Bernd Janetzky

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

Parkinsons 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.

Biomarkers for early diagnosis of Alzheimer disease: ‘ALZheimer ASsociated gene’– a new blood biomarker?

•  Introduction •  Pathogenesis of AD •  Alzheimer pathology and diagnosis •  Biomarkers of AD ‐  CSF biomarkers ‐  Plasma biomarkers ‐  Platelet biomarkers ‐  Structural biomarkers •  ALZheimer ASsociated protein (ALZAS) ‐  ALZAS immunohistochemistry ‐  ALZAS‐mRNA studies ‐  ALZAS as a future biomarker? •  Conclusions

Effect of highly halogenated β-carbolines on dopaminergic cells in culture and on mitochondrial respiration

Pyridoindoles (carbolines) are relatively common indole alkaloids in most diets and in our ecosystem. Besides 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its active metabolite, MPP+, several psychotropic β‐carbolines have been described to exhibit neurotoxic effects on the dopaminergic system. In this work, we have investigated a new class of neurotoxic β‐carbolines, the highly halogenated tetrahydro‐β‐carbolines. The present compounds are derived from the condensation of endogenous tryptamine with the hypnotic drug chloral hydrate or by exposure to the industrial solvent trichloroethylene (which can be metabolized to chloral). These tetrahydro‐β‐carbolines inhibit the mitochondrial respiratory chain, acting as strong inhibitors of Complex I and partial inhibitors of Complex II. They are also neurotoxic to dopaminergic neurons in primary cell culture. Drug Dev. Res. 46:51–56, 1999.

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 Alzheimers 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.

Highly Halogenated Tetrahydro-β-Carbolines as a New Class of Dopaminergic Neurotoxins

The Pictet-Spengler condensation of 2-arylethylamines with aldehydes or α-keto acids (Callaway et al., 1994) is not only the most efficient pathway for the chemical synthesis of tetrahydro-β-carbolines (TH-βCs), but has also been found to play a role in human organisms, leading to endogenous “mammalian alkaloids” (Bringmann, 1979; Buckholtz, 1980; Airaksinen and Kari, 1981a; Airaksinen and Kari, 198 lb; Collins, 1986a; Rommelspacher and Susilo, 1985; Brossi, 1993). During the last three decades, the biological potential of the simple, formaldehyde- and acetaldehyde-derived THβCs tryptoline (1) and eleagnine (2) (see Fig. 1) has been investigated thoroughly because these compounds and related derivatives were regarded as causative links between alcoholism and opioid mania (Davis and Walsh, 1970; Rommelspacher et al., 1984; Myers, 1989). Furthermore, since the merely synthetic neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was found to induce parkinsonism in humans, monkeys, and mice (Burns et al., 1983; Singer et al., 1993), special attention has been focused on the potential role of THβCs as inducers of parkinsonism, closely related in structure, yet occurring in Nature. Indeed, for several β-carbolines toxic behavior towards dopaminergic neurons was demonstrated (Collins et al., 1986b; Albores et al., 1990; Sayre et al., 1991).

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.

P1-367: Nested gene and autoimmune response in Alzheimer’s disease

of these vesicles is severely impaired in APP deficient mice (Kamal et al., 2001). Intriguingly, recent data reported a defect in synapse morphology accompanied by reduction of synaptic vesicle density in mice deficient for APP and its homologue APLP2 (Yang et al., 2005. Neurosci. lett. 384:6671). Together these findings raise the possibility that APP-dependent axonally-transported vesicles could play an important role in regulating the establishment and maintenance of proper synaptic structures. Objective: To test further the nature of APP axonally-transported vesicles and to explore their functional role we are characterizing their protein content and morphology by mass spectrometry and electron microscopy. Methods and Results: To this end, we developed a biochemical approach to purify enriched populations of APP vesicles using subcellular fractionation from mouse brain cortices. We used sucrose density gradients to separate distinct populations of APP vesicles based on their density properties followed by affinity purification using specific APP antibodies attached to magnetic beads. Additionally, we performed Western blot analysis to monitor our fractionation steps and to test for the presence or absence of the APP secretase presenilin in each vesicle population. Parallel silver staining of the gels resulted in a specific protein band pattern when compared to control samples. Finally, we performed electron microscopy and mass spectrometry analysis to confirm the existence of morphologically distinct APP vesicles and to determine their specific protein content. Conclusion: We believe that the isolation and the biochemical and morphological characterization of distinct populations of axonally-transported APP-containing vesicles will shed new light on the normal function of APP in neuronal cells and, ultimately, improve our understanding of the development of Alzheimer’s disease.

Dopamine and Glutamate in Parkinson’s Disease

In 1817 the London physician James Parkinson described in his well-known essay on the shaking palsy, a disease that is characterized by tremor (mainly at rest), muscular rigidity, which leads to difficulties in walking, writing, speaking, and masking of facial expression, bradykinesia, a slowness in initiating and executing movements, and stooped posture and instability. Many of these clinical features are also manifested by other basal ganglia (BG) disorders and, thus, often referred to as parkinsonian syndromes. Today we know three main subgroups of parkinsonian syndromes: To the main group belongs idiopathic Parkinson’s disease (IPD) including the known hereditary forms (e.g., α-synuclein- and PARK-mutations); “parkinsonism-plus” syndromes are characterized by progressive supranuclear palsy, multiple system atrophy, and the Parkinson-amyotrophic lateral sclerosis (ALS)-dementia complex that include parkinsonian movement abnormalities in addition to other neurological deficits. The secondary, symptomatic forms of parkinsonisms are formed by infectious diseases, tumors, metabolic disturbances, drugs, and toxins.

Neurotrophic effects of simplified mastigophorene analogs on mesencephalic dopaminergic cells in primary culture

Mastigophorenes are dimeric isocuparane‐type sesquiterpenes of plant origin which have been found to exhibit neurotrophic properties. In this study, a simplified synthetically produced analog (1) and its dimethylether (2) were screened for potential neurotrophic effects on primary dopaminergic cell cultures in vitro. Dopaminergic cultures were prepared from embryonic mouse mesencephalon and 6‐day serum‐free cultures were exposed to low concentrations (0.05–5.0 μM) of these compounds for 6 days. Although differing by only two O‐methyl groups, the two substances differed in their effects on dopaminergic cells. 1 promoted growth of individual cells at low concentrations, whereas the more lipophilic 2 prevented cell death in culture and also clearly stimulated the growth of neuronal processes, although effects on growth of the total cell area were less pronounced. Effects on mitochondrial respiratory enzymes appear unlikely as 1 and 2 showed inhibitory effects only at concentrations 10–100‐fold higher than those used in cell culture. The results obtained show that synthetically produced substances analogous to neuron‐supportive mastigophorenes of plant origin can serve as neurotrophic substances. Drug Dev. Res. 50:153–156, 2000.

Chemical Modification of the Mitochondrial Complex I Inhibitor 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline: Synthesis and Evaluation of N-Alkanoyl Derivatives

Abstract Several N-alkanoyl derivatives (4-9 and 13-16) of the potent mitochondrial complex I inhibitor TaClo (1-trichlorom ethyl-1,2,3,4-tetrahydro-β-carboline, 2) have been synthesized in order to elucidate the role of hydrophobic portion in the inhibitory action. Using rat brain homogenates or submitochondrial particles, the inhibitory effects of these compounds towards NADH-ubiquinone reductase (complex I) activity indeed appeared to correlate quite strongly with their lipophilic character. An X-ray structure analysis, exemplarily performed for N-acetyl-TaClo (4), revealed the N-substituent of such chlorinated agents to be dramatically pushed out of the β-carboline ring ‘plane’ due to the high steric demand of the huge trichloromethyl group at C-1.