Degeneration and RepairThe small molecule inhibitor PR-619 protects retinal ganglion cells against glutamate excitotoxicityHu, Xinxina,,b,,c,,d,,*; Zhuang, Donglia,,c,,*; Zhang, Ronga,,c; Sun, Xinghuaia,,c; Lu, Qinkangb,,d; Dai, Yia,,cAuthor Information aDepartment of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai bDepartment of Ophthalmology, The Affiliated People’s Hospital of Ningbo University, Ningbo cNHC Key Laboratory of Myopia (Fudan University), Key Laboratory of Myopia, Chinese Academy of Medical Sciences, and Shanghai Key Laboratory of Visual Impairment and Restoration (Fudan University), Shanghai dThe Eye Hospital of Wenzhou Medical University (Ningbo Branch), Ningbo, China *Xinxin Hu and Dongli Zhuang contributed equally to the writing of this article. Received 19 July 2020 Accepted 18 August 2020 Correspondence to Yi Dai, MD, PhD, Department of Ophthalmology, Eye & ENT Hospital, Fudan University, 83 Fenyang Road, Shanghai 200031, China, Tel: +86 021 64377134; fax: +86 021 64377151; e-mail: email@example.com or Qinkang Lu, MD, PhD, Department of Ophthalmology, The Affiliated People’s Hospital of Ningbo University, Ningbo 315000, China Tel:+86 0574 87016888; fax:+86 0574 87017272; e-mail: firstname.lastname@example.org NeuroReport: November 4, 2020 - Volume 31 - Issue 16 - p 1134-1141 doi: 10.1097/WNR.0000000000001522 Buy Metrics Abstract Glutamate excitotoxicity may contribute to the death of retinal ganglion cell (RGC) in glaucoma and other retinal diseases such as ischemia. Deubiquitinating enzyme (DUB) inhibitors are emerging as attractive targets for pharmacological intervention in neurodegenerative diseases. However, the role of PR-619, the broad spectrum DUB inhibitor, on RGCs under different stressful environment remains largely unknown. This study was designed to investigate the role of PR-619 in regulating mitophagy of RGCs under glutamate excitotoxicity. Primary cultured RGCs were incubated with PR-619 or vehicle control in the excitotoxicity model of 100 µM glutamate treatment. Mitochondrial membrane potential was assessed by JC-1 assay. Cytotoxicity of RGCs was measured by LDH activity. Proteins levels of parkin, optineurin, LAMP1, Bax, Bcl-2 and the LC3-II/I ratio were analyzed by western blot. The distribution and morphology of mitochondria in RGCs was stained by MitoTracker and antibody against mitochondria membrane protein, and examined by confocal microscopy. We show here that in the presence of glutamate-induced excitotoxicity, PR-619 stabilized the mitochondrial membrane potential of RGCs, decreased cytotoxicity and apoptosis, attenuated the expression of Bax. Meanwhile, PR-619 promoted the protein levels of Bcl-2, parkin, optineurin, LAMP1 and the LC3-II/I ratio. While knockdown of parkin by siRNA diminished the neuroprotective effect of PR-619 on RGCs. These findings demonstrate that PR-619 exerted a neuroprotective effect and promoted parkin-mediated mitophagy on cultured RGCs against glutamate excitotoxicity. DUB inhibitors may be useful in protecting RGCs through modulating the parkin-mediated mitophagy pathway against excitotoxicity. Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.