Source: IOWA STATE UNIVERSITY submitted to
ROLE OF ASTROCYTES IN PRPSC-INDUCED NEUROTOXICITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0205235
Grant No.
(N/A)
Project No.
IOWV-JEFTI-411-23-07
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 1, 2005
Project End Date
May 30, 2008
Grant Year
(N/A)
Project Director
Jeftinija, S.
Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
VETERINARY MEDICINE
Non Technical Summary
A Prions are infectious pathogens that differ from bacteria, fungi, parasites, virions, and viruses, both with respect to their structure and with respect to the diseases that they cause. B The mechanism of prions neurotoxicity is still unknown. A Using astrocyte or neuron enriched cortical cultures of normal animal in combination with ratiometric imaging with Fura-2 to detect intracellular calcium changes, we will determine whether PrPsc has an early effect on intracellular calcium levels in cultured neurons and interaction between neuronal and noneuronal cell types. B Develop a better method for purification of PrPsc to be used in the culture experiments.
Animal Health Component
25%
Research Effort Categories
Basic
(N/A)
Applied
25%
Developmental
75%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30339991160100%
Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1160 - Pathology;
Goals / Objectives
Prions are infectious pathogens that differ from bacteria, fungi, parasites, virions, and viruses, both with respect to their structure and with respect to the diseases that they cause. The fatal disease scrapie is a transmissible degenerative encephalopathy resulting from infection by prion. A post-translational process converts the cellular prion protein (PrPc) into an abnormal isoform (PrPsc). It is still unknown whether scrapie pathology comes about by neurotoxicity of PrPsc, acute depletion of PrPc, or some other mechanism. The conversion of PrPc to PrPsc and the accumulation of PrPsc in the astrocytes precede neuronal death in the course of the disease. We propose to focus on the astrocyte's role in the neurotoxicity of PrPsc. Interest in the neurobiology of glutamate has greatly increased stemming from the recognition that glutamate is excitotoxic. We have demonstrated glial release of glutamate induced by a fragment of PrPsc consisting of amino acids 106-126 (PrP106-126). By measuring factors associated with excitotoxicity, calcium increase in glial cells and increase in glutamate release, we propose to study the effect of PrPsc.
Project Methods
Using ratiometric imaging technique we found that PrP106-126 (10microM) 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 482nM (n=79) about 6 min after the onset of PrP106-126 application and was followed by a decrease to a plateau which was about a third of the peak concentration at which it stayed for over 30 min. 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 (10microM 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. We have previously show that astrocytes regulate neuronal calcium levels through the calcium-dependent release of glutamate. PrP106-126, fragments of prion protein, have been used as a model to investigate mechanism of degeneration in prion diseases. The toxicity of fragment PrP106-126 on cultured cortical neurons from embrionic and 6-day-old mice require expression of PrPc. Immunocytochemistry using PrPsc antibody revealed presence of PrPc on cultured rat astrocytes. The release of glutamate from neuron-free cultures of neocortical astrocytes was monitored using high-performance liquid chromatography (HPLC). Addition of PrP106-126 at 10micrM caused greater than three-fold increase in the concentration of glutamate and a greater than two fold increase in aspartate. In calcium-depleted saline containing 0.2mM calcium, PrP106-126 failed to to produce a significant increase in the concentration of extracellular EAAs. While PrP106-126 stimulated the release of glutamate and aspartate from astrocyte cultures it did not significantly affect the release of asparagin. These data demonstrate that PrP106-126 causes the selective release of glutamate and aspartate from cultured astrocytes.

Progress 01/01/05 to 12/31/05

Outputs
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.

Impacts
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.

Publications

  • 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.