A Mouse Model with Ablated Asparaginase and Isoaspartyl Peptidase 1 (Asrgl1) Develops Early Onset Retinal Degeneration (RD) Recapitulating the Human Phenotype.
A Mouse Model with Ablated Asparaginase and Isoaspartyl Peptidase 1 (Asrgl1) Develops Early Onset Retinal Degeneration (RD) Recapitulating the Human Phenotype.
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我们先前通过全外显子组分析鉴定了人ASRGL 1（hASRGL 1）中的纯合G178 R突变，该突变导致锥-杆营养不良患者的早发性视网膜变性（RD）。G178 R ASRGL 1在Cos-7细胞中的表达改变了ASRGL 1的定位，而在E.大肠杆菌缺乏产生活性蛋白所需的自催化活性。为了评估受损的ASRGL 1功能在体内对视网膜的影响，我们通过CRISPR/Cas9方法产生了具有c.578_579insAGAAA（NM_001083926.2）突变（Asrgllmut/mut）的小鼠模型。ASGRL 1在Asrgl 1 mut/mut小鼠视网膜中的表达及其天冬酰胺酶活性均未检测到。Asrgl 1 mut/mut小鼠的眼科评价显示，与年龄匹配的野生型小鼠相比，在3个月龄早期观察到暗视视网膜电图（ERG）反应显著且进行性降低，随后在5个月龄左右观察到明视反应降低。与视杆细胞和视锥细胞标记物的免疫染色和RT-PCR分析显示，与年龄匹配的野生型小鼠相比，Asrgl 1 mut/mut小鼠在3个月时视锥细胞外节丢失，视紫红质、Opn 1 sw和Opn 1 mw的表达显著降低。重要的是，Asrgl 1 mut/mut小鼠的视网膜表型与在ASRGL 1中携带G178 R突变的患者中观察到的表型一致，证实了ASRGL 1在视网膜中的关键作用以及ASRGL 1突变在视网膜变性中的贡献。
We previously identified a homozygous G178R mutation in human ASRGL1 (hASRGL1) through whole-exome analysis responsible for early onset retinal degeneration (RD) in patients with cone–rod dystrophy. The mutant G178R ASRGL1 expressed in Cos-7 cells showed altered localization, while the mutant ASRGL1 in E. coli lacked the autocatalytic activity needed to generate the active protein. To evaluate the effect of impaired ASRGL1 function on the retina in vivo, we generated a mouse model with c.578_579insAGAAA (NM_001083926.2) mutation (Asrgl1mut/mut) through the CRISPR/Cas9 methodology. The expression of ASGRL1 and its asparaginase activity were undetectable in the retina of Asrgl1mut/mut mice. The ophthalmic evaluation of Asrgl1mut/mut mice showed a significant and progressive decrease in scotopic electroretinographic (ERG) response observed at an early age of 3 months followed by a decrease in photopic response around 5 months compared with age-matched wildtype mice. Immunostaining and RT-PCR analyses with rod and cone cell markers revealed a loss of cone outer segments and a significant decrease in the expression of Rhodopsin, Opn1sw, and Opn1mw at 3 months in Asrgl1mut/mut mice compared with age-matched wildtype mice. Importantly, the retinal phenotype of Asrgl1mut/mut mice is consistent with the phenotype observed in patients harboring the G178R mutation in ASRGL1 confirming a critical role of ASRGL1 in the retina and the contribution of ASRGL1 mutations in retinal degeneration.
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