Crystallins as Neuroprotective and Neuroregenerative Treatments in Experimental Glaucoma

青光眼是致盲的主要原因，以进行性视网膜神经节细胞（RGC）丧失为特征。降低眼压作为一个关键的危险因素，是目前治疗慢性青光眼的唯一方法。然而，尽管IOP降低，RGCs仍继续发生凋亡。作为中枢神经系统神经元，RGCs缺乏轴突再生的能力，使这种情况更加恶化。更好地理解RGC损伤、细胞凋亡和再生失败的相关机制仍然是一个巨大的挑战。在我们的第一个资助期（PR 1569/1-1）“实验性青光眼治疗中的分子生物标志物及其对凋亡和再生相关信号通路的功能影响”中，我们在功能，细胞和分子水平上详细分析了不同的青光眼模型。我们建立了蛋白质组学图谱，鉴定了分子标记，并研究了与之相关的神经保护和神经再生。晶体蛋白被发现是最引人注目的标记之一。我们破译了晶体蛋白在视网膜和玻璃体样本中表现出非常特殊的表达模式。体内玻璃体内注射α和β 2具有神经保护作用。β - ab2结晶蛋白和表达β - ab2的npc甚至可以促进神经再生。此外，晶体蛋白从培养基中被摄取到细胞中，并通过神经营养和钙依赖途径转运到病变部位，并似乎发挥作用。然而，晶体蛋白——被称为热休克蛋白——由α （a和B）、β （A1/3、A2、A4、B1、B2、B3）和γ （a、B、C、D、E、S）晶体蛋白组成的整个家族。单个晶体蛋白的重要性和相互作用，确切的神经保护和神经再生机制仍然不清楚，必须在它们治疗青光眼的潜力实现之前加以阐明。这一问题将在第二个筹资期得到解决。首先，靶向蛋白质组学将有助于分析整个晶体蛋白家族，包括不太丰富的晶体蛋白及其在再生和退行性途径中的作用。5个关键的晶体蛋白将被鉴定，相应的相关相互作用和信号通路将被破译。在再生和退行性条件下，将通过检查视网膜外植体和分离的RGCs来评估五种可能最强大的神经保护和再生潜力。阻断实验和组织和上清的特定分析将验证信号通路。最后一步将分析两种最有希望的晶体蛋白在体内的神经保护和再生，目的是为青光眼提供新的治疗方案。

Glaucoma is a leading cause of blindness, characterized by progressive retinal ganglion cell (RGC) loss. Lowering of intraocular pressure (IOP), as a key risk factor, is currently the only treatment to slow glaucoma. However, RGCs continue to undergo apoptosis despite IOP lowering . The situation is exacerbated by RGCs, as CNS neurons, lacking the ability to regenerate their axons. A better understanding of the mechanisms and associated with RGC injury, apoptosis and regenerative failure remains an enormous challenge. In our first funding period (PR 1569/1-1) “Molecular biomarkers in the treatment of experimental glaucoma and their functional impact on apoptotic- and regeneration-related signalling pathways”, we analysed different glaucoma models in detail at functional, cellular and molecular levels. We established a proteomic profile, identified molecular markers and looked into neuroprotection and neuroregeneration related to them. Crystallins were found to be one of the most striking markers. We deciphered, that crystallins appeared to show very specific expression patterns in retinal and vitreous samples in the course of the disease. Intravitreal injection of alphaA and betaB2 in vivo exerted neuroprotective effects. BetaB2 crystallin and betaB2 expressing NPCs given intravitreally even promoted neuroregeneration. Besides that crystallins were uptaken from the medium into the cells, transported to the site of lesion and seemed to exert effects via neurotrophic and calcium-dependent pathways. However, crystallins- known as heat-shock proteins- comprise a whole family of alpha (A and B), beta (A1/3, A2, A4, B1, B2, B3) und gamma (A, B, C, D, E, S) crystallins. The importance and interplay of the individual crystallins, the precise neuroprotective and neuroregenerative mechanisms still remain unclear and must be elucidated before their potential in the treatment of glaucoma can be realized. This will be addressed in the second funding period.Firstly, targeted proteomics will help to analyse the whole crystallin family including the less abundant crystallins and their role in regenerative and degenerative pathways. The 5 key crystallins will be identified, corresponding relevant interactions and signalling pathways deciphered. The neuroprotective and regenerative potential of the five presumably most powerful will be assessed by examining retinal explants and isolated RGCs under regenerative and degenerative conditions in vitro. Blocking experiments and specific analysis of the tissue and supernatant will verify the signalling pathways. The last step will analyse neuroprotection and regeneration of the two most promising crystallins in vivo, with the aim of proving novel treatment alternatives for glaucoma.