Ekström P A R, Ueffing M, Zrenner E, Paquet-Durand F
Institute for Ophthalmic Research, University of Tubingen, Rontgenweg 11, 72076 Tubingen, Germany.
Curr Med Chem. 2014;21(30):3478-93. doi: 10.2174/0929867321666140601201337.
The mechanisms of neuronal cell death are still only poorly understood, which has hindered the advancement of therapies for many currently untreatable neurodegenerative diseases. This calls for the development of new methods which reveal critical molecular mechanisms of the celldeath machinery with both high sensitivity and cellular resolution. Using animal models for hereditary neurodegeneration in the retina, we have developed or adapted different biochemical assays to determine the enzymatic activities of calpain, poly-ADP-ribose-polymerase (PARP), and histone deacetylase (HDAC) directly and in situ. Additionally, the enzymatic activity of cGMP-dependent protein kinase (PKG) was assessed indirectly using in situ immunohistological techniques to detect PKG-activity-dependent products. Combining these assays with in situ cell death markers revealed close temporospatial correlations, suggesting causal connections between the PKG, HDAC, PARP and calpain activities and neuronal cell death. Using different pharmacological and genetic manipulations, causality could indeed be demonstrated. Surprisingly, the often dramatic rises in metabolic activities didnot match by corresponding increases in expression, highlighting the importance of analyses of protein activities at the cellular level. The above mentioned studies identified a number of metabolic processes previously unknownto be involved in inherited retinal degeneration. Comparing different animal retinal degeneration models uncovered striking similarities in enzymatic activities, suggesting a generality of the destructive pathways. Taken together, these findings provided a number of novel targets for neuroprotection and as such opened up new perspectives for the therapy of hereditary neurodegeneration in the retina and possibly other parts of the central nervous system.
神经元细胞死亡的机制目前仍知之甚少，这阻碍了许多当前无法治疗的神经退行性疾病治疗方法的进展。这就需要开发新的方法，以高灵敏度和细胞分辨率揭示细胞死亡机制的关键分子机制。利用视网膜遗传性神经退行性变的动物模型，我们开发或改进了不同的生化检测方法，以直接和原位测定钙蛋白酶、聚ADP核糖聚合酶（PARP）和组蛋白脱乙酰酶（HDAC）的酶活性。此外，使用原位免疫组织学技术间接评估环鸟苷酸依赖性蛋白激酶（PKG）的酶活性，以检测PKG活性依赖性产物。将这些检测方法与原位细胞死亡标记物相结合，揭示了紧密的时空相关性，表明PKG、HDAC、PARP和钙蛋白酶活性与神经元细胞死亡之间存在因果关系。通过不同的药理和基因操作，确实可以证明因果关系。令人惊讶的是，代谢活性的经常显著升高与相应的表达增加并不匹配，这突出了在细胞水平分析蛋白质活性的重要性。上述研究确定了一些以前未知参与遗传性视网膜变性的代谢过程。比较不同的动物视网膜变性模型发现酶活性存在惊人的相似性，表明破坏途径具有普遍性。综上所述，这些发现为神经保护提供了许多新靶点，从而为视网膜以及可能中枢神经系统其他部位的遗传性神经退行性变治疗开辟了新的前景。
