Richard B. Parsons

Galantamine inhibits β-amyloid aggregation and cytotoxicity

The ability of galantamine (Reminyl) to inhibit the aggregation and toxicity of the beta-amyloid peptide (Abeta) was investigated. Galantamine showed concentration-dependent inhibition of aggregation of both Abeta 1-40 and Abeta 1-42, as determined by an ELISA method. Electron microscope studies of Abeta 1-40 incubated in the presence of galantamine revealed fibrils that were disordered and clumped in appearance. MTT and lactate dehydrogenase assays, employing SH-SY5Y human neuroblastoma cells, showed that galantamine reduced the cytotoxicity induced by Abeta 1-40. Galantamine also dramatically reduced Abeta 1-40-induced cellular apoptosis in these cells. There is some evidence that galantamine may not be acting purely as a symptomatic treatment. Disease-modifying effects of the drug could be due to an additional effect on Abeta aggregation and/or toxicity.

The expression of nicotinamide N-methyltransferase increases ATP synthesis and protects SH-SY5Y neuroblastoma cells against the toxicity of Complex I inhibitors.

NNMT (nicotinamide N-methyltransferase, E.C. 2.1.1.1) catalyses the N-methylation of nicotinamide to 1-methylnicotinamide. NNMT expression is significantly elevated in a number of cancers, and we have previously demonstrated that NNMT expression is significantly increased in the brains of patients who have died of Parkinsons disease. To investigate the cellular effects of NNMT overexpression, we overexpressed NNMT in the SH-SY5Y cell line, a tumour-derived human dopaminergic neuroblastoma cell line with no endogenous expression of NNMT. NNMT expression significantly decreased SH-SY5Y cell death, which correlated with increased intracellular ATP content, ATP/ADP ratio and Complex I activity, and a reduction in the degradation of the NDUFS3 [NADH dehydrogenase (ubiquinone) iron-sulfur protein 3] subunit of Complex I. These effects were replicated by incubation of SH-SY5Y cells with 1-methylnicotinamide, suggesting that 1-methylnicotinamide mediates the cellular effects of NNMT. Both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone by reversing their effects upon ATP synthesis, the ATP/ADP ratio, Complex I activity and the NDUFS3 subunit. The results of the present study raise the possibility that the increase in NNMT expression that we observed in vivo may be a stress response of the cell to the underlying pathogenic process. Furthermore, the results of the present study also raise the possibility of using inhibitors of NNMT for the treatment of cancer.

High expression of nicotinamide N-methyltransferase in patients with idiopathic Parkinson's disease

We have previously speculated that elevated levels of nicotinamide N-methyltransferase (NNMT), the primary catabolic enzyme of nicotinamide, may result in reduced Complex I activity in idiopathic Parkinsons disease (IPD) in two ways: (1) reduction in the levels of nicotinamide available for nicotinamide adenine dinucleotide synthesis; and (2) increased methylation of compounds such as tetrahydroisoquinolines and beta-carbolines, which are potent Complex I inhibitors. Expression of NNMT was assessed in 91 cerebella (53 IPD, 38 control) using immunohistochemistry coupled with quantitative digital image analysis. Control cerebella showed a distribution of expression ascribed to low, intermediate and high expressors with ratios of 1:2:1 categories. Expression in the parkinsonian cerebella was significantly higher than in the control group (control group median expression 17%, mean expression 16.6%, range 0-51%, standard deviation 11.4%, standard error 1.9%; IPD group median expression 46%, mean expression 53.7%, range 21-100%, standard deviation 23.4%, standard error 3.2%; P<0.0001; unpaired t-test with Welch correction (parametric) and Mann-Whitney U-test (non-parametric)). These results confirm that NNMT expression is elevated in IPD, which may ultimately lead to neurodegeneration via a reduction in Complex I activity.

The aetiology of idiopathic Parkinson's disease.

Agents potentially involved in the aetiology of idiopathic Parkinsons disease are discussed. These include factors regulating dopaminergic neurogenesis (Nurr 1, Ptx-3, and Lmx1b) and related proteins, together with genes involved in familial Parkinsons disease (α synuclein, parkin, and ubiquitin carboxy terminal hydroxylase L1), and endogenous and environmental agents.

Statins inhibit the dimerization of β-secretase via both isoprenoid- and cholesterol-mediated mechanisms

We have previously reported that protein lipidation in the form of palmitoylation and farnesylation is critical for the production of Abeta (amyloid beta-peptide), the dimerization of beta-secretase and its trafficking into cholesterol-rich microdomains. As statins influence these lipid modifications in addition to their effects on cholesterol biosynthesis, we have investigated the effects of lovastatin and SIMVA (simvastatin) at a range of concentrations chosen to distinguish different cellular effects on Abeta production and beta-secretase structure and its localization in bHEK cells [HEK-293 cells (human embryonic kidney cells) transfected with the Asp-2 gene plus a polyhistidine coding tag] cells. We have compared the changes brought about by statins with those brought about by the palmitoylation inhibitor cerulenin and the farnesyltransferase inhibitor CVFM (Cys-Val-Phe-Met). The statin-mediated reduction in Abeta production correlated with an inhibition of beta-secretase dimerization into its more active form at all concentrations of statin investigated. These effects were reversed by the administration of mevalonate, showing that these effects were mediated via 3-hydroxy-3-methylglutaryl-CoA-dependent pathways. At low (1 microM) statin concentrations, reduction in Abeta production and inhibition of beta-secretase dimerization were mediated by inhibition of isoprenoid synthesis. At high (>10 microM) concentrations of statins, inhibition of beta-secretase palmitoylation occurred, which we demonstrated to be regulated by intracellular cholesterol levels. There was also a concomitant concentration-dependent change in beta-secretase subcellular trafficking. Significantly, Abeta release from cells was markedly higher at 50 microM SIMVA than at 1 microM, whereas these concentrations resulted in similar reductions in total Abeta production, suggesting that low-dose statins may be more beneficial than high doses for the therapeutic treatment of Alzheimers disease.

Alternative oxidase rescues mitochondria-mediated dopaminergic cell loss in Drosophila

Mitochondrial dysfunction is commonly observed in degenerative disorders, including Alzheimers and Parkinsons disease that are characterized by the progressive and selective loss of neuronal subpopulations. It is currently unclear, however, whether mitochondrial dysfunction is primary or secondary to other pathogenic processes that eventually lead to age-related neurodegeneration. Here we establish an in vivo Drosophila model of mitochondrial dysfunction by downregulating the catalytic subunit of mitochondrial DNA (mtDNA) polymerase in cholinergic, serotonergic and dopaminergic neurons. The resulting flies are characterized by lowered respiratory chain activity, premature aging, age-related motor deficits as well as adult onset, progressive and cell-type-specific, dopaminergic neurodegeneration. Using this model, we find that associated lethality can be partially rescued by targeting PINK1/parkin signaling or Drp1, both of which have been implicated in mitochondrial dynamics and Parkinsons disease. Bypassing mitochondrial complex III/IV deficiencies with Alternative oxidase (AOX), however, fully restores ATP levels and prevents dopaminergic neurodegeneration. In contrast, ATP levels and neurodegeneration are not rescued when mitochondrial complex I deficiencies are bypassed with NADH-Q oxidoreductase. Our results demonstrate that mtDNA-mediated mitochondrial dysfunction can cause age-related and cell-type-specific neurodegeneration which AOX is able to alleviate and indicate that AOX or its surrogates may prove useful as a therapeutic tool for limiting respiratory chain deficiencies caused by mtDNA decline in healthy aging and neurodegenerative disease.

Toxicity of sulphur-containing compounds to neuronal cell lines.

Cysteine and its metabolites cysteine sulphinic acid (CSA) and taurine (TA) were shown to be toxic to human and rat neuronal cell lines. However, the mechanisms of action of CSA and TA appeared to be different. As MND/ALS patients have high plasma and CSF cysteine levels, they may have increased levels of neurotoxins in vivo, since cysteine was more toxic than CSA in the assay system used. Cysteic acid, homocysteic acid, BMAA and BOAA were also toxic to the cell lines used.

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Human brain cytochrome P450 1B1 : Immunohistochemical localization in human temporal lobe and induction by dimethylbenz(a)anthracene in astrocytoma cell line (MOG-G-CCM)

CYP1B1, a new member of human cytochrome P450 family 1, is involved in the xenobiotic detoxification metabolism and possibly activation of numerous procarcinogens and promutagens. Localization of CYP1B1 in human temporal lobe and its induction in astrocytoma cell line (MOG-G-CCM) by 7,12-dimethylbenz(a)anthracene (DMBA) was investigated using antibodies against human CYP1B1. A single band of approximately 58 kDa size in both human temporal lobe and in MOG-G-CCM was detected by Western blot analysis. Treatment of MOG-G-CCM cells with DMBA resulted in approximately 2.8-fold induction of CYP1B1. CYP1B1 immunoreactivity was detected at the blood-brain interface areas of the temporal lobe as evidenced by co-localization with CD34 antigen. These results suggest that this enzyme may be important in brain xenobiotic metabolism acting as an enzymatic barrier.

The regulation of β-secretase by cholesterol and statins in Alzheimer's disease

Epidemiologists have found a decreased risk of developing Alzheimers disease (AD) in people taking statins (cholesterol biosynthesis inhibitors). We have reported previously that, in cell culture, lovastatin decreases the output of beta-amyloid, a peptide that is toxic to neurones, and is reputably the prime cause of neurodegeneration seen in AD. This report probes the mechanism of statin protection further by finding out how the protease beta-secretase, that releases beta-amyloid from its precursor protein, behaves under changed cholesterol levels induced by statins. We found that, with high cellular cholesterol levels, there is a decrease in glycosylation of mature oligosaccharides in beta-secretase, whereas in the presence of lovastatin, glycosylation progresses further. Moreover, lovastatin does not inhibit beta-secretase in vitro. Thus, the cholesterol and statin effects are due to changes in cellular targeting induced by changed cholesterol gradients. Some of these changes are mimicked by the action of U18666A, a cholesterol-transport inhibitor that produces a defect in cells seen in patients with Neimann Picks disorder.