基因治疗与线粒体热休克蛋白 70 抑制实验性自身免疫性脑脊髓炎的视力丧失和视神经萎缩。
Gene therapy with mitochondrial heat shock protein 70 suppresses visual loss and optic atrophy in experimental autoimmune encephalomyelitis.
机构信息
Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami, Miller School of Medicine, Miami, Florida, United States.
Department of Ophthalmology, University of Florida, Gainesville, Florida, United States.
出版信息
Invest Ophthalmol Vis Sci. 2014 Jul 11;55(8):5214-26. doi: 10.1167/iovs.14-14688.
PURPOSE
To rescue visual loss and optic neuropathy in experimental autoimmune encephalomyelitis (EAE).
METHODS
Encephalomyelitis was induced in mice that received intravitreal injections of AAV2-mtHSP70Flag or AAV2-Cox8-mCherry. Additional mice were injected with AAV2-Cox8-mCherry, but not sensitized for EAE. Visual function was assessed by pattern electroretinograms (PERG) at 1, 3, and 6 months post injection (MPI). Optical coherence tomography (OCT) evaluated the thickness of the inner plexiform layer + nerve fiber layers at 1, 3, and 6 MPI. Retinas and optic nerves (ONs) of mice euthanized 6 MPI were processed for light and electron microscopy. Expression of mtHSP70Flag in the retina and ONs was evaluated by RT-PCR, immunofluorescence, and Western blotting. The activities of respiratory complexes I and III, as well as mitochondrial protein import were quantitated.
RESULTS
Expression: immunofluorescence revealed punctate and perinuclear expression of mtHSP70Flag that colocalized with mitochondrial porin in thy1.2 labeled retinal ganglion cells (RGCs). Immunoblotting and RT-PCR confirmed mtHSP70Flag expression in the retina and ON. Rescue: treatment with mtHSP70Flag resulted in a 44% increase in PERG amplitude and less delays in latency relative to the EAE-mCherry group that also showed progressive inner retinal thinning. At 6 MPI, the almost 50% loss of RGCs and optic nerve axons in EAE mice was suppressed by mtHSP70Flag. In addition, retinas of EAE-mtHSP70Flag mice showed nearly complete rescue of complex I and III activities that was reduced by one-third in the EAE-mCherry retinas. Lastly, reductions in import of COX8-mCherry into mitochondria of mice sensitized for EAE improved by 30% with mtHSP70Flag gene therapy.
CONCLUSIONS
Mitochondrial HSP70 ameliorates mitochondrial dysfunction that culminates in irreversible visual loss and atrophy of the optic nerve in EAE suggesting that it may be useful to prevent irreversible disability in patients with optic neuritis and multiple sclerosis (MS).
目的
挽救实验性自身免疫性脑脊髓炎（EAE）中的视力丧失和视神经病变。
方法
通过玻璃体内注射 AAV2-mtHSP70Flag 或 AAV2-Cox8-mCherry 诱导脑脊髓炎。另外一些小鼠注射了 AAV2-Cox8-mCherry，但未进行 EAE 敏化。在注射后 1、3 和 6 个月（MPI）通过图形视网膜电图（PERG）评估视觉功能。光学相干断层扫描（OCT）评估 1、3 和 6 MPI 时内丛状层+神经纤维层的厚度。在注射后 6 MPI 处死的小鼠的视网膜和视神经（ON）进行光镜和电镜检查。通过 RT-PCR、免疫荧光和 Western blot 评估 mtHSP70Flag 在视网膜和 ON 中的表达。量化呼吸复合物 I 和 III 的活性以及线粒体蛋白的导入。
结果
表达：免疫荧光显示 mtHSP70Flag 呈点状和核周表达，与 th1.2 标记的视网膜神经节细胞（RGC）中的线粒体孔蛋白共定位。免疫印迹和 RT-PCR 证实了视网膜和 ON 中 mtHSP70Flag 的表达。挽救：与 EAE-mCherry 组相比，mtHSP70Flag 治疗导致 PERG 幅度增加 44%，潜伏期延迟减少，EAE-mCherry 组也显示内视网膜逐渐变薄。在 6 MPI 时，EAE 小鼠中几乎 50%的 RGC 和视神经轴突丢失被 mtHSP70Flag 抑制。此外，EAE-mtHSP70Flag 小鼠的视网膜显示出对复合物 I 和 III 活性的几乎完全挽救，而 EAE-mCherry 视网膜的活性减少了三分之一。最后，EAE 敏化小鼠中 COX8-mCherry 导入线粒体的减少通过 mtHSP70Flag 基因治疗改善了 30%。
结论
线粒体 HSP70 改善了线粒体功能障碍，最终导致 EAE 中的不可逆视力丧失和视神经萎缩，表明它可能对预防视神经炎和多发性硬化症（MS）患者的不可逆残疾有用。
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