Abstracts

Rationale: Traumatic brain injury (TBI) accounts for 20% of symptomatic epilepsy in the general population, and 50% in the military. TBI compromises of the blood-brain barrier (BBB), which normally protects the brain from neurotoxic constituents in the blood. We recently identified a novel pathway whereby extravasation of serum albumin into the brain microenvironment following local BBB breakdown activates a TGF-?R mediated signaling cascade and induces albumin uptake in astrocytes. While albumin uptake by astrocytes coincides with activation of the TGF- ?R pathway, and precedes downstream neural hyperexcitability, the mechanism of albumin uptake and the role it plays in epileptogenesis are unknown. We therefore explored mechanisms of uptake in the current project. Methods: Primary hippocampal astrocytic and neuronal cultures were used to quantify albumin uptake in different cell types of the CNS. We developed a quantitative uptake assay using fluorescently conjugated bovine serum albumin, and measured uptake by normalizing fluorescence intensity by total protein content. Cellular compartments were separated with a cytoplasmic and nuclear fractionation kit, and albumin uptake was measured using our fluorescence-based assay as well as western blots. We pharmacologically blocked caveolin-mediated endocytosis using methyl-?-cyclodextrin (MBCD, 30 mM), and TGF-?R signaling with TGF-?R II kinase blocker SB431542 (SB, 50 M).Results: Astrocytes begin taking up albumin by 10 minutes, and our quantitative uptake assay revealed S-shaped uptake kinetics between 10 minutes and 4 hours. In comparison, uptake by neurons did not increase over time, and by 30 minutes there was significantly greater uptake of albumin by astrocytes than neurons (in g albumin/ mg protein; astrocytes: 0.24, n = 3; neurons: 0.08, n = 3; p = 0.007). By 10 minutes, albumin could be observed in the nuclear fraction of astrocytes using both the quantitative uptake assay and western blots. Finally, we determined that albumin uptake is partially dependent on both caveolin-mediated endocytosis and TGF-?R kinase activity. Pharmacological blockade of caveolae with MBCD significantly reduced albumin uptake in astrocytes by ~40 % (in g albumin/ mg protein; astrocytes: 0.49, n = 3; + MBCD: 0.30, n = 3; p = 0.015). Pharmacological blockade of TGF-?R kinase activity reduced albumin uptake in astrocytes by ~50% (in g albumin/ mg protein; astrocytes: 0.52, n = 3; +SB: 0.26, n = 3; p = 0.004).Conclusions: We showed that albumin is selectively taken up by astrocytes, and not by neurons, and that uptake kinetics suggest receptor-mediated endocytosis. Once albumin is taken up by astrocytes, it quickly translocates to the nucleus, indicating possible transcriptional effects. Finally, albumin uptake is dependent on both caveolin-mediated endocytosis and TGF-?R signaling, since pharmacological blockade of these processes reduced uptake. Our research supports a role for astrocytic albumin endocytosis in the development of epilepsy.