Performing Department
VETERINARY MEDICINE
Non Technical Summary
A. Transmissible spongiform encephalopathies (TSEs) form a group of fatal neurodegenerative diseases, also called prion diseases, that cause typical reactive astrocytosis. B. The conversion of PrPc to PrPsc and the accumulation of PrPsc in the astrocytes precede neuronal death in the course of the disease. C. It is unknown whether TSE pathology comes about by neurotoxicity of PrPsc, acute depletion of PrPc , or some other mechanism. The purpose of thei study is to investigate the connection between changes in intracellular calcium and changes in mitochondrial function in PrP106-126 neurotoxicity.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
We have found recently in neurons and astrocytes grown in coculture and in cultures of cortical astrocytes that PrP106-126 causes a delayed rise in intracellular free calcium [Ca2+]i in astrocytes but not in neurons. PrP106-126, a short synthetic peptide derived from PrP, is generally accepted as peptide model for studying the neurotoxicity of PrP protein and provides a useful bridge between in vitro and in vivo systems. Although the exact pathogenic mechanism of PrPsc remains uncertain, it appears that oxidative stress events are involved in TSE pathogenesis leading to the hypothesis that TSEs belong, together with other degenerative disorders, to oxidative stress related diseases Therefore, we propose to investigate the connection between changes in [Ca2+]i and changes in mitochondrial function in PrP106-126 neurotoxicity. The combined effect of high [Ca2+]i with oxidative stress may damage mitochondrial function and has been implicated as a pathophysiological
mechanism in many systems. We will be able to determine whether glial cells play a significant role in PrP106-126 neurotoxicity, and, it may become possible to identify a site of action for drugs to be used in the treatment of prion diseases.
Project Methods
We will use calcium imaging to determine the effect of brief perfusion application of PrP106-126 to cultured astrocytes and neurons. This will include determining dose dependency of the response(s), receptor type involved, extracellular calcium-dependency, and second messenger system contributing. Three separate group of experiments will be performed in order to identify the cell type primarily targeted by the peptide. Group 1: experiments will be done on pure astrocyte cultures. The purity of astrocyte culture will be judged by the GFAP immunostaining. Group 2: experiments will be done on neuronal cultures. Enriched neuron cultures will be obtained following the procedure previously described in methodology section. The purity of the neuronal cultures will be judged by the immunocytochemical analysis using anti-MAP-2 antibody, a specific marker for neurons. Group 3 experiments will be done on mixed glia-neuron cultures. Existance of the neuronal and glial cells in the
culture will be determined by immunocytochemistry of specific neuronal MAP-2 and glial GFAP proteins. Viability of cells will be determined by MTT and nuclear labelling assay. Ethidium bromide and acridine orange are fluorescent intercalating DNA dyes. Acridine orange stains all nuclei green. Ethidium bromide stains nuclei red but is excluded by cells with an intact cell membrane. Double staining with ethidium bromide and acridine orange allows differentiation of live, apoptotic and necrotic cell as previously described. Survival of hippocampal or cortical neurons and astrocytes will be monitored using MTT assay as a measure of cell viability. MTT stock solution will be added to each culture, one tenth of the original culture volume and incubated for 3-4 hours. After incubation medium will be removed and converted dye will be solubilized with acidic isopropanol. Apoptosis is distinguished from necrosis by characteristic morphological and biochemical changes, like: loss of plasma
membrane asymmetry, decrease in mitochondrial membrane potential, DNA fragmentation and chromatin condensation. Changes in a cell membrane permeability will be detected with Vybrant Apoptosis Assay Kit. Recombinant anexin V conjugated with fluorescein isothiocyanate will be added for 30 min. After washing with binding buffer, propidium iodide will be added and the cells will be examined by fluorescence microscopy using filters for FITC detection excitation: 496 nm, emission: 516 nm. PI is the red-fluorescent nucleic dye for necrotic cells. To determine the loss of mitochondrial membrane potential during apoptosis, after treatment with test compounds, cells will be rinsed several times in PBS buffer and then incubated with fluorescent dye for 30 min. Active mitochondria will be stained with Mito Tracker Red dye. Quantification of the data will be done by flow cytometry using BD FACScan and Cell Quest software.