The Role of Autophagy Activation and Its Relation with Ubiquitin-Proteasomes System after Experimental Intracerebral Hemorrhage
|Keywords||ICH iron thrombin autophagy polyubiquitinated proteins p62|
ICH is a subtype of stroke with high morbidity and mortality, resulting in neuronal deathand severe neurological deficits. Brain iron overload and thrombin released from clot causebrain edema, cell death and blood brain barrier (BBB) leakage after ICH. Two majorpathways accomplish regulated protein catabolism: the ubiquitin-proteasome system (UPS)and the autophagy-lysosome system (ALS). Polyubiquitin chains target substrates fordegradation by the proteasome. Dysfunction of the UPS by oxidative stress orpolyubiquitinated proteins overload, reversely results in polyubiquitinated proteinsaggregation.Recent investigations suggested that the sequestosome1/p62as a functional linkbetween these two proteolytic systems. It is stress-responsive and plays an important role inthe proteasomal and/or autophagic clearance of misfolded and aggregation-prone proteins.Our previous studies have indicated that autophagy is activated after ICH, and thrombin playsan important role in autophagy activation after ICH. The role of autophagy after ICH is stillunclear.Our present study was divided in three parts, aiming to investigate whether ICH inducesthe accumulation of polyubiquitinated proteins and upregulation of p62, and the role of ironand thrombin in ICH induced accumulation of polyubiquitinated proteins and the upregulationof p62. Furthermore, we illustrated the effect of inhibition or enhancement of thrombininduced autophagy activation in neuronal protection and intracellular proteins accumulation.Part1: The effect of iron in the accumulation of p62and polyubiquitinated proteinsafter experimental intracerebral hemorrhageObjective: We have shown that iron is an important factor in inducing autophagic celldeath in ICH. In this study, we investigated the role of iron in the accumulation ofpolyubiquitinated protein and the sequestosome1/p62(p62), the link of ubiquitin/proteasomeand autophagy in ICH. Materials and Methods: Male, adult Sprague-Dawley rats were usedand experiments divided into three parts. First, rats received an infusion of100μl autologous whole blood into the right basal ganglia and were killed at1,3,7, or14days for electronmicroscopy (EM), immunohistochemistry and Western blotting analysis. Second, rats had aneedle (Sham) or blood injection and treated with vehicle, minocycline. Rats were killed atday-3and the brains were used for immunostaining and immunoblotting. Third, rats had50μlof saline, FeCl2or FeCl2+minocycline injection and were killed at day-3for immunostainingand immunoblotting. Results: ICH induced the protein aggregation in the ipsilateral basalganglia from day-1to-14in EM, mostly at days3and7. The upregulation of p62andpolyubiquitinated proteins was also peaked at days3and7after ICH. ICH mediated muchhigher p62and polyubiquitinated proteins compared with Sham and that was attenuated byboth minocycline and deferoxamine treatments. Intracerebral infusion of iron resulted in p62and polyubiquitinated proteins upregulation and that was blocked by the coinjection ofminocycline. Conclusions: This research, together with our previous study, illustrate iron isan important factor in ICH-induced accumulation of polyubiquitinated proteins andupregulation of p62, a functional link between ubiquitin/proteasome and autophagy pathways.Part2: The effect of thrombin via PAR-1in the accumulation of p62andpolyubiquitinated proteins after experimental intracerebral hemorrhageObjective: Thrombin activates and plays a role in intracerebral hemorrhage (ICH)-induced autophagy activation. In this study, we investigated the role of thrombin and it’sreceptor, protease-activated receptor-1(PAR-1) in p62and ubiquitinated proteinsaccumulation. Materials and Methods: There are three parts in the study. First, rats receivedan infusion of50μl saline or thrombin into right caudate and were euthanized at days1,3and7for western blotting and electron microscopy examination. Second, rats had intracerebralinfusion of a needle (Sham) or100μl blood±5U hirudin and were killed at day-7for westernblotting. Third, male widetype (WT) and PAR-1knockout (PAR-1-/-) mice had0.5Uthrombin injected into right caudate and were killed at day-7for western blotting. Results:Thrombin induced the accumulation of p62and ubiquitinated proteins, and peaked at day-7.The specific thrombin inhibitor, hirudin attenuated ICH-induced p62and ubiquitinated proteins upregulation. Thrombin mediated more LC3-II conversion, p62and ubiquitinatedproteins accumulation in WT than in PAR-1-/-mice. Conclusions: Thrombin via PAR-1induces p62and ubiquitinated proteins accumulation, and autophagy activation after ICH.Part3: The effect of thrombin in autophagy activation and the accumulation of p62andpolyubiquitinated proteins after experimental intracerebral hemorrhageObjective: We investigated the effect of inhibition or enhancement of thrombin inducedautophagy activation in neuronal protection and intracellular proteins accumulation.Materials and Methods: Primary cultured rat neurons were treated with control or humanthrombin (5U/ml) with or without autophagy inhibitor,3-Methyladenine (3-MA) or inducer,rapamycin. Cells were used for western blotting, MDC staining and cell viabilitydetermination (LDH activity measurement and live/dead cells assay). Results:3-MAattenuated autophagy activation, upregulated p62and ubiquitinated proteins, and increasedthe thrombin induced neuronal death. Rapamycin enhanced autophagy activation,down-regulated p62and ubiquitinated proteins, and attenuated neuronal death. Conclusions:The activation of autophagy attenuates the accumulation of p62and ubiquitinated proteins,and the neuronal death mediated by thrombin.