Transmembrane protein 166 regulates autophagic and apoptotic activities following focal cerebral ischemic injury in rats

Abstract

Transmembrane protein 166 (TMEM166) is a lysosomal/endoplasmic reticulum-associated protein found in various species where it acts as a regulator of programmed cell death, mediating both autophagy and apoptosis. In the present study, we investigated the role of TMEM166 following MCAO injury in rats to determine whether the structural damages following injury were orchestrated in part by TMEM166. One hundred and fifty six male Sprague-Dawley rats were randomly divided into 4 groups: Sham, MCAO, MCAO + control siRNA, MCAO + TMEM166 siRNA. Outcomes were measured including mortality rate, brain edema, BBB disruption, and neurobehavioral testing. Western blotting techniques measured the expression of key proautophagic and apoptotic proteins such as TMEM166. Beclin-1, cleaved casepase-3 and Bcl-2/Bax. The study found that TMEM166 siRNA treatment significantly reduced the mortality rate, cerebral edema, neurobehavioral deficits, and BBB disruption as measured by Evan's blue assay following MCAO injury. Immunohistochemical staining and western blotting analysis demonstrated an increased expressions of TMEM166, Beclin-1, LC3, cleaved casepase-3 and Bcl-2/Bax in the infarcted areas. This study suggests that TMEM166 induces autophagy and apoptosis may in fact play a significant role in cell death following MCAO injury and its mediation may be through the crosstalk of Bcl-2. By blocking the activity of TMEM166 using siRNA, we were able to prevent the cell loss that occured following cerebral ischemia injury. This translated into a preservation of functional integrity and an improvement in mortality. (C) 2012 Elsevier Inc. All rights reserved.