The small molecule inhibitor PR-619 protects retinal ganglion cells against glutamate excitotoxicity

Xinxin Hua,b,c,d,*, Dongli Zhuanga,c,*, Rong Zhanga,c, Xinghuai Suna,c, Qinkang Lub,d and Yi Daia,c

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. NeuroReport 31: 1134– 1141 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.
NeuroReport 2020, 31:1134–1141

Keywords: excitotoxicity, mitophagy, parkin, PR-619, retinal ganglion cell

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 and dThe Eye Hospital of Wenzhou Medical University (Ningbo Branch), Ningbo, China

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 protected] 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: [email protected]
Xinxin Hu and Dongli Zhuang contributed equally to the writing of this article.

Received 19 July 2020 Accepted 18 August 2020

Introduction
Glutamate excitotoxicity has long been suggested to contribute to the death of retinal ganglion cell (RGC) in glaucoma and other retinal diseases such as ischemia [1]. Parkin, an E3 ubiquitin ligase, is a key factor in mitochon- drial quality-control pathway to mediate the selective removal of damaged mitochondria via mitophagy. When the mitochondrial membrane potential is depolarized, parkin is recruited to the outer mitochondrial membrane, leading to the parkin-mediated ubiquitination of mito- chondrial membrane proteins and facilitating mitophagy [2]. Our previous study has indicated that parkin overex- pression exerted a significant protective effect on cultured RGCs against glutamate excitotoxicity [3]. In experi- mental glaucoma rat model, overexpression of parkin played a prominent protective role in RGCs, and partially restored the dysfunction of mitophagy [4]. According to these findings, exploring the small molecular compounds

that may promote parkin mediated mitophagy in RGCs against excitotoxicity is worthy of investigation.
Protein ubiquitination is a reversible posttranslational modification and involves in a variety of cellular func- tions. Accumulating evidences have suggested that deubiquitinating enzymes (DUBs) can counteract par- kin-mediated mitochondrial ubiquitination, and may serve as a key negative regulator of mitophagy [5]. DUB inhibitors are emerging as attractive targets for pharma- cological intervention in neurodegenerative diseases [6]. PR-619, the broad spectrum DUB inhibitor, has been shown to involved in diverse cellular functions: such as autophagy [7], antitumor effect [8] and apoptosis [9], but its role in neurons remained largely unknown. Previous studies indicated that PR-619 led to the activation of autophagy and the recruitment of LC3 in oligodendro- glial cells [7]. However, little is known about the role of PR-619 on RGCs under different stressful environment.

0959-4965 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/WNR.0000000000001522

Hence, the present study was undertaken to investi- gate whether PR-619 can exert a neuroprotective role in RGCs and its mechanism in regulating mitophagy of RGCs under glutamate excitotoxicity.

Materials and methods
Animals
All procedures concerning animals were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and under protocols approved by the Animal Ethics Committee of the Eye and ENT Hospital of Fudan University, China.

Purification and culture of retinal ganglion cells
Our modified two-step panning protocol was performed to isolate purified RGCs as described previously [3]. Briefly, retinal tissues were obtained from 2 to 3-day- old Sprague–Dawley rats and dissociated in 5 mg/ml of papain (Worthington Biochemical, Lakewood, New Jersey, USA). To obtain a suspension of single cells, the tissues were then added in MEM containing 0.1% ovomucoid (Worthington Biochemical). The suspension was centrifuged at 1000 rpm for 10 minutes to separate retinal cells from the ovomucoid solution. Then the ret- inal suspension was resuspended in 0.5 mg/ml BSA in MEM and filtered through Nitex mesh (pore size 40 μm; BD Falcon, Franklin Lakes, New Jersey, USA) twice.
Macrophages and microglial cells were removed by incu- bation of the cell suspension on a panning plate coated with two anti-rat-macrophage antiserum (Millipore Corp., Billerica, Massachusetts, USA) for 1 h at 37°C, shaking each plate every 20 minutes. Nonadherent cells were transferred to two anti-rat-Thy1.1 panning plates (Abcam, Cambridge, Massachusetts, USA) at 37°C for 1 h, shaking each plate every 20 minutes. Then plates were washed three times with D-PBS (Gibco, Grand Island, USA) and swirled moderately vigorously to dislodge non- adherent cells. Adherent cells were released from plates by incubation with 0.25% trypsin (Gibco) for 2 minutes at 37°C. Immediately following treatment, DMEM (Gibco) media with 30% FBS (Gibco) was added to each plate to stop trypsin. After centrifugation at 1000 rpm for 5 min- utes, the cells were seeded on glass coverslips that had been coated with 0.01% poly-D-lysine (Sigma-Aldrich, Sigma-Aldrich, St. Louis, USA).
Purified RGCs were plated at a density of 1 × 106 cells per 24-well plate. Cultures were maintained at 37°C in humidified atmosphere containing 5% CO2 and 95% air
in Neurobasal medium (Gibco) containing supplemen-
tal factors as previously described (Neurobasal medium containing 2% B27, 0.01% BSA, 1 mM L-glutamine, 5 μg/ ml insulin, 1 mM sodium pyruvate, 40 ng/ml triiodothy- ronine, 40 ng/ml thyroxine,60 ng/ml progesterone, 16 μg/ ml putrescine, 40 ng/ml sodium selenite, 60 μg/ml N-acetyl cysteine, 50 ng/ml brain-derived neurotrophic factor, 10 ng/ml basic fibroblast growth factor, 10 ng/

ml ciliary-derived neurotrophic factor, 5 mM Forskolin, 100 units/ml penicillin, 100 mg/ml streptomycin) [3,10]. Immunocytochemical staining of Tubulin and γ-synu- clein were performed after the primary RGCs were cul- tured for 2 days to determine the RGCs purity

Excitotoxicity model
Three days after seeding, 0, 1.17, 4.90, 8.23, 12.8 or 100 µM PR-619 was added to cell culture medium. Then, RGCs were exposed to cell culture medium alone (con- trol) or to cell culture medium containing 100 μM gluta- mate (Sigma-Aldrich) for 3 days in a 37°C, 5% CO2 tissue
culture incubator.

Assessment of cell apoptosis
Apoptosis of RGCs was assessed by Hoechst staining. RGCs on coverslips were fixed with 4% paraformaldehyde (PFA) in PBS for 20 minutes at room temperature, rinsed with PBS, and then were permeabilized with 0.1% Triton X-100 for 20 minutes at room temperature. Cells were then washed thrice and were stained with Hoechst 33342 (1 µg/ ml; Life Technologies, Grand Island, USA) for 10 min- utes at room temperature. Images were taken of randomly using a confocal microscope (Leica SP8). RGCs apoptosis was quantified by having pyknotic nuclei. The number of cells with pyknotic nuclei and the total number of cells were counted. The percentage of apoptotic cells was cal- culated for each control and experimental condition.

Measurement of mitochondrial membrane potential Measurement of mitochondrial membrane potential was performed using the JC-1 Assay Kit (Abcam). Cultured RGCs were harvested at the end of drug exposure, incu- bated with JC-1 (5 μg/ml) dye for 20 minutes at 37°C, and were rinsed twice with EBSS. JC-1 fluorescence intensities of red aggregates (hyperpolarization) and green fluores- cence monomers (depolarization) were read by a fluores- cent plate reader (Tecan; Infinite M1000, Mnnedorf, USA). The ratios of the JC-1 red fluorescence aggregates versus the green fluorescence monomers for each treatment were measured as previously described. All experiments were repeated independently at least three times.

Measurement of cell death and cytotoxicity
The level of RGCs death and cytotoxicity was evaluated by quantitating cytotoxicity based on the measurement of activity of lactate dehydrogenase (LDH) released from damaged cells according to the standard protocol (TaKaRa Biotechnology, Dalian, China). After each treatment, the supernatant media of RGCs is collected and incubated with the reconstituted substrate mix. Absorbance was measured at 490 nm using a microplate reader (Synergy H1, BIOtAK) as we described previously [3].

Mitochondrial distribution
In the presence of glutamate for 3 days in culture, RGCs were loaded with 200nM MitoTracker Red (Molecular

Probes, M7512; Life Technologies) at 37°C for 30 min- utes, and then were washed three times. RGCs on cov- erslips were fixed with 4% PFA in PBS for 10 minutes at room temperature, rinsed with PBS, and then were permeabilized with 0.2% Triton X-100 for 5 minutes. Coverslips were observed by a confocal microscope (Leica SP8). Three randomly-selected fields from one coverslip were included and experiments were per- formed in triplicates, n = 3.

Immunofluorescence analysis
Following the excitotoxicity model, RGCs were fixed with 4% PFA in PBS for 20 minutes, rinsed with PBS, and per- meabilized with 0.1% Triton X-100 in PBS for 20 minutes at room temperature, then washed thrice with PBS. Cells were next blocked with 5% BSA/PBS for 1 h at room tem- perature, and with the primary antibodies against mono- clonal rabbit anti-LC3 (1:200; Abcam) for 16 h at 4°C. After several washes, the RGCs were incubated with Alexa Fluor 488-conjugated goat IgG secondary antibody (1:200; Life Technologies) for 1 h at room temperature then washed with PBS. The RGCs were counterstained with Hoechst 33342 (1 µg/ml; Life Technologies) in PBS. Images were captured by a confocal microscope (Leica SP8).

Western blot analysis
RGCs (n = 3 per group) were mixed with RIPA buffer (Beyotime, Shanghai, China). Each sample was separated by PAGE and electrotransferred onto polyvinylidene dif- luoride membranes. Membranes were blocked with 5% nonfat dry milk at room temperature for 1 h, incubated with polyclonal rabbit anti-parkin (1:1000; Abcam), poly- clonal rabbit anti-Bcl-2 (1:500; Abcam), monoclonal rabbit anti-Bax (1:1000; Abcam), polyclonal rabbit anti-optineu- rin (1:200; Abcam), monoclonal rabbit anti-LC3 (1:2000; Abcam), polyclonal rabbit anti-LAMP1 (1:1000; Abcam)
Fig. 1

and polyclonal rabbit anti-GAPDH (1:2000; Yesen, Shanghai, China) in primary antibody dilution (Beyotime) at 4°C overnight. The membranes were rinsed with 1× TBST (Worthington Biochemical) several times,incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5000; Jackson, West Grove, Pennsylvania, USA), and developed using chemiluminescence detection (SuperSignal West Femto Substrate Trial Kit; Thermo Fisher, Waltham, Massachusetts, USA). Chemiluminescent images were captured using a Kodak Image Station 4000MM PRO (Carestream, Rochester, New York, USA) and analyzed with Image J (National Institutes of Health).

Statistical analysis
Experiments were repeated at least three times. Data were expressed as mean ± SD. One-way analysis of vari- ance and the Bonferroni t-test were used to evaluate study results. P < 0.05 was considered statistically significant.

Results
PR-619 has a positive influence on retinal ganglion cells viability in the absence of glutamate
First, we determined the optimal concentration and
incubation time of PR-619 for RGCs according to the manufacture (Selleck)’s datasheet and the levels of mito- chondrial membrane potential in RGCs. As reflected by JC-1 ratio, the optimal concentration of RP-619 for RGCs was 8.23 μM. And the optimal duration was 30 minutes (Fig. 1a and b).
Because of its poor solubility in water and ethanol, PR-619 was needed to be dissolved in 45 mg/ml DMSO. Thus, 45 mg/ml DMSO was used as vehicle control. Compared with control group in neurobasal medium, RGCs incu- bated with PR-619 showed that the cytotoxicity as meas- ured by LDH activity was significantly decreased (P <

Dose- and time-dependant JC-1 results of RP-619 treatment on RGCs. (a) Dose-dependant JC-1 ratio showed that the optimal concentration of RP-619 for RGCs was 8.23 μM. (b) Time-dependant JC-1 ratio showed that the optimal duration of 8.23 μM RP-619 for RGCs was 30 minutes. n = 3; data are expressed as mean ± SD; *P < 0.05. RGC, retinal ganglion cell.

Effects of RP-619 on retinal ganglion cells (RGCs) under glutamate excitotoxicity. Compared with the control group, RGCs incubated with
PR-619 showed less apoptotic cell death (a), lower level of cytotoxicity (b) and higher level of mitochondrial membrane potential under glutamate excitotoxicity (c). n = 3; data are expressed as mean ± SD; *P < 0.05, **P < 0.01.

Fig. 3

Western blot analysis of RGCs in the control and RP-619 groups under glutamate excitotoxicity. Compared with the control group, the protein levels of parkin (a,d), optineurin (a, c), LAMP1 (a, b) and Bcl-2 (a, e) were significantly increased in RGCs incubated with PR-619 under gluta- mate excitotoxicity. The LC3-II/I ratio (a, g) was also increased in RGCs incubated with PR-619. The protein levels of Bax (a, f) were significantly decreased in RGCs incubated with PR-619 under glutamate excitotoxicity. n = 3; data are expressed as mean ± SD; *P < 0.05, **P < 0.01.
RGC, retinal ganglion cell.
0.01 , Fig. 2b), the percent of apoptotic cell death as meas- ured by Hoechst staining was also decreased (P < 0.05, Fig. 2a), and the protein levels of parkin (P < 0.05) and optineurin (P < 0.01) were increased (Fig. 3a, c and D).

No statistically significant differences were observed for JC-1 ratio (Fig. 2c), LC3-II/I ratio, Bax and Bcl-2 protein levels(P > 0.05, Fig. 3a, e–g). These results suggested that PR-619 has a positive influence on RGC viability.

Impacts of PR-619 on retinal ganglion cells under glutamate excitotoxicity
In the excitotoxicity model of 100 µM glutamate treat-
ment to RGCs as a control group, it induced 26.12% of apoptotic cell. In comparison, RGCs incubated with PR-619 had significantly less apoptotic cell death (10.92%) under glutamate excitotoxicity (P < 0.05, Fig. 2a).
Cytotoxicity was measured by LDH activity also showed the same trend. In the presence of glutamate, LDH release from RGCs increased by 46.32% com- pared with that in the neurobasal medium group (P < 0.05); however, the effect was reversed with PR-619 (P
< 0.05, Fig. 2b).
The control group had a JC-1 ratio of 0.83 ± 0.02, whereas the JC-1 ratio of RGCs incubated with PR-619 was 0.91 ±
0.01. These results implied that PR-619 can increase the level of mitochondrial membrane potential under gluta- mate excitotoxicity (P < 0.05, Fig. 2c).
Compared with the control group, protein levels of Bax were lower in RGCs incubated with PR-619 under glu- tamate excitotoxicity (P < 0.05, Fig. 3a and f), while the protein level of Bcl-2 was higher in RGCs incubated with PR-619 (P < 0.05, Fig. 3a and e). These results indicated that PR-619 can protect RGCs under glutamate excitotoxicity.

Impacts of PR-619 on mitochondrial morphology and mitophagy in retinal ganglion cells under glutamate excitotoxicity
Mitochondria in RGCs were stained with MitoTracker
Red and visualized under confocal microscope (Fig. 4). In the control group under glutamate excitotoxicity, small spherical mitochondria could be observed around nucleus, and few mitochondria in the axon were observed (Fig. 4b). The mean MitoTracker intensity was signifi- cantly increased in RGCs incubated with PR-619 (P < 0.01). More number of large mitochondria was observed around nucleus, and a tubular mitochondrial network was partially restored (Fig. 4f). The above results implied

Fig. 4

Immunofluorescence analysis of RGCs co-stained with MitoTracker and LC3. (a–d) In the control group under glutamate excitotoxicity, small spherical mitochondria (red) could be observed around nucleus, and few mitochondria in the axon were observed. (e–h) PR-619 upregulated the LC3, MitoTracker immunoreactivity, and the colocalization of LC3 with MitoTracker in the axons of the RGCs under glutamate excitotoxicity. RGC, retinal ganglion cell.
Effects of parkin downregulation on RGCs incubated with PR-619 under glutamate excitotoxicity. Compared with siRNA-control group incubated with PR-619, knockdown of parkin incubated with PR-619 showed decreased levels of mitochondrial membrane potential (a) and the protein expression of Bcl-2 (c, e), while the levels of cytotoxicity (b) and the protein expression of Bax were increased (c, d). n = 3; data are expressed as mean ± SD; *P < 0.05. RGC, retinal ganglion cell that PR-619 increased the number of mitochondria while reduced glutamate-induced mitochondrial fragmentation.
Western blot analysis showed that compared with the con- trol group, the protein levels of parkin (P < 0.05, Fig. 3a and d), optineurin (P < 0.05, Fig. 3a and c) and LAMP1 (P < 0.01, Fig. 3a and b) were higher in RGCs incubated with PR-619 under glutamate excitotoxicity. Moreover, the LC3-II/I ratio was also increased in RGCs incubated with PR-619 (P < 0.05, Fig. 3a and g). It is noteworthy that PR-619 upregulated the LC3 immunoreactivity and the colocalization of LC3 with mitochondria in the axons of the RGCs under glutamate excitotoxicity (Fig. 4a and e).

Knockdown of parkin reversed the effect of PR-619 on retinal ganglion cells under glutamate excitotoxicity
To further investigate the relationship between PR-619
and parkin, cultured RGCs were transfected with par- kin siRNA to knockdown parkin gene expression. Both mRNA and protein levels of parkin were decreased in the RGCs transfected with parkin siRNA (P < 0.05). JC-1 assay, LDH assay and western blot analysis were performed to investigate the effect of parkin knockdown on RGCs viability incubated with PR-619. Compared the PR-619 effect observed in the siRNA-control group with 100 µM glutamate treatment, the JC-1 ratio and the protein lev- els of Bcl-2 were statistically significantly decreased (P < 0.05, Fig. 5a, c and e), however, cytotoxicity and the level of Bax were increased (P < 0.05, Fig. 5b–d) in siRNA-par- kin group incubated with PR-619. This result suggested

that knockdown of parkin diminished the neuroprotective effect of PR-619 on RGCs under glutamate excitotoxicity.

Discussion
These results demonstrate that in response to a stressor linked to neurodegeneration (glutamate excitotoxicity), PR-619 stabilized the mitochondrial membrane potential of RGCs, decreased cytotoxicity and apoptosis, attenu- ated the expression of Bax. Meanwhile, PR-619 promoted the protein levels of Bcl-2, parkin, optineurin, LAMP1 and the LC3-II/I ratio. Knockdown of parkin diminished the neuroprotective effect of PR-619 on RGCs under glutamate excitotoxicity.
Ubiquitination of proteins plays a key role in signal transduction pathways and mediates protein stability. It represents a reversible posttranslational modification which could be balanced by the actions of E3-ligases and DUBs [11]. Parkin is an E3 ubiquitin ligase that ubiquit- inates diverse substrates. DUBs could counteract parkin through reversing ubiquitination [5]. RP-619 is a cell-per- meable, broad-range, reversible DUB inhibitor [12]. Our study demonstrated that PR-619 had a neuroprotective effect on RGCs under glutamate excitotoxicity. We also found that PR-619 enhanced the protein level of parkin, while knockdown of parkin abated the neuroprotective effect of PR-619 on RGCs under glutamate excitotoxic- ity. In our previous study, overexpression of parkin has proved to play a protective role on RGCs under gluta- mate-induced excitotoxicity [3]. In line with these find- ings, our results suggest that the mechanism by which
PR-619 mediated RGCs protection against glutamate excitotoxicity may through enhancing parkin expression.
Parkin normally maintains neuronal health by ubiquit- inating its protein substrates [13]. To understand the underlying molecular mechanism involved in the pro- tective effect of PR-619 in RGCs, we further studied downstream pathway of parkin. Previous studies sup- ported an interaction of parkin’s antiapoptotic effects with Bax [14]. As a proapoptotic protein in the BCL-2 family, Bax could induce mitochondrial outer membrane permeabilization and the release of cytochrome c [15]. We provided the in vitro evidence that PR-619 not only promoted the protein levels of parkin, but also stabi- lized the mitochondrial membrane potential, attenuated the expression of Bax in RGCs under glutamate excito- toxicity. Bernardini et al. [16] reported that parkin may inhibit proapoptotic Bax either by limiting recruitment of ubiquitinated Bax mitochondria or by promoting deg- radation of dysregulated Bax. Bcl-2, also as a substrate for parkin, could suppress apoptosis by inhibiting the release of cytochrome c and preventing mitochondrial outer membrane permeabilization. Parkin has been proposed to interact with the C terminus of Bcl-2 and facilitate mono-ubiquitination of Bcl-2, which steadily upregulates the level of Bcl-2 [17]. The current study found that PR-619 promoted the protein levels of Bcl-2 in RGCs under glutamate excitotoxicity. On the basis of above findings, it can be inferred that the antiapoptotic effect of PR-619 on RGCs is mediated by regulating the function parkin and its interaction with Bax and Bcl-2.
When the mitochondrial membrane potential is depolar- ized in damaged or dysfunctional mitochondria, parkin is activated and accumulated to the outer mitochon- drial membrane, which results in the parkin-depend- ent ubiquitination of proteins localized on the outer mitochondrial membrane and mediating mitophagy. Our data showed that optineurin, an autophagy recep- tor [18]¸ which recruited to ubiquitinated mitochondria downstream of parkin was also upregulated in RGCs incubated with PR-619 under glutamate excitotoxicity. Ubiquitinated mitochondria with optineurin then accu- mulates the phagophore protein LC3, resulting in forma- tion of mitophagosomes. LC3-II/I ratio has been widely used as a marker of autophagosome formation [19]. Our study also found that LC3-II/I ratio, LC3 immunoreac- tivity and the colocalization of LC3 with mitochondria was enhanced in RGCs incubated with PR-619 under glutamate excitotoxicity. Finally, mitophagosomes deliv- ers to lysosomes for degradation [20]. In order to evalu- ate whether the increase in LC3-II/I ratio was induced by impaired lysosomal activity or increased autophagic flux, we therefore analyzed the expression of lysoso- mal marker, LAMP1. The result showed that PR-619 increased LAMP1 in RGCs under glutamate excitotox- icity. Taken together, our data indicated that PR-619 may promote parkin-mediated mitophagy in RGCs to cope with glutamate excitotoxicity.
Since PR-619 is a pan-inhibitor of DUBs, the question remains whether specific DUBs exert these protec- tive effects on RGCs. According to the manufacture (Selleck)’s datasheet on PR-619, the 8.23um concentra- tion of PR-619 is the EC50 that can inhibit ubiquitin-spe- cific protease (USP) 15 activity. The close concentration
8.62 µm of PR-619 is the EC50 for USP5. Our dose-de- pendent data showed that the concentration of 8.23um exerted the best protective effect on RGC under gluta- mate excitotoxicity. Cornelissen et al. [21] reported that USP 15 opposed parkin-mediated mitophagy, while USP15 knockdown enhanced mitophagy and rescued the mitochondrial defects of parkin-deficient flies. Recently, the therapeutical potential of USP5 in neurological dis- ease has got increasing attentions [22]. Further studies are needed to investigate the role of specific inhibitor to USP15 or USP5 in regulating mitochondrial quality and protecting the RGCs against glutamate excitotoxicity.
In summary, our findings demonstrate that PR-619 exerted a significant protective effect on RGCs by ways of enhancing parkin-mediated mitophagy against gluta- mate excitotoxicity. DUB inhibitors may serve as ther- apeutic candidates for protecting RGCs under stressful conditions linked with excitotoxicity.

Acknowledgements
This work was supported by the grant from the State Key Program of National Natural Science Foundation of China (No. 81790641), Zhejiang Provincial Natural Science Foundation of China (No. LY20C090001) and Ningbo Science and Technology Project (Nos. 2019C50085 and 2019C50053).

Conflicts of interest
There are no conflicts of interest.

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