Role of PAR in Klk6-mediated astrocyte stellation

To test the hypothesis that PARs play an essential role in Klk6-mediated astrocyte stellation, we utilized a combination of recombinant kallikrein and primary cortical astrocytes derived from wild type mice or those genetically deficient in PAR1 or PAR2. Cortical astrocytes were plated across 12 mm poly-L-lysine (Sigma, St. Louis, MO) coated glass coverslips at a density of 15,000 cells per cm² in serum containing media. After 24 h, media was replaced with defined Neurobasal A media containing 1% N2, 2% B27, 50 U/mL penicillin/streptomycin, 2 mM Glutamax, 1 mM sodium pyruvate, 0.45% glucose, and 50 μM β-mercaptoethanol (Sigma Aldrich, USA). All cells were maintained at 37°C in 95% air and 5% CO₂. Cultures were treated in triplicate with recombinant Klk6 (30, 150 or 300 nM), or vehicle alone, for 24 h prior to fixation with 2% paraformaldehyde. Fixed astrocytes were subsequently permeabilized with 0.1% Triton X-100 and stained to visualize the actin cytoskeleton using Cy3-conjugated Phalloidin (1 U, Life Technologies). Alternatively, β-catenin was localized by immunofluorescence using a combination of a rabbit polyclonal primary antibody (ab16051, Abcam, Cambridge, MA) and an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Labs, West Grove PA). All stained coverslips were slide mounted using Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (Vector Laboratories, Burlingame CA). Five standardized 20X microscopic fields encompassing the poles and center of each coverslip were captured digitally on an Olympus AX70 microscope equipped with SPOT software (Olympus, Center Valley PA). Image J software was used to quantify the extent of astrocyte stellation, membrane or cytosplasmic localization of β-catenin, and nuclei aggregation (Vandell et al. 2008; Scarisbrick et al. 2012b).

Astrocyte stellation was defined as those cells possessing two or more processes at least one cell body in length and expressed as mean fold change compared to vehicle control in each case (Scarisbrick et al. 2012b). Astrocyte process length was measured using Interactive Pathology Laboratory software (BD Biosciences, Franklin Lakes NJ). Cell number in each case was determined by enumeration of DAPI stained nuclei in each microscopic field. The predominant localization β-catenin to the astrocyte plasma membrane or cytoplasm was scored based on immunofluorescence and expressed as fold change over vehicle alone. In each case, cells were treated in triplicate and experiments repeated at least twice. At least 300 cells were scored for each condition without knowledge of the experimental group.