Phatak Nitasha R, Stankowska Dorota L, Krishnamoorthy Raghu R
University of North Texas Health Science Center, North Texas Eye Research Institute, Fort Worth, TX.
Mol Vis. 2016 Aug 16;22:1048-61. eCollection 2016.
Brn3b is a class IV POU domain transcription factor that plays an important role in the development of retinal ganglion cells (RGCs), RGC survival, and particularly axon growth and pathfinding. Our previous study demonstrated that recombinant adenoassociated virus serotype 2 (rAAV-2)-mediated overexpression of Brn3b in RGCs promoted neuroprotection in a rodent model of glaucoma. However, the mechanisms underlying neuroprotection of RGCs in rats overexpressing Brn3b in animal models of glaucoma remain largely unknown. The goal of this study was to understand some of the mechanisms underlying the neuroprotection of RGCs overexpressing Brn3b during intraocular pressure (IOP) elevation in Brown Norway rats.
One eye of Brown Norway rats (Rattus norvegicus) was injected with an AAV construct encoding either green fluorescent protein (GFP; recombinant adenoassociated virus-green fluorescent protein, rAAV-hSyn-GFP) or Brn3b (rAAV-hSyn-Brn3b). Expression of antiapoptotic proteins, including B cell lymphoma/leukemia-2 (Bcl-2) family proteins (Bcl-2 and Bcl-xL), and p-AKT, was observed following immunostaining of rat retinas that overexpress Brn3b. In a different set of experiments, intraocular pressure was elevated in one eye of Brown Norway rats, which was followed by intravitreal injection with AAV constructs encoding either GFP (rAAV-CMV-GFP) or Brn3b (rAAV-CMV-Brn3b). Retinal sections were stained for prosurvival factors, including Bcl-2, Bcl-XL, and p-AKT.
AAV-mediated expression of transcription factor Brn3b promoted statistically significant upregulation of the Bcl-2 protein and increased expression of p-AKT in RGCs of Brown Norway rats. In addition, following IOP elevation, AAV-mediated Brn3b expression also statistically significantly increased levels of Bcl-2 in the RGC layer in Brown Norway rats.
Adenoassociated virus-mediated Brn3b protein overexpression may promote neuroprotection by upregulating key antiapoptotic proteins, including Bcl-2, Bcl-xL, and p-AKT, in animal models of glaucoma.
Brn3b是一种IV类POU结构域转录因子，在视网膜神经节细胞（RGCs）的发育、RGCs存活，尤其是轴突生长和路径寻找中发挥重要作用。我们之前的研究表明，重组腺相关病毒2型（rAAV - 2）介导的RGCs中Brn3b的过表达在青光眼啮齿动物模型中促进了神经保护。然而，在青光眼动物模型中过表达Brn3b的大鼠RGCs神经保护的潜在机制仍 largely未知。本研究的目的是了解在棕色挪威大鼠眼压（IOP）升高期间过表达Brn3b的RGCs神经保护的一些潜在机制。
将编码绿色荧光蛋白（GFP；重组腺相关病毒 - 绿色荧光蛋白，rAAV - hSyn - GFP）或Brn3b（rAAV - hSyn - Brn3b）的腺相关病毒构建体注射到棕色挪威大鼠（褐家鼠）的一只眼睛中。在对过表达Brn3b的大鼠视网膜进行免疫染色后，观察抗凋亡蛋白的表达，包括B细胞淋巴瘤/白血病 - 2（Bcl - 2）家族蛋白（Bcl - 2和Bcl - xL）以及p - AKT。在另一组实验中，升高棕色挪威大鼠一只眼睛的眼压，然后玻璃体内注射编码GFP（rAAV - CMV - GFP）或Brn3b（rAAV - CMV - Brn3b）的腺相关病毒构建体。对视网膜切片进行促存活因子染色，包括Bcl - 2、Bcl - XL和p - AKT。
腺相关病毒介导的转录因子Brn3b表达促进了棕色挪威大鼠RGCs中Bcl - 2蛋白的统计学显著上调以及p - AKT表达的增加。此外，在眼压升高后，腺相关病毒介导的Brn3b表达在棕色挪威大鼠的RGC层中也使Bcl - 2水平有统计学显著增加。
在青光眼动物模型中，腺相关病毒介导的Brn3b蛋白过表达可能通过上调关键抗凋亡蛋白，包括Bcl - 2、Bcl - xL和p - AKT来促进神经保护。
