutathione

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported to undergo preferential oxidation of the cysteine residue at position 106 (Cys-106) under oxidative stress; however, details of the molecular mechanisms are not well known. In the present study, mechanisms of DJ-1 oxidation induced by 6-hydroxydopamine (6-OHDA) were investigated by using SH-SY5Y cells. The treatment of these cells with 6-OHDA caused an obvious acidic spot sift of DJ-1 due to its oxidation. However, when catalase, which is an hydrogen peroxide (H2O2)-removing enzyme, was added during the treatment, it failed to prevent the oxidation induced by 6-OHDA, suggesting that electrophilic p-quinone formed from 6-OHDA, but not H2O2, was responsible for the DJ-1 oxidation. Benzoquinone, another electrophilic p-quinone, also induced DJ-1 oxidation. The intracellular glutathione (GSH) levels were significantly decreased by 6-OHDA, irrespective of the presence or absence of catalase. The inhibition of GSH synthesis by buthionine sulfoximine resulted in a decrease in GSH levels and enhancement of DJ-1 oxidation. The pretreatment of cells with N-acetyl-cysteine prevented the loss of intracellular GSH and subsequently DJ-1 oxidation induced by 6-OHDA. Collectively, these results suggest that electrophilic p-quinone formed from 6-OHDA induces DJ-1 oxidation by decreasing intracellular GSH

Human SOD2 Modification by Dopamine Quinones Affects Enzymatic Activity by Promoting Its Aggregation: Possible Implications for Parkinson’s Disease

Mitochondrial dysfunction and oxidative stress are considered central in dopaminergic neurodegeneration in Parkinson’s disease (PD). Oxidative stress occurs when the endogenous antioxidant systems are overcome by the generation of reactive oxygen species (ROS). A plausible source of oxidative stress, which could account for the selective degeneration of dopaminergic neurons, is the redox chemistry of dopamine (DA) and leads to the formation of ROS and reactive dopamine-quinones (DAQs). Superoxide dismutase 2 (SOD2) is a mitochondrial enzyme that converts superoxide radicals to molecular oxygen and hydrogen peroxide, providing a first line of defense against ROS. We investigated the possible interplay between DA and SOD2 in the pathogenesis of PD using enzymatic essays, site-specific mutagenesis, and optical and high-field-cw-EPR spectroscopies. Using radioactive DA, we demonstrated that SOD2 is a target of DAQs. Exposure to micromolar DAQ concentrations induces a loss of up to 50% of SOD2 enzymatic activity in a dose-dependent manner, which is correlated to the concomitant formation of protein aggregates, while the coordination geometry of the active site appears unaffected by DAQ modifications. Our findings support a model in which DAQ-mediated SOD2 inactivation increases mitochondrial ROS production, suggesting a link between oxidative stress and mitochondrial dysfunction

Homeostatic mechanisms in dopamine synthesis and release: a mathematical model

<p>Abstract</p> <p>Background</p> <p>Dopamine is a catecholamine that is used as a neurotransmitter both in the periphery and in the central nervous system. Dysfunction in various dopaminergic systems is known to be associated with various disorders, including schizophrenia, Parkinson's disease, and Tourette's syndrome. Furthermore, microdialysis studies have shown that addictive drugs increase extracellular dopamine and brain imaging has shown a correlation between euphoria and psycho-stimulant-induced increases in extracellular dopamine <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. These consequences of dopamine dysfunction indicate the importance of maintaining dopamine functionality through homeostatic mechanisms that have been attributed to the delicate balance between synthesis, storage, release, metabolism, and reuptake.</p> <p>Methods</p> <p>We construct a mathematical model of dopamine synthesis, release, and reuptake and use it to study homeostasis in single dopaminergic neuron terminals. We investigate the substrate inhibition of tyrosine hydroxylase by tyrosine, the consequences of the rapid uptake of extracellular dopamine by the dopamine transporters, and the effects of the autoreceoptors on dopaminergic function. The main focus is to understand the regulation and control of synthesis and release and to explicate and interpret experimental findings.</p> <p>Results</p> <p>We show that the substrate inhibition of tyrosine hydroxylase by tyrosine stabilizes cytosolic and vesicular dopamine against changes in tyrosine availability due to meals. We find that the autoreceptors dampen the fluctuations in extracellular dopamine caused by changes in tyrosine hydroxylase expression and changes in the rate of firing. We show that short bursts of action potentials create significant dopamine signals against the background of tonic firing. We explain the observed time courses of extracellular dopamine responses to stimulation in wild type mice and mice that have genetically altered dopamine transporter densities and the observed half-lives of extracellular dopamine under various treatment protocols.</p> <p>Conclusion</p> <p>Dopaminergic systems must respond robustly to important biological signals such as bursts, while at the same time maintaining homeostasis in the face of normal biological fluctuations in inputs, expression levels, and firing rates. This is accomplished through the cooperative effect of many different homeostatic mechanisms including special properties of tyrosine hydroxylase, the dopamine transporters, and the dopamine autoreceptors.</p

Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders

The recent identification of multiple new genetic causes of neurological disorders highlights the need for model systems that give experimental access to the underlying biology. In particular, the ability to couple disease-causing mutations with human neuronal differentiation systems would be beneficial. Gene targeting is a well-known approach for dissecting gene function, but low rates of homologous recombination in somatic cells (including neuronal cells) have traditionally impeded the development of robust cellular models of neurological disorders. Recently, however, CRISPR/Cas9 gene editing technologies have expanded the number of systems within which gene targeting is possible. Here we adopt as a model system LUHMES cells, a commercially available diploid human female mesencephalic cell line that differentiates into homogeneous mature neurons in 1-2 weeks. We describe optimised methods for transfection and selection of neuronal progenitor cells carrying targeted genomic alterations using CRISPR/Cas9 technology. By targeting the endogenous X-linked MECP2 locus, we introduced four independent missense mutations that cause the autism spectrum disorder Rett syndrome and observed the desired genetic structure in 3-26% of selected clones, including gene targeting of the inactive X chromosome. Similar efficiencies were achieved by introducing neurodevelopmental disorder-causing mutations at the autosomal EEF1A2 locus on chromosome 20. Our results indicate that efficiency of genetic “knock-in” is determined by the location of the mutation within the donor DNA molecule. Furthermore, we successfully introduced an mCherry tag at the MECP2 locus to yield a fusion protein, demonstrating that larger insertions are also straightforward in this system. We suggest that our optimised methods for altering the genome of LUHMES cells make them an attractive model for the study of neurogenetic disorders