Companion Diagnosis for Retinal Neuroprotective Treatment by Real-Time Imaging of Calpain Activation Using a Novel Fluorescent Probe.,Bioconjugate Chemistry

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Companion Diagnosis for Retinal Neuroprotective Treatment by Real-Time Imaging of Calpain Activation Using a Novel Fluorescent Probe.
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2020-08-25 , DOI: 10.1021/acs.bioconjchem.0c00435
Toshifumi Asano ₁ , Yuri Nagayo ₂ , Satoru Tsuda ₁ , Azusa Ito ₁ , Wataru Kobayashi ₃ , Kosuke Fujita ₃ , Kota Sato _{1,

4} , Koji M Nishiguchi ₅ , Hiroshi Kunikata _{1,

3} , Hiroyoshi Fujioka ₆ , Mako Kamiya ₂ , Yasuteru Urano ₂ , Toru Nakazawa _{1,

3,

4,

5,

7}

Affiliation

Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-l-leucyl-l-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase cytotoxicity assay, retinal sections, and electroretinograms. Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N-methyl-d-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a noninvasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.

中文翻译：

通过使用新型荧光探针对钙蛋白酶激活进行实时成像，对视网膜神经保护性治疗进行伴随诊断。

在动物研究中，钙蛋白酶激活可诱导视网膜神经节细胞（RGC）死亡，而钙蛋白酶抑制可抑制RGC死亡。但是，钙蛋白酶在人类视网膜疾病中的作用尚不清楚。这项研究研究了一种新策略，以研究基于实时成像的钙蛋白酶的作用。我们合成了一种新型的针对钙蛋白酶的荧光探针，乙酰基-1-亮氨酰-1-甲硫氨酸-羟甲基若丹明绿（Ac-LM-HMRG），并将其用于钙蛋白酶激活的实时成像。Ac-LM-HMRG的毒性通过乳酸脱氢酶细胞毒性测定，视网膜切片和视网膜电图进行评估。在这里，我们进行了大鼠模型中钙蛋白酶激活的实时成像。首先，我们给予Ñ甲基d-天冬氨酸（NMDA）诱发视网膜损伤。二十分钟后，我们进行了玻璃体内注射Ac-LM-HMRG。然后用无创共聚焦扫描激光检眼镜完成实时成像。还使用相同的实时成像方法检查了SNJ-1945对钙蛋白酶激活的抑制作用。Ac-LM-HMRG没有毒性作用。NMDA损伤后，实时成像中的Ac-LM-HMRG阳性细胞数量显着增加，而SNJ-1945显着降低了Ac-LM-HMRG阳性细胞的数量。Ac-LM-HMRG实时成像能够快速量化NMDA诱导的钙蛋白酶激活和SNJ-1945的抑制作用。用作辅助诊断系统的这项技术可能有助于研究开发新的神经保护疗法。

更新日期：2020-09-16

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