Mario E. Götz

Oxidative stress: free radical production in neural degeneration.

It is not yet established whether oxidative stress is a major cause of cell death or simply a consequence of an unknown pathogenetic factor. Concerning chronic diseases, as Parkinsons and Alzheimers disease are assumed to be, it is possible that a gradual impairment of cellular defense mechanisms leads to cell damage because of toxic substances being increasingly formed during normal cellular metabolism. This point of view brings into consideration the possibility that, besides exogenous factors, the pathogenetic process of neurodegeration is triggered by endogenous mechanisms, either by an endogenous toxin or by inherited metabolic disorders, which become progressively more evident with aging. In the following review, we focus on the oxidative stress theory of neurodegeneration, on excitotoxin-induced cell damage and on impairment of mitochondrial function as three major noxae being the most likely causes of cell death either independently or in connection with each other. First, having discussed clinical, pathophysiological, pathological and biochemical features of movement and cognitive disorders, we discuss the common features of these biochemical theories of neurodegeneration separately. Second, we attempt to evaluate possible biochemical links between them and third, we discuss experimental findings that confirm or rule out the involvement of any of these theories in neurodegeneration. Finally, we report some therapeutic strategies evolved from each of these theories.

The Relevance of Iron in the Pathogenesis of Parkinson's Disease

Abstract: Investigations that revealed increased levels of iron in postmortem brains from patients with Parkinsons disease (PD) as compared to those from individuals not suffering from neurological disorders are reported. The chemical natures in which iron predominates in the brain and the relevance of neuromelanin for neuronal iron binding are discussed. Major findings have been that iron levels increase with the severity of neuropathological changes in PD, presumably due to increased transport through the blood‐brain barrier in late stages of parkinsonism. Glial iron is mainly stored as ferric iron in ferritin, while neuronal iron is predominantly bound to neuromelanin. Iron overload may induce progressive degeneration of nigrostriatal neurons by facilitating the formation of reactive biological intermediates, including reactive oxygen species, and the formation of cytotoxic protein aggregates. There are indications that iron‐mediated neuronal death in PD proceeds retrogradely. These results are also discussed with respect to their relevance for disease progression in relation to cytotoxic α‐synuclein protofibril formation.

Oxidative stress: a role in the pathogenesis of Parkinson’s disease

The degeneration of nigro-striatal dopaminergic neurons is considered to be a predominant pathogenetic factor of Parkinsons disease (PD). However, the etiology of this degeneration is not known. Hypotheses assume accumulation of endogenous and/or exogenous toxins as trigger of the disease. An increase in the concentration of free radicals has been suggested to be toxic to cells, especially when combined with certain metals like free iron or copper. The role of melanin in the degenerative process is not clear, but autoxidative reactions such as the oxidation of dopamine (DA) to melanin generating radicals and toxic metabolites seem to enhance the vulnerability of neurons in the substantia nigra (SN). Disappearance of melanin in the SN, increase of total iron and ferric iron, extreme decrease of glutathione (GSH) levels, reduced activity of enzymes involved in the detoxification of hydrogen peroxide, hydroxyl and superoxide radicals (peroxidases, catalase, glutathione peroxidase), an increase of monoamine oxidase B (MAO B) activity and the substantial increase of malondialdehyde, a marker of lipid peroxidation, in the SN seem to indicate a role of an oxidative stress syndrome in the SN causing or aggravating PD.

Monoamine oxidase-inhibition and MPTP-induced neurotoxicity in the non-human primate: comparison of rasagiline (TVP 1012) with selegiline.

Summary. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce parkinsonism in man and non-human primates. Monoamine-oxidase B (MAO-B) has been reported to be implicated in both MPTP-induced parkinsonism and Parkinsons disease, since selegiline (L-deprenyl), an irreversible MAO-B inhibitor, prevents MPTP-induced neurotoxicity in numerous species including mice, goldfish and drosophyla. However, one disadvantage of this substance relates to its metabolism to (−)-methamphetamine and (−)-amphetamine. Rasagiline (R-(+)-N-propyl-1-aminoindane) is a novel irrevesible MAO-B-inhibitor, which is not metabolized to metamphetamine and/or amphetamine. The present study compared the effects of high doses of selegiline and rasagiline (10 mg/kg body weight s.c.) on MPTP-induced dopaminergic neurotoxicity in a non-human primate (Callithrix jacchus) model of PD. Groups of four monkeys were assigned to the following six experimental groups: Group I: Saline, Group II: Selegiline/Saline, Group III: Rasagiline/Saline, Group IV: MPTP/Saline, Group V: Rasagiline/MPTP, Group VI: Selegiline/MPTP. Daily treatment with MAO-B-inhibitors (either rasagiline or selegiline, 10 mg/kg body weight s.c.) was initiated four days prior to MPTP-exposure (MPTP-HCl, 2 mg/kg body weight subcutaneously, separated by an interval of 24 hours for a total of four days) and was continued until the end of the experiment, i.e. 7 days after the cessation of the MPTP-injections, when animals were sacrificed. MPTP-treatment caused distinct behavioural, histological, and biochemical alterations: 1. significant reduction of motor activity assessed by clinical rating and by computerized locomotor activity measurements; 2. substantial loss (approx. 40%) of dopaminergic (tyrosine-hydroxylase-positive) cells in the substantia nigra, pars compacta; and 3. putaminal dopamine depletion of 98% and its metabolites DOPAC (88%) and HVA (96%). Treatment with either rasagiline or selegiline markedly attenuated the neurotoxic effects of MPTP at the behavioural, histological, and at the biochemical levels. There were no significant differences between rasagiline/MPTP and selegiline/MPTP-treated animals in respect to signs of motor impairment, the number of dopaminergic cells in the substantia nigra, and striatal dopamine levels. As expected, both inhibitors decreased the metabolism of dopamine, leading to reduced levels of HVA and DOPAC (by >95% and 45% respectively). In conclusion, rasagiline and selegiline at the dosages employed equally protect against MPTP-toxicity in the common marmoset, suggesting that selegiline-derived metabolites are not important for the neuroprotective effects of high dose selegiline in the non-human MPTP-primate model in the experimental design employed. However, unexpectedly, high dose treatment with both MAO-inhibitors caused a decrease of the cell sizes of nigral tyrosine hydroxylase positive neurons. It remains to be determined, if this histological observation represents potential adverse effects of high dose treatment with monoamine oxidase inhibitors.

Altered redox state of platelet coenzyme Q10 in Parkinson's disease

Summary.Background. The reduced form of coenzyme Q10 (CoQ10) acts as a lipophilic antioxidant and participates in electron and proton transport of the respiratory chain in the inner mitochondrial membrane. An alteration in CoQ10 redox state may thus reflect a change in membrane electron transport and the effectiveness of defense against toxic reactive oxygen species such as hydrogen peroxide and superoxide. In Parkinsons disease alterations in the activities of complex I have been reported in substantia nigra and platelets. Deficiency of mitochondrial enzyme activities could affect electron transport which might be reflected by the platelet CoQ10 redox state. Method. We have determined concentrations of the reduced and oxidized forms of CoQ10 and the activity of monoamine oxidase B in platelets isolated from parkinsonian patients and age- and gender-matched controls. Results. Platelet CoQ10 redox ratios (reduced CoQ10 to oxidized CoQ10) and the ratio of the reduced form, compared with total platelet CoQ10, were significantly decreased in de novo parkinsonian patients. Platelet CoQ10 redox ratios were further decreased by L-DOPA treatment (not significant), whilst selegiline treatment partially restored CoQ10 redox ratios. Monoamine oxidase activities in non-selegiline treated patients were similar to controls. Interpretation. Our results either suggest an impairment of electron transport or a higher need for reduced forms of CoQ10 in the platelets of even de novo parkinsonian patients. However, the CoQ10 redox ratio was not correlated to disease severity, as determined by the Hoehn and Yahr PD disability classification, suggesting that this parameter may not be useful as a peripheral trait marker for the severity of PD but as an early state marker of PD.

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease.

In the last decade an important role for the progression of neuronal cell death in Alzheimers disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS). Levels of HNEGSH conjugates calculated as the sum of three chromatographically separated diastereomers were determined in hippocampus, entorhinal cortex, substantia innominata, frontal and temporal cortex, as well as cerebellum from patients with AD and controls matched for age, gender, postmortem delay and storage time. Neither age, nor postmortem delay, nor storage time did correlate with levels of HNEGSH conjugates which ranged between 1 and 500 pmol/g fresh weight in the brain areas examined. The brain specimen from patients with clinically and neuropathologically probable AD diagnosed according to criteria of the consortium to establish a registry for AD (CERAD) show increased levels of HNEGSH in the temporal and frontal cortex, as well as in the substantia innominata. Classification of disease severity according to Braak and Braak, which takes into consideration the amount of neurofibrillary tangles and neuritic plaques, revealed highest levels of HNEGSH in the substantia innominata and the hippocampus, two brain regions known to be preferentially affected in AD. These results substantiate the link between conjugates of glutathione with a product of lipid peroxidation and Alzheimers disease and justify further studies to evaluate the role of HNE metabolites as potential biomarkers for disease progression in AD.

Regulation of glutathione and cell toxicity following exposure to neurotropic substances and human immunodeficiency virus-1 in vitro.

The aim of the present study was to assess the toxic potential of drugs of abuse and other neuropharmacological agents in the pathogenesis of AIDS dementia complex (ADC), the neurological complication of AIDS. Neuroblastoma and glioblastoma cell lines expressing the dopamine transporter, as well as primary macrophages exposed to human immunodeficiency virus-1 (HIV-1), were used to investigate the possibility of any synergistic effect between the mode of toxicity of such substances and virus exposure. The drugs of abuse used in our experiments were cocaine and morphine, which exert their action, among others, on the dopaminergic system. Effects were compared to treatment with dopamine itself and a typical dopaminergic drug used pharmaceutically, selegiline. In macrophage cultures, glutathione (GSH) was upregulated strongly after treatment with dopamine, morphine or selegiline, and this effect was enhanced when cells were pre-exposed to virus. This upregulation is discussed as a compensatory reaction to an oxidative signal. When hydrogen peroxide plus iron sulfate was used as a strong oxidant in macrophages, GSH concentrations decreased as a result of cell injury. Cell numbers remained constant in all treatment groups. In contrast, in both neuroblastoma and glioblastoma cell lines, the modulation of GSH concentrations by neurotropic substances was accompanied by significant cell loss, which was exacerbated by HIV-1 pretreatment. Selegiline did not change cell numbers when incubated alone. However, when incubated following treatment with HIV-1 cell death was highly significant. Ascorbic acid (AA), included as antioxidant, totally restored cell loss in cultures treated with dopamine. However, no effect was observed in combined treatment of AA and morphine or selegiline. The results demonstrate a synergistic role in cellular toxicity due to neurotropic substances and HIV-1, and suggest that neuropharmacological agents may contribute to the pathogenesis of ADC.

Hippocampal level of neural specific adenylyl cyclase type I is decreased in Alzheimer's disease.

Previous studies reported disruption of adenylyl cyclase (AC)-cyclic AMP (cAMP) signal transduction in brain of Alzheimers disease (AD). We also demonstrated that basal and stimulated AC activities in the presence of calcium and calmodulin (Ca(2+)/CaM) were significantly decreased in AD parietal cortex. In the present study, we examined the amounts of Ca(2+)/CaM-sensitive types I and VIII AC, and Ca(2+)/CaM-insensitive type VII AC in the postmortem hippocampi from AD patients and age-matched controls using immunoblotting. The specificities of the anti-type VII and VIII AC antibodies were confirmed by preabsorption with their specific blocking peptides. We observed a significant decrease in the level of type I AC and a tendency to decrease in the level of type VIII AC in AD hippocampus. On the other hand, the level of type VII AC showed no alteration between AD and controls. A body of evidence from the studies with invertebrates and vertebrates suggests that types I and VIII AC may play an essential role in learning and memory. Our finding thus firstly demonstrated that a specific disruption of the Ca(2+)/CaM-sensitive AC isoforms is likely involved in the pathophysiology in AD hippocampus.

Effect of lipoic acid on redox state of coenzyme Q in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and diethyldithiocarbamate

We investigated the effects of a combined treatment of male C57Bl/6 mice with diethyldithiocarbamate and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the absence or presence of different forms of lipoic acid (Thioctacid TR; commonly used for treatment of diabetic polyneuropathies) on levels and redox states of alpha-tocopherol and coenzyme Q in vivo and on activities of various enzymes of energy metabolism ex vivo. Treatment of mice with diethyldithiocarbamate plus MPTP resulted in a decrease in dopamine (67%) and its major metabolites dihydroxyphenylacetic acid (38%) and homovanillic acid (37%) in striatum. alpha-Tocopherol levels were unaltered in striatum; however, the reduced forms of coenzyme Q were decreased in frontal cortex and hippocampus following diethyldithiocarbamate plus MPTP. In frontal cortex activity of NADH dehydrogenase was significantly inhibited by diethyldithiocarbamate plus MPTP ex vivo, suggesting that the neurotoxic metabolite of MPTP, 1-methyl-4-phenylpyridinium ion, is acting in brain regions other than striatum as well. Lipoic acid, administered 6 times, each at 90 min prior to MPTP, could not restore dopamine in striatum but in contrast maintained a normal ratio of the reduced form to the oxidized form of coenzyme Q, suggesting an interaction of lipoic acid with energy metabolism which seems, however, not only to be due to an activation of pyruvate dehydrogenase.

BLOOD TRANSFERRIN AND FERRITIN IN ALZHEIMER'S DISEASE

In the present study we found a significant correlation between severity of dementia of Alzheimers type (DAT) and both transferrin and ferritin serum levels. Levels of transferrin in serum of 41 DAT patients tended to be lower than those of 19 age-matched controls, while levels of ferritin were not significantly different in DAT patients compared to controls. These results are interpreted in line with previous findings of higher brain ferritin and lower brain transferrin levels in DAT and are a circumstantial support for the oxygen radical hypothesis of degenerative brain disease.