The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures

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Abstract

A characteristic feature of neuritic plaques in Alzheimer's disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and β-amyloid deposition. To explore the role of astrocytes in in vitro β-amyloid neurotoxicity, we studied the effect of β-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes. We evaluated two characteristic aspects of in vitro β-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree. We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component. Our results support the hypothesis that β-amyloid–astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimer's disease.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder consisting of a progressive cognitive decline, due to neuronal loss or impairment. It is characterized by neuropathological features consisting of extracellular β-amyloid (Aβ) protein deposits, associated with distrophic neurites, degenerating and apoptotic neurons showing filamentous aggregates of hyperphosphorylated tau protein (neurofibrillary tangles), as well as reactive astrocytes and microglia. However, the role of glial activation in the neurodegenerative progression in AD is not clear.

In vitro studies have clearly shown that Aβ is toxic to neurons and induces apoptosis [4]. Astrocytes could contribute to neurodegeneration, since Aβ-activated astrocytes overexpress factors such as interleukin 1β, nitric oxide, and S100β [3], [6], [9], which are all potentially neurotoxic to neurons. However, the actual role of astrocyte-derived factors in Aβ neurotoxicity is still to be clarified. Recently, our group demonstrated that Aβ-treated astrocytes can induce neuronal apoptosis, suggesting a contribution of astrocytes to Aβ-neurotoxicity [8]. In line with this observation, we intended to further explore the contribution of astrocytes to in vitro Aβ neurotoxicity. For this purpose, we compared the effects of Aβ treatment in two different in vitro models obtained from rat embryos: pure hippocampal neuronal cultures and mixed cultures of hippocampal neurons seeded on a layer of astrocytes. We found that in the presence of astrocytes, Aβ is more effective in decreasing neuronal viability and damaging the neuritic tree.

Section snippets

Materials and methods

Pure neuronal cultures were obtained from the hippocampi of Wistar rat embryos at day 18 (Charles River, Germany), as already described [8]. Briefly, cells were dissociated and plated on poly-l-lysine-coated plastic coverslips in Minimum Essential Medium containing 10% fetal calf serum (MEM/FCS; Gibco, Burlington, Ontario); after 2 h, the medium was replaced with Neurobasal Medium supplemented with B27 (NBM/B27; Gibco); at 1 day in vitro (DIV), 5 μM arabinosylcytosine was added to prevent glial

Results

The treatment with Aβ induced a cytotoxic effect both in mixed and in pure neuronal cultures, as assessed by MTT assay. However, while pure neuronal cultures showed a significant cell loss only at the highest concentration (30 μM) and were insensitive to the middle (3 μM) and low (0.3 μM) concentrations, mixed cultures showed a toxic effect in a dose-dependent manner, which was significant down to the lowest concentration (Fig. 1). This observation could suggest that in the presence of

Discussion and conclusion

The toxicity of amyloid peptides to neurons in culture has been thoroughly described and is one of the main arguments supporting the pathogenetic role of Aβ in the development of AD [1], [12]. Recently, the possible contribution of other cell types, such as glial cells, has been the object of considerable interest [11]. Here we have investigated if astrocytes have a role in Aβ neurotoxicity by comparing Aβ-induced toxicity in pure neuronal and in mixed astrocytic/neuronal cultures. As

Acknowledgements

The work was partly supported by the Italian Ministry of Health (1% funds).

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