Zoe M. Smith

N -Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease

Huntington’s disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington’s disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.

Kinetics and selectivity of permanganate chemiluminescence : a study of hydroxyl and amino disubstituted benzene positional isomers

Examination of the chemiluminescence reactions of dihydroxybenzenes, aminophenols and phenylenediamines with acidic potassium permanganate has provided a new understanding of the relationships between analyte structure, reaction conditions, kinetics of the light-producing pathway and emission intensity, with broad implications for this widely utilised chemiluminescence detection system. Using a permanganate reagent prepared in a polyphosphate solution and adjusted to pH 2.5, large differences in the rate of reaction with different positional isomers were observed, with the meta-substituted forms reacting far slower and therefore exhibiting much lower chemiluminescence intensities in flow analysis systems. The preliminary partial reduction of permanganate to form significant concentrations of Mn(III) increased the rate of reaction with all analytes tested, resulting in comparable or (in the case of aminophenol and phenylenediamine) even greater emission intensities for the meta-isomers, demonstrating the opportunity to tune the selectivity of the reagent towards certain classes of compound or even specific positional isomers of the same compound. Using more acidic permanganate reagents, in which polyphosphates are not required, the discrepancy between the chemiluminescence intensities was still observed, but was less prominent due to the generally faster rates of reaction. The enhancement of these chemiluminescence reactions by on-line addition of formic acid or formaldehyde can in part also be attributed to the generation of significant pools of the key Mn(III) precursor to the emitting species.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Autocatalytic Chemiluminescence Sheds New Light on the Classic Permanganate–Oxalate Reaction

The emission of light from the permanganate-oxalate reaction enables monitoring of intermediates not accessible through traditional spectrophotometric interrogation. Despite the inherent complexity of the underlying chemical reactions and equilibria, the emission intensity-time profile was characterized by a simple model combining previously independent minimalistic descriptions of chemiluminescence and autocatalysis. The generation of the electronically excited [Mn(II)]* emitter and the acceleration of the reaction even in the presence of high initial concentrations of Mn(II) (under conditions that preclude accumulation of colloidal Mn(IV)) provide new evidence for the reduction of manganese species by a reactive radical intermediate as a supplementary positive feedback loop to the formation of Mn(II).

Ethanol as an alternative to formaldehyde for the enhancement of manganese(IV) chemiluminescence detection

Previous applications of manganese(IV) as a chemiluminescence reagent have required the use of formaldehyde to enhance the emission intensity to analytically useful levels. However, this known human carcinogen (by inhalation) is not ideal for routine application. A wide range of alternative enhancers have been examined but to date none have been found to provide the dramatic increase in chemiluminescence intensities obtained using formaldehyde. Herein, we demonstrate that ethanol offers a simple, safe and inexpensive alternative to the use of formaldehyde for manganese(IV) chemiluminescence detection, without compromising signal intensity or sensitivity. For example, chemiluminescence signals for opiate alkaloids using 50-100% ethanol were 0.8-1.6-fold those using 2M formaldehyde. This innocuous alternative enhancer is shown to be a particularly effective for the direct detection of thiols and disulfides by manganese(IV) chemiluminescence, which we have applied to a simple HPLC procedure to determine a series of biomarkers of oxidative stress.

Analytically useful blue chemiluminescence from a water-soluble iridium(III) complex containing a tetraethylene glycol functionalised triazolylpyridine ligand.

We examine [Ir(df-ppy)2(pt-TEG)](+) as the first highly water soluble, blue-luminescent iridium(III) complex for chemiluminescence detection. Marked differences in selectivity were observed between the new complex and the conventional [Ru(bpy)3](2+) reagent, which will enable this mode of detection to be extended to new areas of application.

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimers, Parkinsons, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.

Deficiency of selenoprotein S, an endoplasmic reticulum resident oxidoreductase, impairs the contractile function of fast twitch hindlimb muscles

Selenoprotein S (Seps1) is an endoplasmic reticulum (ER) resident antioxidant implicated in ER stress and inflammation. In human vastus lateralis and mouse hindlimb muscles, Seps1 localization and expression were fiber-type specific. In male Seps1+/- heterozygous mice, spontaneous physical activity was reduced compared with wild-type littermates ( d = 1.10, P = 0.029). A similar trend was also observed in Seps1-/- knockout mice ( d = 1.12, P = 0.051). Whole body metabolism, body composition, extensor digitorum longus (EDL), and soleus mass and myofiber diameter were unaffected by genotype. However, in isolated fast EDL muscles from Seps1-/- knockout mice, the force frequency curve (FFC; 1-120 Hz) was shifted downward versus EDL muscles from wild-type littermates ( d = 0.55, P = 0.002), suggestive of reduced strength. During 4 min of intermittent, submaximal (60 Hz) stimulation, the genetic deletion or reduction of Seps1 decreased EDL force production ( d = 0.52, P < 0.001). Furthermore, at the start of the intermittent stimulation protocol, when compared with the 60-Hz stimulation of the FFC, EDL muscles from Seps1-/- knockout or Seps1+/- heterozygous mice produced 10% less force than those from wild-type littermates ( d = 0.31, P < 0.001 and d = 0.39, P = 0.015). This functional impairment was associated with reduced mRNA transcript abundance of thioredoxin-1 ( Trx1), thioredoxin interacting protein ( Txnip), and the ER stress markers Chop and Grp94, whereas, in slow soleus muscles, Seps1 deletion did not compromise contractile function and Trx1 ( d = 1.38, P = 0.012) and Txnip ( d = 1.27, P = 0.025) gene expression was increased. Seps1 is a novel regulator of contractile function and cellular stress responses in fast-twitch muscles.