Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in industrialized countries. Present cell culture models for PD rely on either primary cells or immortal cell lines, neither of which allow for long-term experiments on a constant population, a crucial requisite for a realistic model of slowly progressing neurodegenerative diseases. We differentiated SH-SY5Y human dopaminergic neuroblastoma cells to a neuronal-like state in a perfusion culture system using a combination of retinoic acid and mitotic inhibitors. The cells could be cultivated for two months without the need for passage. We show, by various means, that the differentiated cells exhibit, at the molecular level, many neuronal properties not characteristic to the starting line. This approach opens the possibility to develop chronic models, in which the effect of perturbations and putative counteracting strategies can be monitored over long periods of time in a quasi-stable cell population.

PrP Sc-like prion protein conformer in sudden infant death syndrome brain.

Nothing is known about the pathophysiology of sudden infant death syndrome (SIDS). Here we show the presence of misfolded prion protein (PrP(Sc)-like) in extracts of various sections of the brains of two SIDS victims. DNA sequence information for one of these (death at 12 days) revealed two nucleotide variants in the protein coding region of the PrP gene. This may be a key finding in the understanding of SIDS pathology, and may suggest ways for identifying risk factors for SIDS in newborn infants.

Coenzyme Q10 reduces the toxicity of rotenone in neuronal cultures by preserving the mitochondrial membrane potential.

Defects in mitochondrial energy metabolism due to respiratory chain disorders lead to a decrease in mitochondrial membrane potential (DeltaPsim) and induce apoptosis. Since coenzyme Q10 (CoQ10) plays a dual role as an antioxidant and bioenergetic agent in the respiratory chain, it has attracted increasing attention concerning the prevention of apoptosis in mitochondrial diseases. In this study the potential of CoQ10 to antagonize the apoptosis-inducing effects of the respiratory chain inhibitor rotenone was explored by video-enhanced microscopy in SH-SY5Y neuroblastoma cells. The cationic fluorescent dye JC-1 which exhibits potential-dependent accumulation in mitochondria was used as an indicator to monitor changes in DeltaPsim. The relative changes in fluorescence intensity after incubation with rotenone for 15 minutes were calculated. Pre-treatment with CoQ10 (10 or 100 microM) for 48 h led to a significant reduction of rotenone-induced loss of DeltaPsim. These results suggest, that cytoprotection by CoQ10 may be mediated by raising cellular resistance against the initiating steps of apoptosis, namely the decrease of DeltaPsim. Whether these data may provide new directions for the development of neuroprotective strategies has to be investigated in future studies.

A broader horizon of Alzheimer pathogenesis: ALZAS--an early serum biomarker?

Recently, a novel risk gene protein expressed in elderly patients with the diagnosis of Alzheimer disease (AD) was discovered on chromosome 21 within the APP (amyloid precursor protein) region. This 79 amino acid protein, ALZAS (Alzheimer Associated Protein) contains the beta-amyloid peptide 1-42 fragment, the APP transmembrane signal, and a unique 12 amino acid c-terminal which is not present in any known allele of the APP gene. Reverse transcription-PCR revealed that the transcript of ALZAS was expressed in cortical and hippocampal regions of human Alzheimer disease brain as well as in leukocytes derived from AD patients. Most specifically, an endogenous antibody was found in patients with confirmed AD, in patients with depression, and in subjects suggested to have presymptomatic AD, where it was directed against epitopes within the intron encoded amino acid c-terminal sequence.

Increased hippocampal DNA oxidation in serotonin transporter deficient mice.

The serotonin transporter (5HTT) is the molecule responsible for the high-affinity reuptake of 5HT from the synaptic cleft. Mice lacking the 5HTT exhibit highly elevated extracellular concentrations of 5HT. We assessed whether the glutathione detoxification system is altered in 5HTT-deficient mice. While levels of reduced and oxidized glutathione were unchanged, glutathione metabolising enzymes showed a differential pattern of modulation. Glutathione peroxidase was reduced in frontal cortex, brainstem, and cerebellum of 5HTT-deficient mice, though not to a statistically significant extent, while a putative isoform of the detoxifying enzyme glutathione-S-transferase pi was decreased in a number of brain regions, especially in brainstem. At the level of the DNA, we found an increase of oxidative DNA adducts in the hippocampus of 5HTT-deficient mice. Given the importance of the hippocampus in learning and memory, this may be the most important neurochemical consequence of the absence of the 5HTT.

Pergolide protects dopaminergic neurons in primary culture under stress conditions.

Dopamine agonists are an important therapeutic strategy in the treatment of Parkinson's disease. They postpone the necessity for and reduce the required dose of L-3,4-dihydroxyphenylalanine (L-DOPA) medication thus protecting against the development of motor complications and potential oxidative stress due to L-DOPA metabolism. In primary cultures from mouse mesencephalon we show that pergolide, a preferential D(2) agonist enhanced the survival of healthy dopaminergic neurons at low concentrations of 0.001 microM. About 100 fold higher concentrations (0.1 microM) were necessary to partially reverse the toxic effects of 10 microM 1-methyl-4-phenylpyridinium (MPP(+)). Pergolide was equally effective in preventing the reduction of dopamine uptake induced by 200 microM L-DOPA. Furthermore, between 0.001-0.1 microM it also reduced lactate production thus promoting aerobic metabolism. The present findings suggest that pergolide protects dopaminergic neurons under conditions of elevated oxidative stress.

Novel mitochondrial DNA mutations in Parkinson's disease.

Despite the recent discovery of several chromosomal gene mutations in familial Parkinson's disease (PD) the genetic background for idiopathic PD remains to be elusive. Since the discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) action on dopaminergic neuronal cells and the specific decrease of mitochondrial complex I activity in substantia nigra of PD patients mitochondrial biochemistry and genetics emerged to become Pandora's box in the pathogenesis of PD. One approach was to establish the potential role of defective mitochondrial DNA (mtDNA). As complex I genes are the most vulnerable part of mtDNA we analyzed the mitochondrial MTND1 and MTND2 genes of 10 substantia nigra and 85 platelet samples from PD patients. We were uneventful to detect heteroplasmic base changes even applying techniques able to visualize mutations with low percentage of heteroplasmy but here we report novel homoplasmic base changes. These results add further evidence that there are no inherited disease specific mtDNA mutations, hence individual homoplasmic mutations or very low grade heteroplasmic mutations in the vicinity of mitochondrial metabolism and oxidative stress may contribute to selective neuronal vulnerability in PD.

Protection of dopaminergic neurons in primary culture by lisuride.

Dopamine agonists play an important role in the treatment of Parkinson's disease by reducing the administration of L-3,4-dihydroxyphenylalanine (L-DOPA). The enzymatic and non-enzymatic conversion of L-DOPA is suspected to increase oxidative stress, which leads to the degeneration of dopaminergic neurons in Parkinson's disease. In primary mouse mesencephalic cultures we show that the dopamine D1/D2 receptor agonist lisuride, in a concentration range of 0.001-1 microM, enhances the survival of dopaminergic neurons, protects against toxicity induced by L-DOPA or 1-methyl-4-phenylpyridinium ion (MPP+) and stimulates 3H-dopamine uptake. Lisuride also reduces anaerobic metabolism during incubation with L-DOPA. The present findings suggest that lisuride may have trophic/survival-promoting properties and potentially reduces oxidative stress.

Chronic alcohol consumption and cerebral indices of oxidative stress: is there a link?

BACKGROUND: It is still difficult to define the biochemical mechanisms that cause alterations in neuronal function and plasticity and neuronal cell loss in the brains of alcohol-dependent patients. METHODS: To evaluate the extent of cerebral alcohol-induced oxidative stress ex vivo, we investigated the levels of glutathione (GSH), its oxidation product glutathione disulfide (GSSG, produced by GSH-peroxidases), and the activities of catalase and superoxide dismutases (SOD). In addition, selected brain regions from up to 22 subjects (versus controls) were studied post mortem to compare the amount of oxidized DNA-base 8-hydroxy-2'-deoxyguanosine (8-OHdG) with levels of deoxyguanosine (dG) in mitochondrial and nuclear DNA. RESULTS: The most prominent findings showed significantly decreased GSH/(GSH+2GSSG) molar redox (oxidation-reduction) ratios in the corpus mamillare and cerebellum, which appeared due to an increase in GSSG caused by chronic alcohol intake. Catalase activity was increased in only the frontal cortex, whereas decreased catalase activity was found in the corpus callosum. In contrast, neither copper-zinc-superoxide dismutase (CuZnSOD) and manganese-superoxide dismutase (MnSOD) activities nor 8-OHdG/dG molar ratios were altered, although a tendency toward higher OHdG/dG ratios in temporal and parietal cortex from alcohol-dependent patients could be detected when mitochondrial DNA was analyzed selectively. CONCLUSIONS: We propose that decreased brain GSH/(GSH+2GSSG) molar redox (oxidation-reduction) ratios in alcohol-dependent patients may reflect neural impairment due to increased peroxide production after chronic alcohol consumption. However, future experiments, investigating the activities of enzymes and cofactors involved in GSH synthesis and metabolism in the human brain, will have to validate the specificity of these results for oxidative stress.

Mitochondrial dysfunction--a pathogenetic factor in Parkinson's disease.

The cause of Parkinson's disease is still unknown. Nonetheless, there are some generally accepted hypotheses with respect to the cascade of dopaminergic cell degeneration. One of the factors is a decrease in respiratory chain complex I activity. This enzyme abnormality is only found in substantia nigra pars compacta. It is not currently known whether this is due to a genetic abnormality of the nuclear or mitochondrial genome or to an exo- or endotoxin. To date, no specific abnormality of the mitochondrial genome has been detected, although ageing leads to deletions in up to 5% of all mitochondrial genome molecules in the brain. There is controversy whether this complex I defect is also detectable in muscle or blood cells. It is our conviction that the considerable overlap between blood cells from normal controls and patients with Parkinson's disease means that such measurements are not distinctive for the two conditions. A decrease in complex I activity in postmitotic cells may be one of the crucial factors for cell death.

Bromal-derived tetrahydro-beta-carbolines as neurotoxic agents: chemistry, impairment of the dopamine metabolism, and inhibitory effects on mitochondrial respiration.

The mammalian alkaloids tryptoline (1) and eleagnine (2) as well as the highly halogenated (X = F, Cl, Br) tetrahydro-beta-carbolines (THbetaCs) 3-5, structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 6), were found to have a common feature of inducing a severe impairment of the nigrostriatal dopamine metabolism and inhibiting complex I of the mitochondrial respiratory chain highly selectively. Within the series of compounds tested, 1-tribromomethyl-1,2,3,4-tetrahydro-beta-carboline ('TaBro', 5), which was prepared in high yields from the biogenic amine tryptamine ('Ta', 7) and the unnatural aldehyde bromal ('Bro', 8) by a Pictet-Spengler cyclization reaction, turned out to be the most potent toxin in vitro and in vivo. As demonstrated by voltammetric measurements on rats, for all the THbetaCs 1-5 investigated, intranigral application of a single dose of 10 microg resulted in a significant reduction of the dopaminergic activity in the striatum, with the strongest effect being observed for TaBro (5). Using rat brain homogenates, again 5 (IC50 = 200 microM) as well as its dehydrohalogenation product 11 (IC50 = 150 microM) exhibited the most pronounced inhibitory potential on mitochondrial respiration. The halogen-free THbetaCs 1 and 2 as well as the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP+), by contrast, showed only a moderate inhibition at concentrations in the millimolar range (e.g. for MPP+: IC50 = 3.5 mM). For an elucidation of the role of hydrophobic portion in the inhibitory action against complex I activity, several N-acyl derivatives (15-21) of 5 were synthesized and tested. An X-ray diffraction study on the 3-dimensional structure of trifluoroacetylated highly halogenated THbetaCs (12-14) revealed the tetrahydropyrido part to adopt a nearly planarized half-chair conformation. Because of the steric demand of the trihalogenmethyl moiety (CF3 < CCl3 < CBr3), the N-substituent is dramatically pushed out of that ring 'plane'.

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

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 Parkinson's 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.

Acute MPTP treatment produces no changes in mitochondrial complex activities and indices of oxidative damage in the common marmoset ex vivo one week after exposure to the toxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to cause a Parkinsonian syndrome in man and non-human primates. Hypotheses concerning the pathogenetic mechanisms of MPTP toxicity on nigro-striatal dopaminergic neurons relate to impairment of mitochondrial function and oxidative stress. However, surprisingly few primate studies addressed these issues ex vivo. Thus, the present study assessed the enzyme activities of the respiratory chain, GSH/GSSG and ubiquinol/ubiquinone content in the MPTP primate model (common marmoset, Callithrix jacchus; 2 mg MPTP-hydrochloride/kg body wt were injected subcutaneously (s.c.) on four consecutive days; animals were sacrificed 7 days after last MPTP exposure). Activities of respiratory chain enzymes were measured in crude homogenates of the caudate nucleus, because the probable toxic metabolite of MPTP, MPP+, is transported into dopaminergic neurons via the dopamine uptake system in striatal synapses and mitochondria are concentrated in axonal terminals. Since MPP+ can damage membranes of axonal terminals of nigro-striatal neurons we measured GSH/GSSG contents in the putamen and ubiquinol/ubiquinone concentrations in the substantia nigra and putamen as indices of oxidative damage. At the time of sacrifice MPTP-induced deficits comprised severe behavioural Parkinsonian symptoms, profound depletion of striatal dopamine and its major metabolites as well as pronounced loss of nigro-striatal neurons. Despite these severe lesions, acute MPTP treatment had no effect on any of the enzymes of the respiratory chain in the caudate nucleus and indices of oxidative damage in both the substantia nigra and putamen. These results suggest that factors other than mitochondrial impairment and/or oxidative stress may be involved in MPTP neurotoxicity in primates. Alternatively, early compensatory mechanisms and/or transient effects could account for the reported results and will be discussed.

Investigations on the point mutations at nt 5460 of the mtDNA in different neurodegenerative and neuromuscular diseases.

Point mutations of the mitochondrial genome are often considered to be the cause of certain neurodegenerative disorders and mitochondrial myopathies. Recently, there has been a report on Alzheimer's disease (AD) point mutations at position 5460 of the mitochondrial genome located within the ND2 gene. Using allele-specific PCR with a sensitivity of detection of less than 1% mutated mtDNA, we investigated postmortem brain samples from 48 patients with AD and blood samples of 15 patients with clinically diagnosed AD. In addition, we investigated tissue samples of patients with different neuromuscular disorders and patients with Downs syndrome. Independent of the tissue analysed very few of all the tested samples of patients showed a point mutation at nt 5460 with a base substitution from G to A. Two out of 19 brain and 48 blood samples from controls carried this mutation. The G to T transversion was not found in any of the so far tested samples. Our results do not support the previously reported significant high frequency of these mutations in AD.A polymorphism seems more likely.

Iron-dependent enzymes in Parkinson's disease.

There is increasing evidence for a mitochondrial disturbance in Parkinson's disease (PD). We present data which favor a defect of complex I of the respiratory chain only in substantia nigra pars compacta. Evidence from literature supports the view that this complex I defect is specific for Parkinson's disease. Questions like feasibility of postmortem biochemical analyses, impairment of the mitochondrial genome, and evidence for the complex I defect as a causative event for PD are discussed in detail. We consider complex I defect to play a most important role in the cell death of dopaminergic neurons.

TaClo as a neurotoxic lead: improved synthesis, stereochemical analysis, and inhibition of the mitochondrial respiratory chain.

"TaClo", a highly halogenated tetrahydro-beta-carboline derived from the biogenic amine tryptamine ("Ta") and the synthetic hypnotic chloral ("Clo"), has to be considered as a dopaminergic neurotoxin potentially occurring in vivo. For the preparation of TaClo on a large scale, an improved synthetic pathway was elaborated. The distinct neurotoxic activity of TaClo warrants its intensive study also under stereochemical aspects. For this reason, an analytic device for the separation and stereochemical attribution of its two enantiomers, (R)-TaClo and (S)-TaClo, was developed, based on chromatography on a chiral HPLC phase. Elucidation of the absolute configuration was achieved by CD spectroscopy and confirmed by X-ray crystallography. TaClo exhibits highly selective in vitro inhibition of complex I of the mitochondrial respiratory chain, the required concentrations being much lower than those needed for related halogen-free beta-carbolines or for MPP+ (1-methyl-4-phenyl-pyridinium ion), the active metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Furthermore, TaClo as a novel lead structure stimulated chemical and neuropharmacological investigations also on related highly halogenated beta-carbolines. Thus, some of the tested compounds--both potential TaClo metabolites and unnatural derivatives--showed even an enhanced inhibition of the mitochondrial respiration in vitro.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline, a new inhibitor of complex I.

The neurotoxic agent MPP+ is an artificial substance producing a syndrome very similar to that of idiopathic Parkinson's disease. There are also naturally occuring neurotoxic substances under discussion like the group of isoquinoline and beta-carboline alkaloids. All these substances are more or less powerfull inhibitors of complex I of the mitochondrial oxidative phosphorylation. This study examined the effect of 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a putative in vivo condensation product of chloralhydrate and tryptamine, on the oxidative phosphorylation system compared to MPP+. Similar to MPP+, TaClo inhibits only the electron transfer from complex I towards ubiquinone. Demonstrating a 10-times more effective inhibition than MPP+, complex I activity is fully inhibited by 800 microM TaClo in brain homogenates and submitochondrial particles. By extending the preincubation time from 5 to 30 min complex I is already inhibited by 400 microM TaClo. Other derivates of TaClo as N-methyl-TaClo demonstrate an even greater inhibitory effect on complex I and especially on complex II activities.

Respiratory chain and mitochondrial deoxyribonucleic acid in blood cells from patients with focal and generalized dystonia.

An increasing number of neurodegenerative diseases seem to be associated with or even due to disturbances of cerebral energy metabolism. One generally accepted example is complex I deficiency in substantia nigra from patients with Parkinson's disease. Reports on a complex I defect in platelets from patients with dystonia led us to check for disturbances of the respiratory chain or of the mitochondrial genome in isolated mitochondria from patients with focal or generalized dystonia. We could not confirm the idea of mitochondrial disturbance in platelets from patients with dystonia because we did not find abnormal enzyme activities or any deletions of the mitochondrial genome. Thus, we do not think that blood cells such as platelets can serve as markers for neurodegenerative disorders such as dystonia.