Progress 01/01/05 to 12/31/05
PrP106-126 stimulates cultured astrocytes: The level of astrocyte activation was determined by increase in intracellular calcium. To ask whether internal calcium might play a role in PrP106-126-induced glutamate release from astrocytes, we used ratiometric imaging technique to monitored internal astrocyte calcium ([Ca2+]i). Cells were loaded with the membrane permeant calcium indicator Fura-2AM. In resting conditions, the cytoplasmic calcium level of astrocyte was 102 plus/minus 9nM (n=105) PrP106-126 (10 microM) reliably raised the cytoplasmic level of free calcium in 75% of cells tested (n=79 of 105). This increase of intracellular calcium reached a peak level of 482 plus/minus 48nM (n=79) about 6 min after the onset of PrP106-126 application and was followed by a decrease to a platoau which was about a third of the peak concentration at wich it stayed for over 30 min. PrP106-126 did not cause significant change in intracellular calcium when calcium was removed from
the external saline (p > 0.1, paired t-test) . The release of glutamate from neuron-free cultures of neocortical astrocytes was monitored using high-performance liquid chromatography (HPLC). The basal release of of glutamate and aspartate into superfusate produced levels of 26 plus/minus 2nM (mean plus/minus sem, n=12) and 12 plus/minus 2 nM, respectively. Addition of PrP106-126 at 10 microM caused greater than three-fold increase in the concentration of glutamate to 76 plus/minus 8nM and a greater than two fold increase in aspartate to 34 plus/minus 4nM (n=6, p <0.02, paired t-test). To investigate whether extracellular calcium might play a role in PrP106-126-induced release of EAAs from cultured astrocytes, cells were bathed in calcium-depleated saline containing 0.2mM calcium with the addition of 1mM EGTA to yeald an estimated free extracellular calcium level of 26nM. We found that it was necessary to have some calcium in the bathing medium, otherwise cells detached from the
culture substrate. In this calcium-depleated saline, PrP106-126 failed to to produce a significant increase in the concentration of extracellular EAAs (p>0.05, n=4, Student's t test). While PrP106-126 stimulated the release of glutamate and aspartate from astrocyte cultures it did not significantly affect the release of asparagine. The basal level of asparagin was 12 plus/minus 2nM compared to 14 plus/minus 4 nM in the presence of PrP106-126 (n=6, p>0.5). These data demonstrate that PrP106-126 causes the selective release of glutamate and aspartate from cultured astrocytes. PrP106-126 potentiates stimulatory effect of ATP on cultured astrocytes Previously we have demonstrated that ATP elevates intracellular calcium levels of astrocytes which evokes the calcium-dependent release of excitatory amino acids glutamate and aspartate. In order to investigate possible potentiation by PrP106-126 of response of glial cells to ATP, we exposed cultures to PrP106-126 (10 microM for 5 min) 10 to 20
min prior to application of ATP. Responses of PrP pretreated astrocytes to ATP were potentiated. Our data suggest that PrP106-126 neurotoxicity in culture may be mediated by glutamate released from astrocytes.
The conversion of PrPc to PrPsc and the accumulation of PrPSC in the astrocytes precede neuronal death in the course of the disease. These observations suggest a role for astrocytes in pathogenesis of prion diseases. We propose to focus on the role of astrocyte in the neurotoxicity of PrPSC. The advances in understandings of fundamental biology of prion diseases may open the possibilities for the prevention and treatment of these unusual diseases and also suggest applications in more common neurodegenerative disorders.
- Lee JS, Mayes MS, Stromer MH, Scanes CG, Jeftinija S, Anderson LL. 2004. Number of secretory vesicles in growth hormone cells of the pituitary remains unchanged after secretion. Exp Biol Med (Maywood). 229:632-639.
- Anderson, L.L., S. Jeftinija, and C. Scanes 2004., Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches. Exp Biol Med (Maywood). 2004 229:291-302.