Crystallins as Neuroprotective and Neuroregenerative Treatments in Experimental Glaucoma

青光眼是导致失明的主要原因，其特征是进行性视网膜神经节细胞(RGC)丢失。降低眼压(IOP)是目前减缓青光眼的唯一治疗方法，是一个关键的危险因素。然而，尽管眼压降低，视网膜节细胞仍继续发生凋亡。视网膜节细胞作为中枢神经系统神经元，缺乏再生轴突的能力，这加剧了这种情况。更好地了解RGC损伤、凋亡和再生衰竭的机制及其相关因素仍然是一个巨大的挑战。在我们的第一个资助期(PR 1569/1-1)“分子生物标记物在实验性青光眼治疗中的作用及其对凋亡和再生相关信号通路的功能影响”中，我们从功能、细胞和分子水平详细分析了不同的青光眼模型。我们建立了蛋白质组图谱，确定了分子标记，并研究了与它们相关的神经保护和神经再生。晶体蛋白被发现是最显著的标志之一。我们破译出，在疾病过程中，晶体蛋白似乎在视网膜和玻璃体样本中显示出非常特殊的表达模式。玻璃体内注射αA和βB2具有神经保护作用。玻璃体内注射BetaB2晶体蛋白和表达NPC的BetaB2甚至可以促进神经再生。此外，晶体蛋白被从培养液中吸收进入细胞，运输到病变部位，似乎通过神经营养和钙依赖途径发挥作用。然而，晶体蛋白-被称为热休克蛋白-包括α(A和B)、β(A1/3、A2、A4、B1、B2、B3)和伽马(A、B、C、D、E、S)晶体蛋白的整个家族。单个晶体蛋白的重要性和相互作用，以及确切的神经保护和神经再生机制仍不清楚，必须在其在青光眼治疗中的潜力得以实现之前被阐明。首先，有针对性的蛋白质组学将有助于分析整个晶体蛋白家族，包括含量较低的晶体蛋白及其在再生和退化途径中的作用。将确定5个关键的晶体蛋白，破译相应的相关相互作用和信号通路。通过在体外再生和退化条件下检查视网膜外植体和分离的RGC，将评估这五个可能最强大的RGC的神经保护和再生潜力。阻断实验和对组织和上清液的具体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，目的是证明青光眼的新治疗方案。

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.