Dénes Zádori

Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells

Reliable in vitro assays are essential for study of the effects of neurotoxic compounds such as beta-amyloid peptides (Abeta). The MTT assay has been used in cultures of different cells, e.g. SH-SY5Y neuroblastoma cells, for the quantitative measurement of Abeta toxicity. In our laboratory differentiated SH-SY5Y cells were used in the MTT assay. Cell differentiation with 10 microM all-trans-retinoic acid resulted in a constant cell number. The cells possess highly developed neurites and exhibit high sensitivity against Abeta. Owing to the constant cell number in differentiated SH-SY5Y cultures the decrease of the redox activity is directly proportional to the neurotoxicity of the substances, no correction is needed. The results of the MTT assay of Abeta peptides on differentiated SH-SY5Y cells displayed a good correlation also with the in vivo results. The present experiments reveal an effective assay for the study of potentially neurotoxic compounds.

Kynurenines in chronic neurodegenerative disorders: future therapeutic strategies

Parkinson’s, Alzheimer’s and Huntington’s diseases are chronic neurodegenerative disorders of a progressive nature which lead to a considerable deterioration of the quality of life. Their pathomechanisms display some common features, including an imbalance of the tryptophan metabolism. Alterations in the concentrations of neuroactive kynurenines can be accompanied by devastating excitotoxic injuries and metabolic disturbances. From therapeutic considerations, possibilities that come into account include increasing the neuroprotective effect of kynurenic acid, or decreasing the levels of neurotoxic 3-hydroxy-l-kynurenine and quinolinic acid. The experimental data indicate that neuroprotection can be achieved by both alternatives, suggesting opportunities for further drug development in this field.

Valproate ameliorates the survival and the motor performance in a transgenic mouse model of Huntington's disease.

Huntingtons disease (HD) is one of the chronic devastating neurodegenerative disorders. The pathophysiological processes clearly involve both excitotoxicity and reduced gene transcription due to the decreased level of histone acetylation, accompanied by the loss of gamma-aminobutyric acidergic (GABAergic) medium-sized spiny neurons in the striatum as a pathological hallmark of HD. Thus, the antiepileptic drug valproate, which has proved GABAergic, antiexcitotoxic and histone deacetylase inhibitor effects, might be of value by exerting a beneficial neuroprotective effect. We have now tested this drug in the N171-82Q transgenic mouse model of HD, following its chronic intraperitoneal administration in a daily dose of 100 mg/kg. Valproate significantly prolonged the survival of the transgenic mice and significantly ameliorated their diminished spontaneous locomotor activity, without exerting any noteworthy side-effect on their behaviour or the striatal dopamine content at the dose administered. The beneficial effect of valproate is probably explained by its complex pharmacological activity. As several previous clinical trials carried out with valproate did not indicate any positive effect in HD, it is worth considering the design of new studies based on a well-planned treatment regime with higher dose, using valproate in monotherapy or in combination therapy with a high number of participating patients.

Neuroprotective effects of a novel kynurenic acid analogue in a transgenic mouse model of Huntington's disease.

Huntington’s disease (HD) is a progressive neurodegenerative disorder, the pathomechanism of which is not yet fully understood. Excitotoxicity is known to be involved in the development of HD and antiglutamatergic agents may, therefore, have beneficial neuroprotective effects. One of these agents is the tryptophan metabolite kynurenic acid (KYNA), which is an endogenous NMDA receptor antagonist. However, its pharmacological properties rule out its systemic administration in CNS disorders. We have tested a novel KYNA analogue, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride, in the N171-82Q transgenic mouse model of HD. The analogue exhibited several significant effects: it prolonged the survival of the transgenic mice, ameliorated their hypolocomotion, prevented the loss of weight and completely prevented the atrophy of the striatal neurons. The beneficial effects of this KYNA analogue are probably explained by its complex anti-excitotoxic activity. As it did not induce any appreciable side-effect at the protective dose applied in a chronic dosing regime in this mouse model, it appears worthy of further thorough investigations with a view to eventual clinical trials.

Mitochondrial disturbances, excitotoxicity, neuroinflammation and kynurenines: Novel therapeutic strategies for neurodegenerative disorders

A mitochondrial dysfunction causes an abatement in ATP production, the induction of oxidative damage and the propagation of cell death pathways. It is additionally closely related to both glutamate excitotoxicity and neuroinflammation. All of these interconnected aspects of a cellular dysfunction are involved in the pathogenesis of numerous neurological disorders, including those with an acute (e.g. ischemic stroke) or a chronic (e.g. Huntingtons disease) onset. Both acute and chronic neurodegenerative disorders have been demonstrated to involve multiple imbalances of the kynurenine pathway metabolism in the pathogenesis of the disease. As regards neuroactive compounds featuring in the pathway, quinolinic acid is a specific agonist of N-methyl-d-aspartate receptors, and a potent neurotoxin with additional and marked free radical-producing and lipid peroxidation-inducing properties. The toxic effects of 3-hydroxy-L-kynurenine are mediated by free radicals. Besides the possibility of increasing brain kynurenic acid concentrations, L-kynurenine may have vasoactive properties, too. Kynurenic acid has proven to be neuroprotective in several experimental settings, but in consequence of its pharmacokinetic properties it is not applicable as systemic administration in human cases. The aim of this short review is to emphasize the common features of cerebral ischemia and Huntingtons disease and to highlight therapeutic strategies targeting the kynurenine pathway.

Glutamatergic Dysfunctioning in Alzheimer's Disease and Related Therapeutic Targets

The impairment of glutamatergic neurotransmission plays an important role in the development of Alzheimers disease (AD). The pathological process, which involves the production of amyloid-β peptides and hyperphosphorylated tau proteins, spreads over well-delineated neuroanatomical circuits. The gradual deterioration of proper synaptic functioning (via GluN2A-containing N-methyl-D-aspartate receptors, NMDARs) and the development of excitotoxicity (via GluN2B-containing NMDARs) in these structures both accompany the disease pathogenesis. Although one of the most important therapeutic targets would be glutamate excitotoxicity, the application of conventional anti-glutamatergic agents could result in further deterioration of synaptic transmission and intolerable side-effects. With regard to NMDAR antagonists with tolerable side-effects, ion channel blockers with low affinity, glycine site agents, and specific antagonists of polyamine site and GluN2B subunit may come into play. However, in the mirror of experimental data, only the application of ion channel blockers with pronounced voltage dependency, low affinity, and rapid unblocking kinetics (e.g., memantine) and specific antagonists of the GluN2B subunit (e.g., ifenprodil and certain kynurenic acid amides) resulted in desirable symptom amelioration. Therefore we propose that these kinds of chemical agents may have therapeutic potential for present and future drug development.

Mitochondrial disturbances, tryptophan metabolites and neurodegeneration: Medicinal chemistry aspects

Neurodegenerative disorders, e.g. Parkinsons, Huntingtons and Alzheimers diseases are distinct clinical and pathological entities sharing a number of leading features in their underlying processes. These common features involve the disturbances in the normal functioning of the mitochondria and the alterations in the delicate balance of tryptophan metabolism. The development of agents capable of halting the progression of these diseases is in the limelight of neuroscience research. This review highlights the role of mitochondria in the development of neurodegenerative processes with special focus on the involvement of neuroactive kynurenines both as pathological agents and potential targets and tools for future therapeutic approaches by providing a comprehensive summary of the main streams of rational drug design and giving an insight into present clinical achievements.

Syntheses, Transformations and Pharmaceutical Applications of Kynurenic Acid Derivatives

The syntheses and transformations of 4-hydroxyquinoline-2-carboxylic acid, kynurenic acid, are reviewed, and special attention is paid to the pharmacological activities and pharmaceutical applications of its derivatives.

Endogenous neuroprotection in chronic neurodegenerative disorders: with particular regard to the kynurenines

•  Introduction •  The role of mitochondrial impairment and oxidative stress in neurodegeneration ‐  Background ‐  Parkinson’s disease ‐  Huntington’s disease •  Glutamate excitotoxicity in neurodegeneration ‐  Background ‐  Parkinson’s disease ‐  Huntington’s disease •  The kynurenine pathway ‐  Historical overview ‐  The biosynthesis of neuroactive kynurenines ‐  Neuroactive kynurenines: sites of action •  Neuroactive kynurenines in neurodegenerative disorders ‐  Parkinson’s disease ‐  Huntington’s disease •  Endogenous neuroprotection in Parkinson’s and Huntington’s diseases ‐  Background ‐  L‐carnitine ‐  L‐carnosine ‐  Coenzyme Q10 ‐  Creatine ‐  Cysteamine/cystamine ‐  Eicosapentaenoic acid ‐  α‐lipoic acid ‐  Pyruvate ‐  Taurine ‐  Tocopherol ‐  L‐KYN/KYNA •  Concluding remarks

Evaluating biomarkers of neuronal degeneration and neuroinflammation in CSF of patients with multiple sclerosis-osteopontin as a potential marker of clinical severity

Biomarkers capable of predicting the clinical course and the rate of disease progression in multiple sclerosis are currently unavailable. Our objective was to examine if the levels of proteins associated with axonal and neuronal degeneration (Tau, p-Tau and β-amyloid(1-42)) and T-cell-mediated autoimmunity (osteopontin) are altered in the cerebrospinal fluid (CSF) of MS patients, and to assess their potential in reflecting the clinical severity and predicting the progression and clinical evolution of early MS. The CSF samples collected from patients presenting with different clinical forms of MS were evaluated by enzyme-linked immunosorbent assays. The patients were regularly followed-up and their clinical status was re-evaluated 5 years after sampling. The results demonstrated that while CSF levels of Tau, p-Tau and β-amyloid(1-42) did not differ between MS and Control groups, the levels of osteopontin were significantly elevated in MS patients. This increase was associated with the presence of a relapse and correlated with clinical severity, which findings were independent of age and blood-CSF barrier function. However, none of the examined protein levels differed significantly between groups with different clinical evolutions and no positive correlations with clinical progression could be detected. We conclude that Tau, p-Tau and β-amyloid(1-42) are inappropriate as biomarkers in MS. This is the first report on CSF osteopontin as an independent marker of clinical severity in definite MS.