Somarajan Bindu I, Khanday Mudasir A, Mallick Birendra N
School of Life Sciences, Jawaharlal Nehru University , New Delhi , India.
Front Neurol. 2016 Mar 7;7:25. doi: 10.3389/fneur.2016.00025. eCollection 2016.
Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate intrinsic pathway for inducing neuronal apoptosis in REMS-deprived rat brain.
许多神经退行性疾病都与快速眼动睡眠(REMS)缺失有关;然而,其机制尚不清楚。由于REMS缺失会提高大脑中的去甲肾上腺素(NA)水平,并诱导神经元凋亡和变性,在本研究中,我们已经阐明了参与REMS剥夺(REMSD)相关的NA诱导神经元凋亡的细胞内分子途径。通过经典的花盆法对大鼠进行6天的REMS剥夺;设置合适的对照组并评估对凋亡标志物的影响。此外,通过以下两种方式研究了NA的作用:一是腹腔注射(i.p.)NA能α1肾上腺素能受体拮抗剂哌唑嗪(PRZ),二是通过局部微量注射酪氨酸羟化酶小干扰RNA(TH-siRNA)下调蓝斑(LC)神经元中的NA合成。免疫印迹估计显示,促凋亡蛋白的表达,即Bcl2相关死亡促进蛋白、凋亡蛋白酶激活因子-1(Apaf-1)、细胞色素c、半胱天冬酶9、半胱天冬酶3在REMS剥夺的大鼠脑中升高,而半胱天冬酶8水平未受影响;PRZ处理不允许这些促凋亡因子升高。此外,REMSD增加了细胞色素c的表达,如果在自由活动的正常大鼠REMSD期间通过体内向LC微量注射TH-siRNA来阻断LC神经元的NA合成,则可防止这种情况发生。透射电子显微镜再次证实了线粒体损伤,其显示线粒体明显肿胀,嵴解体,染色体浓缩,并沿核膜聚集,而在PRZ处理的大鼠中所有这些变化均被阻止。结合本研究的结果以及早期报告,我们提出,在REMSD时,由于LC中NA能REM-OFF神经元不会停止放电且这些神经元中TH上调,大脑中的NA水平会升高。这种升高的NA作用于α1肾上腺素能受体,损害线粒体,导致细胞色素c释放,从而激活内在途径,诱导REMS剥夺的大鼠脑神经元凋亡。