Deimination as an astrocytic marker following temperature incubation

ALGECIRAS M; SERRA HM; SANJOY K. BHATTACHARYA

Congreso; Neuroscience 2013; 2013

Purpose: To determine whether temperature fluctuation elicits an increase in deimination (conversion of protein bound arginine into citrulline) in astrocytes and whether elevated deimination levels can serve as an astrocytic activation marker. Astrocyte activation will be assessed by measuring the expression of a repertoire of established astrocytic markers, including GFAP. Methods: Isolated brain cortex astrocytes obtained from C57BL/6J mice (about 5000 per plate) were cultured and subjected to different incubation temperatures for one hour, followed by a stabilization period of 23 hours at 37°C. The exposed and control astrocytes were evaluated for deimination levels as well as for levels of other established markers of astrocyte activation using immunohistochemical, Western blot and ELISA analyses. Results: Optimal growth of astrocytes occurred at 37°C. Hypothermia (31°) treatment showed very few loss of cells (2%) as measured by Tunnel assay. However, 33% cell death was found after hyperthermic incubation (41°) for 1 hour. Decreased GFAP, deimination and peptidylarginine deiminase type 2 (PAD2) levels were found in cells subjected to hypothermia. An increase in some astrocyte activation markers such as Aquaporin4, S100β and Thrombospondin, however, was found in astrocytes subjected to hypothermia. The astrocytes subjected to hyperthermia showed an increase in deimination and PAD2 levels. Furthermore their levels were higher (approximately 3-fold) than for the cells subjected to hypothermia. Hyperthermia was accompanied by an increase in some astrocytic markers such as GFAP, aldehyde dehydrogenase 1 family member L1 (ALDH1L1), J131 and isolevuglandin ( ISO[4]LGE2 ). Mass spectrometric analysis revealed some unique proteins associated with each treatment condition. Conclusion: The level of deimination undergoes a shift on either side of optimal temperature incubation (37°C) for astrocytic growth. Level of deimination correlates with astrocyte activation markers when cells are subjected to temperature treatment.