HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice. HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice

Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of accurate and color vision and ultimately blindness. In RP, a vast number of mutations are affecting the structure and function of rod photoreceptors while cones remain mutation-free. Once majority of rods have degenerated cones are dying secondarily due to the increased oxidative stress, inflammation and loss of structural and nutritional support normally provided by rods. Here we demonstrated that secondary cone cell death in animal models for RP is governed by an increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at a late stage of the disease, when majority of rods have already degenerated, is sufficient to delay cone death and support long-term cone survival. Surviving cones are retaining functionality and are mediating light-driven ganglion cell responses. RNA-seq analysis of surviving cones demonstrated that HDAC inhibition affords multi-level protection trough regulation of different prosurvival pathways including MAPK, PI3K-AKT and autophagy. These study suggest a unique possibility for targeted pharmacological protection of both primary degenerating rods and mutation-free secondary dying cones and creates hope to maintain vision in RP patients independent of the disease stage. Overall design: 2000 to 5000 frozen sorted-cells were lysed in approximatively 10µl of lysis buffer and cDNA synthesis was performed using the SMART-Seq v4 Ultra Low Input RNA Kit (catalog 634888, Takara Bio). First-strand cDNA synthesis was performed using 20 to 50% of the input and was followed by full-length double-strand cDNA amplification using 17 PCR cycles. Quality of the resulting cDNA was validated using Bioanalyzer and High Sensitivity DNA Kit (Agilent) as well as Qubit dsDNA HS fluorometric quantification (ThermoFisher Scientific). NGS libraries were prepared using 150 pg of cDNA input and the Nextera XT DNA Library Preparation Kit (catalog FC-131-1024, Illumina) with 11 cycles of PCR.

遗传性视网膜疾病（如色素性视网膜炎（Retinitis Pigmentosa, RP））引发的视锥细胞（cone photoreceptor）死亡，会导致精确视觉与色觉丧失，最终造成失明。在色素性视网膜炎中，大量突变会损害视杆细胞（rod photoreceptor）的结构与功能，而视锥细胞并无此类突变。当大部分视杆细胞发生退行性病变后，视锥细胞会因氧化应激加剧、炎症反应以及视杆细胞原本提供的结构与营养支持缺失而继发性死亡。本研究证实，色素性视网膜炎动物模型中的继发性视锥细胞死亡，由组蛋白去乙酰化酶（histone deacetylases, HDACs）活性升高所调控。在疾病晚期，即大部分视杆细胞已发生退行性变时，单次玻璃体内注射（intravitreal injection）组蛋白去乙酰化酶抑制剂，即可有效延缓视锥细胞死亡并维持其长期存活。存活的视锥细胞仍保留功能，并可介导光驱动的神经节细胞反应。对存活视锥细胞的RNA测序（RNA-seq）分析显示，组蛋白去乙酰化酶抑制剂可通过调控丝裂原活化蛋白激酶（MAPK）、PI3K-AKT及自噬等多种促存活通路，实现多层次的保护作用。本研究提示，可针对原发性退行性变的视杆细胞与无突变的继发性死亡视锥细胞进行靶向药物保护，为维持色素性视网膜炎患者的视力带来了希望，且该方案不受疾病分期限制。

整体实验设计：将2000至5000个经冷冻分选的细胞置于约10μl裂解缓冲液中裂解，使用SMART-Seq v4超低起始量RNA试剂盒（SMART-Seq v4 Ultra Low Input RNA Kit，货号634888，宝生物工程（Takara Bio））完成cDNA合成。以20%至50%的起始产物进行第一链cDNA合成，随后通过17轮PCR扩增获得全长双链cDNA。采用安捷伦（Agilent）的生物分析仪（Bioanalyzer）与高灵敏度DNA试剂盒（High Sensitivity DNA Kit），以及赛默飞世尔科技（ThermoFisher Scientific）的Qubit dsDNA HS荧光定量检测法对所得cDNA的质量进行验证。以150pg cDNA为起始量，使用Nextera XT DNA文库制备试剂盒（Nextera XT DNA Library Preparation Kit，货号FC-131-1024，因美纳（Illumina））并经11轮PCR扩增，制备下一代测序（next-generation sequencing, NGS）文库。