Zhang Nan, Li Ying, Zhang Xian, Chrenek Micah A, Wang Jiaxing, Girardot Preston E, Sellers Jana T, Brenner Charles, Cui Xiangqin, Geisert Eldon E, Nickerson John, Boatright Jeffrey H
Atlanta Veterans Administration Center for Visual and Cognitive Rehabilitation, Decatur, GA, United States.
Department of Ophthalmology, Zhongnan hospital of Wuhan University, Wuhan, Hubei, China.
bioRxiv. 2024 Dec 4:2024.12.03.626460. doi: 10.1101/2024.12.03.626460.
The aim of this study was to test whether oral administration of nicotinamide riboside (NR), the nicotinamide adenine dinucleotide (NAD+) precursors, protect retina ganglion cells (RGCs) from neurodegeneration in DBA/2J (D2) mice, which is a widely used mouse model of age-related inherited glaucoma.
Oral NR or NAM administration (NR low dose: 1150mg/kg; NR high dose: 4200mg/kg; NAM low dose group: 500mg/kg; NAM high dose: 2000mg/kg of body weight per day) essentially started when D2 mice were 4 or 9 months old and continued up to 12 months old. Control cohort identically received food/water without NAM or NR. Intraocular pressure (IOP) was measured every month until experiment completion. Pattern electroretinography (PERG) was recorded. Retinas were harvested for whole mount immunofluorescence staining with RGCs marker Brn3a and imaged by fluorescent confocal microscopy. Optic nerves were harvested for axon staining and quantification. Retinal NAD+ levels were enzymatically assayed.
NR oral supplementary treatment started at 4 months old robustly increased retinal NAD+ levels in D2 mice (NR vs. vehicle: 273.7±23.59% vs. 108.70±12.10%, <0.001). In aged vehicle group (12 months old), there was significantly diminution of the P1 and N2 components of PERG response compare with naïve group (naïve vs. vehicle: P1: 7.82± 0.70uV vs 1.63± 0.17uV, <0.0001; N2: -13.29± 0.83uV vs. -3.22± 0.27uV, <0.0001; Kruskal-Wallis test with Dunn's multiple comparison test). NR treatment preserved aged D2 visual function when mice were 9 and 12 months old. In addition, long-term NR high dose treatment significantly protected against total RGCs loss and optic nerve atrophy (RGC: NR vs. vehicle: 1412±62.00vs 475.2±94.68 cells/field, <0.00001; axon numbers: NR vs. vehicle: 23990±1159 vs 8573±1160, n=41-53, <0.0001). Furthermore, long-term NR supplementation prevent iris depigmentation and delayed IOP elevation.
NR oral supplementary treatment significantly preserved RGC and axon numbers, potentially preserves retinal function via elevated retinal NAD+ level in aged D2 mice. Interestingly, NR treatment also prevented iris atrophy, delayed IOP elevation associated with this glaucoma model. NR oral supplementation thus treated several aspects of murine pigment dispersion glaucoma. Given parallels between this model and glaucoma in human, out data indicate that NR is worth exploring as a therapeutic candidate in treatment of glaucoma.
本研究旨在测试口服烟酰胺核糖（NR）这种烟酰胺腺嘌呤二核苷酸（NAD+）前体，是否能保护DBA/2J（D2）小鼠的视网膜神经节细胞（RGCs）免受神经退行性变，D2小鼠是一种广泛应用的年龄相关性遗传性青光眼小鼠模型。
口服NR或烟酰胺（NAM）（NR低剂量：1150mg/kg；NR高剂量：4200mg/kg；NAM低剂量组：500mg/kg；NAM高剂量：2000mg/kg体重/天）基本在D2小鼠4或9个月大时开始，持续至12个月大。对照组同样接受不含NAM或NR的食物/水。每月测量眼压直至实验结束。记录图形视网膜电图（PERG）。收获视网膜用于用RGCs标志物Brn3a进行全层免疫荧光染色，并通过荧光共聚焦显微镜成像。收获视神经用于轴突染色和定量分析。酶法测定视网膜NAD+水平。
4个月大时开始的NR口服补充治疗显著提高了D2小鼠的视网膜NAD+水平（NR组与载体组：273.7±23.59%对108.70±12.10%，<0.001）。在老年载体组（12个月大）中，与未处理组相比，PERG反应的P1和N2成分显著降低（未处理组与载体组：P1：7.82±0.70μV对1.63±0.17μV，<0.0001；N2：-13.29±0.83μV对-3.22±0.27μV，<0.0001；Kruskal-Wallis检验及Dunn多重比较检验）。当小鼠9个月和12个月大时，NR治疗保留了老年D2小鼠的视觉功能。此外，长期NR高剂量治疗显著预防了RGCs总数的丢失和视神经萎缩（RGCs：NR组与载体组：1412±62.00对475.2±94.68个细胞/视野，<0.00001；轴突数量：NR组与载体组：23990±1159对8573±1160，n = 41 - 53，<0.0001）。此外，长期补充NR可预防虹膜色素脱失并延缓眼压升高。
NR口服补充治疗显著保留了RGCs和轴突数量，可能通过提高老年D2小鼠视网膜NAD+水平来保留视网膜功能。有趣的是，NR治疗还预防了虹膜萎缩，延缓了与该青光眼模型相关的眼压升高。因此，NR口服补充治疗改善了小鼠色素性青光眼的多个方面。鉴于该模型与人类青光眼之间的相似性，我们的数据表明NR作为青光眼治疗的候选药物值得探索。
