Multi-omics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6b rd1/rd1 mouse model of retinal degeneration

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multi-omics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial over-activation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration. We generated rd1-GFP mice by mating the Pde6brd1/rd1 mice to TgNrlp-EGFP (WT) (Akimoto et al., 2006) mice, used flow cytometry to purify GFP+ rods at P2, P4, P6, P8 and P10 and performed RNA-seq analysis. Isolated cells were lysis with TRIzol LS and the total RNA were isolated. RNAseq data were generated using TruSeq Stranded mRNA Sample Prep Kit (Illumina) and 125 base pair-end reads were generated on HiSeq 2500 platform (Illumina).视网膜疾病具有广泛的遗传异质性与复杂的病因学特征，其起病时间与严重程度各不相同。在视网膜退行性病变中，超过200个基因的突变均可导致光感受器（photoreceptor）功能异常或细胞死亡。为阐明启动或驱动细胞死亡的分子通路，我们采用时序多组学策略，在一种广泛应用的Pde6b^rd1/rd1（rd1）小鼠——一种由PDE6B突变引发的常染色体隐性遗传性色素性视网膜炎（retinitis pigmentosa）模型——的病变发生前，对新生及发育阶段光感受器的分子与细胞事件开展了检测。对rd1小鼠视网膜内新生及发育阶段视杆细胞的转录组分析（transcriptome profiling）显示，合成代谢通路与能量代谢相关基因出现早期下调。对rd1小鼠视网膜的定量蛋白质组学分析显示，钙信号通路与氧化磷酸化过程发生早期改变，具体表现为复合物I电子传递出现特定的部分旁路，该变化早于细胞死亡的发生。与此同时，我们检测到中枢碳代谢出现异常，包括糖酵解、磷酸戊糖途径与嘌呤生物合成相关组分的失调。体外氧消耗实验与透射电子显微镜（transmission electron microscopy）验证了rd1视杆细胞早期且进行性的线粒体应激，以及线粒体结构与功能异常。上述数据揭示线粒体过度激活及相关代谢改变是早期病理的决定性因素，并提示异常钙信号可加剧线粒体应激。因此，本研究为视网膜退行性病变中光感受器细胞死亡的早期驱动因素——线粒体损伤与代谢紊乱——提供了一套机制性框架。我们通过将Pde6b^rd1/rd1小鼠与TgNrlp-EGFP（野生型(WT)，Akimoto等，2006）小鼠杂交，构建了rd1-GFP小鼠；利用流式细胞术在P2、P4、P6、P8及P10时期分选GFP阳性的视杆细胞，并进行RNA测序（RNA-seq）分析。分离得到的细胞经TRIzol LS试剂裂解以提取总RNA。测序文库采用TruSeq Stranded mRNA Sample Prep Kit（Illumina）构建，测序数据在HiSeq 2500平台（Illumina）上以125碱基对双端读长模式生成。