|Art der Förderung:||Standard Projekt|
|Institution:||Universität Heidelberg Zentrum für Molekularbiologie|
|Projektleiter:||Prof. Dr. Roland Brandt|
|Laufzeit:||01. November 1999 - 31. Oktober 2000|
A critical event during Alzheimer's disease (AD) is a massive death of subsets of CNS neurons which finally leads to the severe dementia characteristic for the disease. Although the precise mechanisms leading to neuronal death during AD are unknown, it is assumed that cytoskeletal abnormalities and malfunction critically contribute. A prime candidate in this cascade represents the microtubule- associated protein tau which aggregates into neurofibrillary tangles in affected neurons and of which several mutations have recently been identified in patients with neurodegenerative diseases others than AD exhibiting tau pathology. Unfortunately, no animal or cell culture model with tau pathology exists so far which would allow to screen for drug5 capable of suppressing neuronal death in disease. It is possible that such a model requires the use of a human system and/or a characteristic processing of tau which does not occur in standard animal models such as mice.
Aim of the proposed project is to establish a human neuronal culture model that exhibits tau pathology and is suitabte to l a high-throughput screening of potentially useful therapeutics. Terminally differentiated and postmitotic human model neurons will be cultured in a microplate format in chemically defined medium and cell survival will be analyzed c olorimetrically. Conditions will be established where the neurons develop neurodegeneration reflective of affected neurons during AD. This includes to induce cytoskeletal pathology by infecting the neurons with tau mutants simulating an AD-like tau modification. In addition, extracellular factors or additional treatments will be tested for their potential to potentiate tau-mediated effects. This includes to test for the effect of added AB-fibrills, heparin or oxidative stressors in the culture system. The assay will be optimized with respect to time and culture conditions and will be used to analyze the effect of potentially useful drugs which interfere with neuronal survival. If the system proofs to be useful for screening purposes, stably transfected hNT neurons which express AD-like tau mutants from a neuronal promotor will be produced and the screening-assay simplified.
A critical event during Alzheimer's disease (AD) is a massive death of subsets of CNS neurons which finally leads to the severe dementia characteristic for the disease. Although the precise mechanisms leading to neuronal death during AD are unknown, it is assumed that cytoskeletal abnormalities and malfunction critically contribute. A prime candidate in this cascade represents the microtubule associated protein tau which is characteristically modified during the disease process, aggregates into neurofribrillary tangles in affected neurons and of which several mutations have been identified in patients with FTDP-17, a neurodegenerative disease also exhibiting tau pathology.
Unfortunately, no animal model with tau pathology reflecting the selective accumulation of modified tau in affected brain regions of AD exists so far. Furthermore, neural cell culture models that would allow to analyze for a potential toxic effect of tau modifications and would allow to screen for drugs capable of suppressing neuronal death in disease are not available. It is possible that such a model requires the use of a human system and/or a characteristic processing of tau which does not occur in standard animal models such as mice. Aim of the project was to establish a culture model that exhibits degeneration mediated by tau protein with disease-like tau modifications. Furthermore, such a culture model should be in an appropriate format to allow high-troughput screening for potentially useful therapeutics.
To achieve this, a mirotiter-plate based cell survival assay using neural cell lines expressing wildtype human tau protein and a mutated tau construct that mimics aspects of a disease-like tau modification was established. We could show that expression of tau with a disease-like modification leads to a significantly increased death of differentiated neural cells compared to wildtype tau expressing or control cells indicating cytotoxic activity of the tau modification. Addition of drugs with cytotoxic potential significantly decreased the relative survival of cells expressing modified tau protein. This suggests that the modification sensitizes the cells for toxic effects of other agents. In addition, we could show that the cytotoxic activity is closely associated with an increase in the rate of programmed cell death (apoptosis). [n order to confirm these results using a human neuronal system, terminally differentiated and postmitotic human model neurons were employed. Also in this system, overexpression of tau constructs with disease-like tau modifications led to a significant increase in neurodegeneration and an increased rate of apoptotic neurons. The data provide novel evidence that tau modifications directly lead to the degeneration of neurons by a mechanism that involves an induction of programmed cell death. Thus, drugs that inhibit tau modification or reduce the neurotoxic effect of modified tau protein could help to increase the survival of neurons during AD. The microtiter-plate based assay system which we have developed in the course of this project could be very valuable to identify therapeutics capable of suppressing neuronal death in AD and thus preventing the development of dementia. Currently, experiments are in progress to further analyze the intracellular mechanisms that mediate neuronal death and to determine a potential interference with other factors known to be involved in AD.
Fath, T., Eidenmüller, J., Brandt R. (2002). Tau-Mediated Cytotoxicity in a Pseudohyperphosphorylation Model of Alzheimer’s Disease. The Journal of Neuroscience, 22(22):9733–9741.
Shahani, N., Brandt R. (2002). Functions and malfunctions of the tau proteins. Cellular and Molecular Life Sciences (CMLS), 59(10):1668-1680.
Brandt, R. (2001). Cytoskeletal mechanisms of neuronal degeneration. Cell Tissue Res., 305(2):255-65.