Phosphorylation of GFAP is Associated with Injury in the Neonatal Pig Hypoxic-Ischemic Brain

Abstract

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in the astrocytecytoskeleton that plays an important role in the structure and function of the cell. GFAP can be phosphorylated at six serine (Ser) or threonine (Thr) residues but little is known about the role of GFAP phosphorylation in physiological and pathophysiological states. We have generated antibodies against two phosphorylated GFAP (pGFAP) proteins: p8GFAP, where GFAP is phosphorylated at Ser-8 and p13GFAP, where GFAP is phosphorylated at Ser-13. We examined p8GFAP and p13GFAP expression in the control neonatal pig brain and at 24h and 72h after an hypoxic-ischemic (HI) insult. Immunohistochemistry demonstrated pGFAP expression in astrocytes with an atypical cytoskeletal morphology, even in control brains. Semi-quantitative western blotting revealed that p8GFAP expression was significantly increased at 24h post-insult in HI animals with seizures in frontal, parietal, temporal and occipital cortices. At 72h post-insult, p8GFAP and p13GFAP expression were significantly increased in HI animals with seizures in brain regions that are vulnerable to cellular damage (cortex and basal ganglia), but no changes were observed in brain regions that are relatively spared following an HI insult (brain stem and cerebellum). Increased pGFAP expression was associated with poor neurological outcomes such as abnormal encephalography (EEG) and neurobehaviour, and increased histological brain damage.Phosphorylation of GFAP may play an important role in astrocyte remodelling during development and disease and could potentially contribute to the plasticity of the central nervous system. 3

Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker

“…GFAP protein is highly regulated by protein kinases (such as protein kinase–A, calmodulin dependent kinase II and PK-C), with many phosphorylation sites mapped to the N-terminal domain: T7 (PKA), S8 (PKA, PKC, cdk2), S13 (CAMPKII, PK, PKC), S17 (CAMPKII), S38 (CAMPKII, PKA, PKC) and S289 (CAMPKII) (Figure 2; Table 1) [14,38-41]. One of the key phosphorylation pathways of GFAP appears to involve the G-protein-coupled mGluR receptor leading to calcium influx and CAMPKII activation [40,42].…”

“…In addition, since most of these phosphorylation sites reside at the N-terminal head domain, phosphorylation of GFAP has a negative effect on filament assembly. GFAP phosphorylation is elevated after hypoxic-ischemia in neonatal pig brain [38]. …”

Developmental Changes in Expression of GABA_A Receptor Subunits α₁, α₂, and α₃ in the Pig Brain

“…The pig is an excellent model for human brain growth [18,21,22] and is frequently used in preclinical research models of neonatal hypoxic brain injury and treatment [27][28][29][30][31][32] . The major brain growth spurt of the pig occurs in the late prenatal to postnatal period as in humans, and Fig.…”

Examining the GABA_A receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

“…Problems with control proteins have been demonstrated previously [1,18,19]. A number of solutions have been proposed including total protein stains, stain-free detection, and loading a separate blot with samples at lower µg loads to detect a control protein (such as β-actin) within its linear range [2,20,21].…”

“…Generally normalization using total protein stains is sensitive and inexpensive. However, there are complications: toxicity, difficulty, and time to capture and document, changes to binding due to time of staining, and variability in the consistency of staining across the membrane have all been reported [18,22]. An alternative is stain-free technology that allows monitoring of the quality of separation and transfer efficiency via UV induced fluorescence in the gel.…”