Elsevier

Brain Research

Volume 1424, 18 November 2011, Pages 60-66
Brain Research

Research Report
Thrombin-induced autophagy: A potential role in intracerebral hemorrhage

https://doi.org/10.1016/j.brainres.2011.09.062Get rights and content

Abstract

Autophagy occurs in the brain after intracerebral hemorrhage (ICH) and thrombin contributes to ICH-induced brain injury and cell death. In this study, we investigated whether thrombin may activate autophagy (in vivo and in cultured astrocytes) and its potential role in ICH. Autophagy was examined using electron microscopy, conversion of light chain 3(LC3) from the LC3-I form to LC3-II, cathepsin D Western blotting and monodansylcadaverine (MDC) staining to detect autophagic vacuoles. 3-Methyladenine (3-MA) was used as an autophagy inhibitor. In vivo, we found that intracaudate injection of thrombin increased conversion of LC3-I to LC3-II, cathepsin D levels, and formation of autophagic vacuoles in the ipsilateral basal ganglia. ICH-induced upregulation of LC3-I to LC3-II conversion and cathepsin D levels was reduced by a thrombin inhibitor, hirudin. In cultured astrocytes, thrombin enhanced the conversion of LC3-I to LC3-II and increased MDC-labeled autophagic vacuoles. 3-MA inhibited thrombin-induced autophagic vacuole formation and exacerbated thrombin-induced cell death. These results indicate that thrombin activates autophagy in the brain and that thrombin has a role in ICH-induced autophagy.

Highlights

► Thrombin causes autophagy in brain and cultured astrocytes. ► Hirudin, an inhibitor of thrombin, reduces intracerebral hemorrhage-induced autophagy. ► 3-Methyladenine, an inhibitor of autophagy, attenuates autophagic vacuole accumulation after thrombin exposure. ► 3-Methyladenine aggravates thrombin-induced cell death.

Introduction

Thrombin, a serine protease generated by the cleavage of prothrombin, is an essential component of the coagulation cascade. As such, it is produced in the brain immediately after a cerebral hemorrhage to induce hemostasis. However, thrombin has multiple effects in brain injury. Thus, evidence indicates that thrombin contributes to early brain injury following intracerebral hemorrhage (ICH) and cerebral ischemia (Xi et al., 2003, Xi et al., 2006). In contrast to these early effects, thrombin is also associated with brain recovery after ICH (Yang et al., 2008).

Autophagy is a cellular degradation process in which cellular proteins and organelles are sequestered in double membrane vesicles known as autophagosomes, delivered to lysosomes, and digested by lysosomal hydrolases (Wang and Klionsky, 2003). Autophagy plays an important role in cellular homeostasis, and it is involved in a number of human diseases (Adhami et al., 2006, Komatsu et al., 2006, Rubinsztein et al., 2005). We have demonstrated that autophagy occurs after ICH and iron has a role (Gong et al., 2011, He et al., 2008) and most autophagic brain cells are astrocytes (He et al., 2008). It is known that thrombin and iron are two major factors causing brain injury after ICH (Xi et al., 2006). However, it is unclear whether thrombin also causes autophagic cell death after ICH and whether modifying thrombin-induced autophagy might affect brain injury or recovery after ICH. The purpose of the current study was, therefore, to investigate whether thrombin causes autophagy in brain (in vivo) and astrocytes (in vitro). This was examined using electron microscopy and three markers of autophagy. Light chain 3 is a marker for the detection of autophagosomes. Light chain 3 has two forms: type I (LC3-I) is cytosolic and type II (LC3-II) is membrane-bound. During autophagy, LC3-II is increased by conversion from LC3-I (Kabeya et al., 2000). Cathepsin D is a protein known to mediate autophagy (Araki et al., 2006, Klionsky et al., 2008) and monodanysylcadaverine (MDC) staining is a marker of autophagic vacuoles.

Section snippets

Thrombin increased the conversion of LC3-I to LC3-II and upregulated cathepsin D in brain

The time-course study showed that ratio of LC3-II to LC3-I (Western blotting) in the ipsilateral basal ganglia was increased by thrombin injection (Fig. 1). The conversion of LC3-I to LC3-II in the ipsilateral basal ganglia was significantly higher in the thrombin-treated group at day 1 (ratio: 0.8 ± 0.1 vs. 0.2 ± 0.1 in the saline group, p < 0.01, Fig. 1) or day 3 (ratio: 0.9 ± 0.1 vs. 0.4 ± 0.2 in the saline group, p < 0.05, Fig. 1). Thrombin also induced upregulation of cathepsin D (Fig. 2). The levels

Discussion

In the current study, we found: 1) thrombin causes autophagy in brain and cultured astrocytes; 2) hirudin, an inhibitor of thrombin, reduces ICH-induced autophagy; and 3) 3-MA, an inhibitor of autophagy, reduces MDC-labeled vacuoles accumulation after thrombin exposure and aggravates thrombin-induced cell death. The results suggest that thrombin has a role in autophagy after ICH.

Thrombin is a serine protease and an essential component in the coagulation cascade. It is produced immediately in

Animal preparation and intracerebral infusion

The University of Michigan Committee on the Use and Care of Animals approved the protocols for these studies. Male Sprague–Dawley rats (weighing 275 to 350 g, Charles River Laboratories, Portage, MI, USA) were anesthetized with pentobarbital (45 mg/kg, i.p.). A polyethylene catheter (PE-50) was then inserted into the right femoral artery to monitor arterial blood pressure and blood gasses, and to obtain blood for intracerebral blood infusion. Rectal temperature was maintained at 37.5 ± 0.5 °C using

Acknowledgments

This study was supported by grants , , from the National Institutes of Health (NIH) and 0840016N from American Heart Association (AHA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH and AHA.

References (25)

  • Y. He et al.

    Autophagy after experimental intracerebral hemorrhage

    J. Cereb. Blood Flow Metab.

    (2008)
  • Y. Kabeya et al.

    LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

    EMBO J.

    (2000)
  • Cited by (49)

    • MicroRNA-23b alleviates neuroinflammation and brain injury in intracerebral hemorrhage by targeting inositol polyphosphate multikinase

      2019, International Immunopharmacology
      Citation Excerpt :

      Previous studies have shown that inhibition of neuroinflammation can effectively ameliorate ICH-induced neuronal cell death and alleviate ICH-induced injury, making it a therapeutic strategy for ICH [10–14]. Recently, studies have demonstrated that autophagy, a process of cell self-eating for energy recycling, is implicated in the process of ICH [15,16], and inhibition of autophagy can perform a protective role in ICH, which can relieve neuroinflammation and neuronal death [17–19]. However, molecular mechanisms underlying the regulation between autophagy and neuroinflammation of ICH remain to be identified.

    • Astrocytic thrombin-evoked VEGF release is dependent on p44/42 MAPKs and PAR1

      2019, Biochemical and Biophysical Research Communications
    • Autophagy and stroke

      2018, Autophagy and Cardiometabolic Diseases: From Molecular Mechanisms to Translational Medicine
    View all citing articles on Scopus
    View full text