TY - JOUR
T1 - Companion Diagnosis for Retinal Neuroprotective Treatment by Real-Time Imaging of Calpain Activation Using a Novel Fluorescent Probe
AU - Asano, Toshifumi
AU - Nagayo, Yuri
AU - Tsuda, Satoru
AU - Ito, Azusa
AU - Kobayashi, Wataru
AU - Fujita, Kosuke
AU - Sato, Kota
AU - Nishiguchi, Koji M.
AU - Kunikata, Hiroshi
AU - Fujioka, Hiroyoshi
AU - Kamiya, Mako
AU - Urano, Yasuteru
AU - Nakazawa, Toru
N1 - Funding Information:
This study was supported by the Acceleration Transformative Research for Medical Innovation Set-up Scheme, from the Japan Agency for Medical Research and Development (AMED), Japan. This work was supported by the Public Trust Suda Memorial Fund for Glaucoma Research. We thank Mr. Tim Hilts for editing this document. We also thank Senju Pharmaceutical Co., Ltd. for the generous gift of SNJ-1945 and technical assistance.
PY - 2020/9/16
Y1 - 2020/9/16
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.bioconjchem.0c00435
DO - 10.1021/acs.bioconjchem.0c00435
M3 - Article
C2 - 32840357
AN - SCOPUS:85091127305
VL - 31
SP - 2241
EP - 2251
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
SN - 1043-1802
IS - 9
ER -