Experimental Approaches for Biochemical Analysis of Glial Fibrillary Acidic Protein and Its Disease-associated Variants

My research focused on GFAP changes in affected astrocyte and how their dysfunction leads to Alexander disease. We share easy to follow methods to study GFAP. Disease related versions of GFAP are harder to study because they do not assemble properly, tend to clump together, and have unusual modifications.

To begin, place glow discharge form bar and carbon coated copper grids in a glow discharge cleaning system. Clean the grids for 45 seconds at 20 milli amperes. Deliver the assembly mixtures onto the glottis charged grid.

Remove excess liquid by wicking the edge of the grid with a piece of blotting paper. Then wash the grids with distilled water. Stain the grid with 20 microliters of 1%urinal acetate for 60 seconds.

Remove the excess staining solution and allow the grid to air dry for 30 seconds. Examine the prepared grids using a transmission electron microscope in high resolution mode at an accelerating voltage of 100 kilovolts. Extract brain tissues from Alexander disease rats, using a deuce homogenizer, containing 10 milliliters of 10 buffer.

Centrifuge the brain homogenates at 76, 000 G at four degrees Celsius for 20 minutes. Then sequentially extract the resulting pellet with 10 milliliters of Triton X-100 buffer, sucrose buffer, high salt buffer, and urea buffer. Collect the supernatant fraction and dialyze it against Q column buffer.

Now load the dialyzed sample onto an anion exchange column in an NGC chromatography system. Elute the bound proteins using a linear gradient of zero to 0.5 molar sodium chloride in Q buffer at a flow rate of one milliliter per minute. Pool the glial fibrillary acidic protein containing fractions and dialyze them against S column buffer.

Now apply the dialyzed sample to a cation exchange column and elute the bound proteins using a linear gradient of zero to one molar sodium chloride in S buffer at a flow rate of one milliliter per minute. Analyze the eluded fractions by SDS page and Coomassie blue staining. Collect those fractions containing purified glial fibrillary acidic protein.

Wild type GFAP formed uniform 10 nanometer filaments in vitro, whereas the R239H mutant produced dense aggregated structures, under low speed centrifugation, most wild type GFAP remained in the supernatant, while the majority of R239H GFAP was found in the pellet fraction. Under high speed centrifugation, nearly all wild type GFAP and R239H GFAP were found in the pellet fractions confirming efficient sedimentation. Following hydrogen peroxide treatment, wild type GFAP formed multiple high molecular weight bands, which were reduced to monomers by DTT.

In the absence of oxidative stress, R239H GFAP formed a high molecular weight band around 180 kilodaltons, which required high concentrations of dithiothreitol to convert to a monomeric form. Quantification confirmed that a higher proportion of R239H GFAP remained in high molecular weight forms compared to wild type GFAP. Immuno blotting confirmed that native GFAP from R237H rat brain was ubiquitinated as shown by overlapping signals for GFAP and ubiquitin.

Electron microscopy revealed that GFAP from Alexander disease rat brain failed to form filaments. In low speed centrifugation, most GFAP from Alexander disease rat brains remained in the supernatant, while under high speed centrifugation, it sedimented into the pellet fraction. Quantification confirmed a shift in GFAP distribution from supernatant to pellet between low speed and high speed centrifugation.

We still lack a clear picture of how mutant GFAP alters filaments, drive aggregation, and impairs cells. Our protocol directly investigate these mechanisms. We have developed a standardized method to purify both normal and mutant GFAP, making it much easier to study the protein and its role in disease.

This protocol streamlines preparing normal and mutant GFAP to clarify how filament assembly, aggregation, and modifications contribute to Alexander disease.