Characterization of neuronal dystrophy induced by fibrillar amyloid β: implications for Alzheimer’s disease
Section snippets
Primary neuronal cultures
Rat cortical cultures were established from embryonic day 17 fetuses as described (Busciglio et al., 1993). The cells were plated on poly-L-lysine-coated dishes or glass coverslips at a density of 10 000 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% horse serum for 2 h. The medium was then replaced with DMEM plus a 1:1 mixture of N2 and B27 supplements (Gibco BRL).
Aβ treatment
Peptide treatments were performed as previously described (Busciglio et al., 1995, Pigino et al., 2001). In brief,
Characterization of Aβ-induced neuronal dystrophy
Cortical neurons were treated with 20 μM fibrillar Aβ from days 5 to 9 in culture and the development of neuronal dystrophy was analyzed starting at day 1 after the initiation of the treatment. The cultures were fixed at the indicated time points, immunostained with an antibody against the neuronal-specific protein β-tubulin class III, and analyzed by immunofluorescence microscopy. Neuronal cells were placed in three categories according to the degree of dystrophy: non-dystrophic, mildly
Discussion
The characterization of the cellular and molecular mechanisms leading to neuronal dysfunction in AD is essential for the development of effective therapies. These experiments demonstrate that treatment of cortical neurons with low micromolar concentrations of fibrillar Aβ induces neuritic dystrophy without affecting neuronal viability. At higher Aβ concentrations, close to 80% of the neurons in the culture exhibit dystrophic features after 4 days of incubation with fibrillar Aβ (Fig. 3). The
Acknowledgements
Supported by grants from the University of Connecticut Health Center, the Alzheimer’s Association and NIH (HD38466) to J.B.
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