Effects of amyloid (1-42) peptide on cortical neuron/glia cultures from parkin null mice. Role of autophagy and glutathione homeostasis

Abstract

The production and aggregation of amyloid beta peptides (A-beta) has been linked to the development and progression of Alzheimer's disease (AD). A-beta 1-42 is increased in patients affected by AD, both in familial and sporadic cases. Parkin mutations impair ubiquitin-proteasome pathways and produce familial Parkinson's disease (PD) in humans. Parkin may play a role in the processing of A-beta 1-42. Transgenic animals were obtained from 129SV/C57BL6 wild type (+/+) or parkin null (-/-) mice (Itier et al. 2003). Neuronal and glial cortical primary cultures were derived from littermate -/- and +/+ embryos obtained from homozygous colonies previously generated by heterozygous parkin -/+ intercross. The genotype was confirmed by PCR analysis of tail tissue and by WB analysis of parkin in the cultures (Casarejos et al. 2005; Solano et al. 2008). Neuronal and glial cortical primary cultures were obtained at 16 days of gestation and grown in Neurobasal medium supplemented with B27, during 7 days. The treatment of mixed neuron/glia cultures with A-beta 1-42, 4.4 uM, for 48 h induced significant cell death by necrosis (trypan blue) and apoptosis (TUNEL). These effects were both more pronounced in WT than in PK-KO neurons. In WT cultures the treatment with A-beta 1-42 reduced both the total number of cells as well as the number of astrocytes, expressed as the area of GFAP+ cells. However, the treatment with A-beta 1-42 increased more the percentage of microglia in PK-KO than in WT cultures. The temporal profile of activation of ERK 1/2 and AKT proteins showed a rapid increase of phospho-proteins with a maximum increase of activation 10 minute after treatment with A-beta 1-42 in PK-KO glial cells. Pre-treatment with an inhibitor of GSH synthesis and the inhibition of autophagy, reverted the increased resistance to A-beta of the PK-KO cultures. Neuron/glia PK-KO cultures are more resistant to A-beta induced toxicity than WT cultures. Supported by: FIS 2010/172, Laín Entralgo NDG07/4, CAM 02/02/2006, and CIBERNed 2006/05/059 and 2010.