Characterization of hippocampal slice cultures as model systems for neurodegenerative processes in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is the most common cause of dementia and a growing health care problem worldwide. The disease affects different brain regions, including hippocampus, enthorhinal cortex, amygdala, neocortex and certain basal forebrain nuclei. Macroscopically, shrinkage of gyri and widening of sulci characterize the AD brain. Histopathological changes consist of deposition of extracellular senile plaques, composed of the amyloid-beta peptide (Abeta), and intracellular neurofibrillary tangles (NFTs), composed of hyperphosphorylated forms of the microtubule-binding protein tau, concomitant with a progressive loss of neurons. Inflammatory processes are believed to contribute to the pathophysiology of AD and may play an important role in the disease progression. One of the cytokines upregulated in AD is interleukin 1 (IL-1). IL-1 seems to be overexpressed early in the plaque formation process, possibly by activated microglia.

Organotypic slice cultures of the roller-drum and membrane techniques were applied to develop model systems of AD. The major advantages with these cultures are the well-preserved phenotypic cellular and structural organization as well as possibilities for long-term culturing. The following studies were performed using organotypic hippocampal cultures to characterize key pathophysiological components of AD: In study 1 roller-drum cultures were used for evaluating the effect of Abeta25-35 on different nerve cell populations and glial cells. AP25-35 gave rise to fibrillar aggregates, a time- and concentrationdependent reduction in the number of NMDA-R1 immunolabeled pyramidal cells and damaged GAD65 immunopositive interneurons. Also, neurons immunopositive for phosphorylated tau and GFAP-labeled astrocytes were affected, while GABAimmunopositive interneurons and glial cells seemed unaffected. In study 2 roller-drum cultures were used to investigate mechanisms involved in neuronal cell death induced by IL-1beta. IL-1beta induced a concentrationdependent loss of NMDA-R1 immunoreactive pyramidal neurons, which could be inhibited by trolox and MK-801. These findings suggest the involvement of free radicals and NMDA receptor-mediated processes in IL-1beta-induced neurodegeneration. In study 3 the neurodegenerative effect of lipopolysaccharide (LPS) in roller-drum cultures was investigated. The LPS-exposure caused an increased density of CD11b positive cells, indicating the occurrence of reactive microgliosis. A rapid loss of both NMDA-R1 and GABAimmunoreactive cells was induced by the LPS exposure. Concomitant with the pyramidal cell loss an increased number of p53/NMDA-R1 and NMDA-R1/TUNEL double-labeled cells were found, suggesting that apoptotic events were involved in the neurodegeneration. In study 4 the relation between Abeta and tau phosphorylation was characterized in a membrane organotypic culture system. Western blotting analysis showed that exposure to Abeta25-35, increased the degree of tau phosphorylation at Tau [pS396] and [pS199] epitopes. Also protein levels of active GSK3beta [py216] were increased, while Cdk5 and p35 did not change significantly.

In this thesis different components of the amyloid cascade hypothesis were characterized in phenotypically advanced model systems. Links between Abeta neurotoxicity, tau phosphorylation and alterations in activated kinases were established (study 1 and 4) as well as a role of inflammatory processes in neurodegeneration, using IL-1beta (study 2) and LPS (study 3) as inducers.

The developed models may serve as a useful platform for detailed characterization of key components AD pathophysiology and their interdependence in the disease process. Additional opportunities include the benefits of organotypic cultures as a crucial step between simple in vitro assays and in vivo models in the evaluation of disease modifying therapies for AD.