p21 inhibitor UC2288 ameliorates MPTP induced Parkinson’s disease progression through inhibition of oxidative stress and neuroinflammation


 BackgroundParkinson's disease (PD) is a neurodegenerative disease characterized by the early prominent death of dopaminergic neurons and a decrease of dopamine levels. Dopamine depletion leads to several motor dysfunctions, including resting tremor, muscular rigidity, bradykinesia and postural instability. Our previous study determined that knockout of parkin, a gene of PD degrade p21, suppresses neurogenesis which is critical for a neurodegenerative disease. MethodsThus, we investigated the effect of UC2288, an inhibitor of p21, for its therapeutic effect on PD. We found that UC2288 attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral impairment in Rota-rod and Pole test as well as dopamine depletion.ResultsMoreover, UC2288 recovered the number of TH positive cells, but decreased the number of GFAP and Iba-1 positive cells accompanied the decrease of BAX and cleaved caspase3 as well as iNOS and COX-2 expression. In cultured neurons, UC2288 recovered MPP+-induced neuronal cell death in a concentration dependent manner. We also found that UC2288 decreased the p21 reactive cell number, oxidative neuronal damages, cytokines product in vivo and cultured neurons. In a mechanism study, we found that UC2288 significantly decreased the activation of ERK and p38 kinase pathway in the mitogen-activated protein kinase (MAPK) pathway. In addition, 1-10 μM concentration of ERK kinase inhibitor U0126 recovered MPP+-induced neuronal cell death. However, ERK kinase inhibitor U0126 further decreased cell viability with the increase of H2O2.ConclusionThese results indicated that the administration of UC2288 exerted neuroprotective effects on the death of dopaminergic neurons through the suppression of oxidative stress and neuroinflammation via ERK pathway inhibition.

concentration dependent manner. We also found that UC2288 decreased the p21 reactive cell number, oxidative neuronal damages, cytokines product in vivo and cultured neurons. In a mechanism study, we found that UC2288 significantly decreased the activation of ERK and p38 kinase pathway in the mitogen-activated protein kinase (MAPK) pathway. In addition, 1-10 μM concentration of ERK kinase inhibitor U0126 recovered MPP+-induced neuronal cell death. However, ERK kinase inhibitor U0126 further decreased cell viability with the increase of H 2 O 2 .

Conclusion
These results indicated that the administration of UC2288 exerted neuroprotective effects on the death of dopaminergic neurons through the suppression of oxidative stress and neuroinflammation via ERK pathway inhibition.

Background
Parkinson's disease (PD) is a common chronic neurodegenerative disease characterized by muscle tremor, as well as decreased mobility and coordination capacity 12, . The pathogenesis of PD is associated to the degeneration of dopaminergic neurons 3 . This degeneration of dopaminergic neurons reduces the dopamine content in cerebral cortex 3 . Although L-dopa therapy has been the mainstay of PD therapy since 1969 4 , limitations of its efficacy and side effects have necessitated the development of new target oriented drugs 5 . Moreover, as dopaminergic drugs cannot easily penetrate the Brain-blood-barrier, they need to be systemically administered at a high dose.
Therefore, other treatment strategies such as aromatic amino acid decarboxylase (AADC), glutamic acid decarboxylase (GAD) and glial cell line-derived neurotrophic factor (GDNF) gene therapy have all been suggested 6-8 .
Reactive Oxygen Species (ROS) may result in significant damage to cell structures. In PD, ROS is a major cause of neuronal death. In animal models of PD-like symptoms, ROS causes the activation of microglial cells which subsequently attack neighboring dopaminergic neurons 9 . Vitamin E scavengers several ROS, thus inhibiting lipid peroxidation ameliorates the severity of PD 10 . A combination of ascorbate and α-tocopherol has also used for patients with early PD 9 . The neuroprotective effects of CoQ 10  Several studies have demonstrated the involvement of the mitogen activated protein kinase (MAPK) pathway in the development of PD. The p38 MAPK activation was increased in a 6-hydroxydopamine (6-OHDA) induced mouse PD model, and it was also implicated in the mechanism of neuronal cell death 13-15 . α-Synuclein, a marker of PD, activated p38, ERK, and JNK pathways to produce IL-1β and TNF-α, and thus promoted neuroinflammation in human microglial cells 16 . Moreover, rotenone induced the activation of JNK and p38 MAPK caused apoptosis in dopaminergic SH-SY5Y cell death 17 .
However, the inhibition of JNK attenuated neuronal apoptosis and increased dopamine levels in both in vitro and in vivo models of PD 18 . It has also been reported that mice deficient in JNK3 were more resistant to dopaminergic neuron survival by MPTP than their corresponding wild-type 19 . Treatment of dopaminergic cells with 6-OHDA activated ERK1/2 pathway, as well as pharmacological inhibition of the ERK pathway enhanced neuronal survival 20 . Many studies have also demonstrated that ROS can induce the activation of the MAPK pathways in neuronal cell death 152122,, .
Parkin, a ubiquitin E3 ligase, ubiquitinates proteins in order to regulate a variety of cellular processes, with its mutation begin the second most commonly known cause of PD 23 . Parkin knockout mice display normal performance on behavioral tests, but they experience disrupted memory and leak dopamine from the nerve 24 . Overexpression of parkin protected acute α-synuclein induced dopaminergic neurons in primary culture 25 and in a rat brain 26 . In PD, the expression of p21 ras was increased in MPTP-induced mice brains 27 . Moreover, increased p21 degradation was formed in lymphocytes as a result of PD 2829, . It has also been reported that simvastatin inhibited p21 activation and prevented the loss of dopaminergic neurons in a mouse PD model 27 . Our previous study found that parkin knockout in a mouse inhibits neurodevelopment via the degradation of p21 1 . Further RNA seq results revealed that p21 is closely related to both with SNAP25 and BDNF, as well as parkin in the brain of PD patient 1 . A recent study also indicated that the mutation of reduced solubility and E3 ligase activity resulted in progressive neuronal degeneration through the accumulation of toxic proteins 30 . It was also found that parkin expression protects 6-OHDA induced neuronal damages 31 . Therefore, it is possible that knockout parkin could not degrade p21, and its accumulation may inhibits neurogenesis causing the progression of neurodegenerative disease such as PD. Thus, in the present study, we investigated whether a p21 inhibitor could be effective for the treatment of PD and its action mechanisms.

Modeling method
MPTP was used to establish the PD mice (C57BL6) model. Each mouse in MPTP group received intraperitoneal injection of MPTP 20 mg/kg for 4 times in 7 days, while the same amount of normal saline was injected to each mouse in CON group. UC2288 group each mouse was injected with 10 mg/kg UC2288 and MPTP + UC2288 group treated MPTP (20 mg/kg) with UC2288 (10 mg/kg) for 4 6 times in 7 days by intraperitoneal injection under the guideline of National Institute of Toxicological Research Korea and Drug Administration for the human care and use of laboratory animals and approved by IACUC of Chungbuk National University CBNUR-1117-18. 1-2 h later after the last injection, the behavioral tests and biochemical experiments were conducted in succession.

Behavior Tests
Behavior changes were tested by Rota rod, Pole and gait tests as described elsewhere 32 . Motor performance and coordination were examined using the Rota rod treadmill (MED Associates Inc., St. Albans, VT.), consisting of a 3.6-cm diameter cylindrical treadmill connected to a computer controlled stepper motor as described previously 33 . The pole test trials were performed using the roughsurfaced wooden pole three times per animal and average values from three examinations were used for each animal. A gait test trial was performed on a bright runway (4.5 cm wide) and a dark goal box (20 × 17 × 10 cm) and stride length was measured as the distance between successive paw prints.
Data was presented as the average of five strides for each animal.

Cresyl Violet Staining
The brains were post fixed in 4% paraformaldehyde for 24 h at 4 ℃, and then transferred to 30% sucrose solutions. 25-µm brain sections were thoroughly washed with phosphate-buffered saline (PBS), and then remove the excess fixative agent and then transferred to gelatin-coated slide glasses and stained with 0.1% cresyl violet (2-5 min) for the purpose of identifying cortical layers and cytoarchitectural features of the isocortical region. After this, the sections were washed in distilled water then dehydrated through ascending grades of ethanol, 50, 70, 90, and 100% ethanol for 2 min in each grade followed by 10 min immersion in a 1:1 mixture of absolute alcohol and xylene. They were cleared in xylene for 5-10 min and mounted in mounting medium (Cytoseal™ XYL; Thermo Scientific, Pittsburgh, CA).

Oxidative Stress Assay
The MDA concentration in brain tissue and neuronal cells were determined based on thiobarbituric acid (TBA) reactivity (Cell Biolabs, Inc. San Diego, CA) by the manufacture's guidance. Briefly, trichloroacetic acid with HaCaT keratinocytes mixture were centrifuged, and then supernatant was 7 related with TBA. The reactingr color was measured at 532 nm with a spectrophotometer. H 2 O 2 binds to molybdenic acid complex was assessed using a commercially available kit (DCFH-DA, Sigma Aldrich) specifically as a H 2 O 2 content at 405 nm, and the H 2 O 2 content was then calculated. GSH-Px activity was determined by the velocity method using a GSH-Px kit (DCFH-DA, Sigma Aldrich). The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 412 nm as GSH-Px activity. SOD production of ARBEC was assessed with a superoxide anion assay kit (Catalog Number CS1000, Sigma, USA). The cells were grown in none serum medium, and they were Scientific, Waltham, USA), and 1 mmol/L lactic acid (Sigma-Aldrich, UK) at a density of 6 × 10 5 cells per cm 2 . The percentage of neuronal cells in our culture system is more than 90% judged with cell morphology and immunostaining measurement. Cells were incubated at 37 °C and 95% humidity with 5% CO 2 . When the cells reached ∼80% confluence, the cells were then treated either with freshly prepared MPP + in DMSO (100 nM final concentration).

TUNEL Assay
Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used for TUNEL assay according to the manufacturer's instructions. briefly, after fixation of 25-mm cryosections with 4% paraformaldehyde, 0.1% NaBH4 and 0.1 Triton X-100 was treated The slides were then incubated for at least 1 h with mixture reaction buffer containing deoxynucleotidyl transferase and FITC-dUDP (Roche, Reinach, Switzerland). For 40, 60-diamidino-2-phenylindole dihydrochloride (DAPI) staining, the slides were then incubated for 15 min at room temperature in the dark with a mounting medium for fluorescence containing DAPI (Vector Laboratories, Cambridgeshire, UK). The tissues were then examined through a fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany), and the nuclei were visualized via DAPI staining.

Statistical Analysis
GraphPad Prism 4 software (GraphPad Software, La Jolla, CA) was used for statistical ananlysin. A oneway analysis of variance (ANOVA) was applied for assessment of difference among the graph. Once the significant was found, the differences were further analyzed by the Dunnett's test. Data are presented as mean ± SD, a value of P < 0.05 was considered to be statistically significant.

Effect of UC2288 on behavioral impairments by MPTP
We examined the effect of UC2288 on behavior disorders by MPTP. The rota-rod test was conducted in order to test coordination capability. The MPTP treated group significantly decreased latency on the rota-rod. However, the latency in the MPTP with UC2288 (10 mg/kg) co-treated mice (44.7 ± 49.5 s) was significantly increased compared to that in the MPTP treated mice (94.8 ± 32.5 s) (Fig. 1A). Next, we performed the pole test, which measures the time descending from the top of the pole to the floor.
The time is considered to reflect bradykinesia. The control and UC2288 treated groups did not show any significant differences as far as behavior disorders. However, the time to descend was significantly decreased in the MPTP with UC2288 (10 mg/kg) treated mice (22.8 ± 6.49 s) compared to that in the MPTP treated mice (41.0 ± 1.0 s) (Fig. 1A). When the stride length test was performed, the results showed that the stride length of the hind limb was lengthened by more in the MPTP with UC2288 co-treated mice (4.9 ± 0.6 cm) than in the MPTP treated mice (4.5 ± 0.3 cm) (Fig. 1A).
However, there was no significant difference found in terms of behavior disorders between MPTP treated mice and MPTP with UC2288 co-treated mice.

Effect Of UC2288 On MPTP-induced Apoptosis In Mice Brains
Cresyl violet staining was performed in order to assess the cell death in the striatum. We counted the number of stained cells indicated by cresyl violet. The average number of stained cells in the UC2288 treated mice brains was 691.658 /cm 2 , compared to that (390.29 /cm 2 ) in MPTP treated mice ( Fig. 1B). This result indicated that UC2288 suppressed MPTP-induced neurological cell death.
Western blot data for BAX and cleave-caspase3 (a pro-apoptotic protein) expression was also decreased in the MPTP and UC2288 treated mice brain compared to the expression in MPTP treated mice brains (Fig. 1C).

Effect Of UC2288 On ROS Generation And Lipid Peroxidation
It was well known that MPTP induces an oxidized lipid by forming a high ROS level, which is a critical factor for neuronal cell death. Therefore, we examined ROS generation and lipid oxidation in the MPTP treated mice brains. The ROS level was elevated by MPTP, however, the elevated ROS was reduced by the treatment of UC2288 (10 mg/kg) ( Fig. 2A). There was no significant difference in the ROS levels between the control and UC2288 (10 mg/kg) treated mice groups. We also determined lipid peroxidation through measuring the MDA level. The MDA levels were also significantly higher in the MPTP treated group, but these levels were also reduced by UC2288 treatment. There was no significant difference in MDA level between the control and UC2288 (10 mg/kg) treated mice groups.
Moreover, the GSH/GSSG ratio was also increased by UC2288 treatment compared to MPTP treated mice ( Fig. 2A).

Effect Of UC2288 On Cytokine Level
TNF-α, IL-6, and IL-1β all play important roles in neurodegenerative diseases, and these cytokines could be released by the oxidative stress of neuronal cells. We thus examined the cytokine levels.
MPTP treatment raised TNF-α, IL-6 and IL-1β levels in MPTP treated mice brain. However, TNF-α, and IL-6 release were both significantly decreased by UC2288 treatment in MPTP treated mice brains, but IL-1β was not (Fig. 2B). There was no significant difference in cytokine level between control and UC2288 (10 mg/kg) treated mice brains.

Effect Of UC2288 On Dopaminergic Neurodegeneration
Abundant TH-positive fibers in the striatum and substantia nigra were found in the control and UC2288 treated groups, whereas the number of TH-positive neurons was significantly lowered in the striatum and substantia nigra of the MPTP treated mice brain. However, UC2288 treatment recovered the number of TH-positive neurons in MPTP treated mice. (Fig. 3A). In addition, the GFAP and Iba-1 reactivity was also reduced following treatment of UC2288 in MPTP treated mice (68.79 vs 53.43 cells/mm 2 ) (Fig. 3A). Treatment of UC2288 recovered dopamine level in the MPTP treated brain, which was associated with the recovery of TH positive cells (Fig. 3B).
UC2288 prevented activation of MAPK pathway in the MPTP treated mice brain We speculated that MPTP-induced ROS generation might related to the activation of the MAPK pathway. Increased activation of the MAPK pathway in MPTP treated mice was found (Fig. 4A).
However, UC2288, a p21 inhibitor suppressed the phosphorylation of p38 and ERK in the MPTP treated mice brains (Fig. 4A). In addition, the MPTP-elevated expressions of iNOS and COX-2 were also decreased by UC2288 (Fig. 4B).

UC2288 Reduced ROS And Suppressed Cell Death In Vitro
Treatment of MPTP causes the degeneration of dopaminergic neurons. Exposure for 24 h to MPTP up to 100 µM causes cell death in cultured neurons. MTT assay results revealed that UC2288 (at concentrations of 10, 20 and 50 nM) treatment with MPTP significantly increased the neuronal cell viability in a concentration-dependent manner (Fig. 5A). In order to investigate apoptosis of neuronal cells, we performed a TUNEL assay in the primary cultured neuronal cells. In the MPTP group, TUNELpositive apoptotic cells were significantly higher in the MPTP treated group, but the number of apoptotic cells was decreased by UC2288 treatment (Fig. 5B). As shown in Fig. 5C, whole-cell intracellular ROS production was also quantified as an index of MPTP-induced oxidative stress.
Exposure for 24 h to 100 µM MPTP caused increase H 2 O 2 and decreased GSH levels, while treatment with UC2288 returned their levels to their previous values (Fig. 5C).

UC2288 inhibited p21 expression and suppressed MAPK pathway in vitro
We co-treated UC2288 and MPP + in primary cultured neurons. There was no significant difference with UC2288 (50 nM) treatment alone. However, treatment of MPP + increased p-p21 level, but UC2288 reduced its expression (Fig. 6A). Moreover, the expression of MAPK family; (p-ERK, p-JNK, p-P38, and iNOS and COX-2) was increased by MPP+, but their expression was decreased by UC2288 treatment in primary cultured neurons (Fig. 6B). Of the MAPK family, the MPP+-induced phosphorylation of ERK was most significantly decreased by UC2288 (Fig. 6B) Inhibition of the ERK pathway abolished the recovering effect of p21 inhibitor on MPP + induced ROS and IL-6 and cell death in vitro Exposure for 24 h to MPP+ (100 µM) causes cell death in cultured neurons. However, inhibition of ERK kinase (U0126) increased cell viability. The inhibition of ERK most dramatically reduced cell death in 5 and 10 µM (Fig. 7A). However, the abolishment of the ERK pathway by U0126 treatment further increased H 2 O 2 levels, while decreasing cell survival, but no changed IL-6 level (Fig. 7B).

Discussion
PD is one of the most prevalent neurodegenerative disease, and is characterized by typical motor manifestations including resting tremor, rigidity, bradykinesia, and postural instability 2 . Loss of dopaminergic neurons in the substantia nigra and the depletion of dopamine in the striatum are associated with these motor manifestations 35 . However, the specific mechanisms underlying these actions are clear, making it difficult to study ways to cure the disease. Previously, we showed that p21, a substrate of parkin, was not degraded in Park KO mouse through the loss of the ubiquitination ability of parkin, and thus reduced neurogenesis as well as BDNF, SNAP25 expression level compared to WT 1 . Moreover, a study showed that MPP + treatment resulted in the induction of p21 WAF1/Cip1 expression, which caused cell death in dopaminergic neurons 36 . Since the inhibition of neurogenesis 13 is critical for the development of neurodegenerative diseases, we speculated that the inhibition of p21 could attenuate neuronal damages, which could have therapeutically positive effect on Parkinson's disease. We used an MPTP (20 mg/kg)-induced mouse PD model 37 . Our present data showed that UC2288 treatment reduced PD behavioral disorder and dopamine depletion, indicating that p21 could be useful for the treatment of PD. However, the mechanisms behind its effects remain unclear. Moreover, it is interesting to note that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells 45 . In addition, p21 also inhibited nigral activation of p21 ras and p21 rac as well as the level of GSH, and also significantly protected the MPTP-induced nigrostriatal neurons via the increase of GSH 46 . Our studies also showed that treatment of UC2288 recovered MPTP induced decreases of GSH. We also observed that UC2288 treatment reduced MDA and H 2 O 2 levels but recovered GSH levels. Thus, these data suggest that the anti-oxidative effect of p21 inhibitor could contribute to the therapeutic effect on PD.
Neuroinflammation such as astrogliosis and microgliosis is one of the prominent characteristics of PD 47 . Upon activation by brain injury and inflammatory stimuli, astrocytes and microglia excreted 14 amounts of proinflammatory factors such as cytokines and reactive oxygen species (ROS). The accumulation of these factors was recognized as causing the progressive loss of dopaminergic neurons 4849, . Moreover, microglia and astrocytes were activated in PD models 50 . Thus, the inhibition of neuroinflammation exhibits promising therapeutic potential for PD. Therefore several anti-inflammatory agents have been suggested to inhibit the progression of the disorder 51-53 .
In the present study, p21 inhibitor reduced the expression of iNOS, TNF-α and IL-1β levels as well as activation of astrocytes and microglia in a mouse PD model 46 . In support of our data, it was reported that p21 knockout prevented angiotensin II-induced NF-κB activation through reduced ROS generation in vitro and in vivo 54 , and that LPS induced neuroinflammation 55 . Thus, the antineuroinflammatory effects of p21 inhibitor could be also associated with neuroprotective and anti-PD effects.
Several studies have showned that MAPK phosphorylation was activated in a cell-specific manner in the MPTP mouse model 56 . In addition, the activation of p38 kinase and c-Jun N-terminal kinase (JNK) caused neuronal cell death 57 . Further, recent studies have demonstrated that p38 MAPK and JNK were activated by neurotoxicants, stress, or inflammation, and that these activations may be responsible for the development of PD. Thus, the blocking of the p38 MAPK and JNK pathways may aid the development of therapeutic effects for PD patients 5859, . A previous study also indicated that homocysteine-NMDA receptor induced activation of p38 and ERK induced neuronal cell death 6061, .
Moreover, treatment of ERK kinase inhibitor reduced neuronal cell death against oxidative stress 62 .
The inhibition of JNK attenuated neuronal cell death and inflammation-induced cerebral malaria 63 . In our study, p-ERK, p-JNK and p38 were activated by the administration of MPTP, but UC2288 treatment reduced their phosphorylation. Accompanying the prevention of MAPK was activation, and UC2288 treatment suppressed neuronal cell death in MPTP treated mice brains and cultured neuronal cells.
BAX and cleavead-caspase3 levels were also reduced by UC2288. In addition, treatment of p-ERK, p-JNK and p-p38 inhibitors would recover MPP+-induced cell death with the most effectiveness by ERK kinase inhibitor. However, surprisingly, the abolishment of the ERK pathway further decreased cell survival with increased H2O2. This may be due to the inhibition of the prevented ERK pathway. Taken together, these data indicated that the MAPK pathway (and perhaps ERK pathway) could be associated with the preventive effects of p21 inhibitor. Combined with the findings of previous studies 1 , our present data conclusively suggest that p21 may be a target of PD, and that p21 inhibition could be beneficial for stalling the progression of PD.

Conclusion
In conclusion, our systematic study investigated the effect of UC2288, a inhibitor of P21, on the

Consent for publication
Not applicable.

Availability of data and material
All data generated and analyzed during the current study are included in this published article and its supplementary files.

Competing interests
Not applicable.   Effect of UC2288 on ROS generation, lipid peroxidation and inflammatory cytokines level.
(A) MDA levels in the brain striatum site were measured using a TBARS assay kit. H2O2 levels in the brain striatum site were measured using an H2O2 assay kit. GSH/GSSG levels in the brain striatum site were measured using a GSH/GSSG Ratio Detection Assay Kit. (B) Serum levels of cytokines were measured by ELISA. Inflammatory cytokines (TNF-α, IL-6, and IL-1β) were also examined. Each value is presented as mean±SD from eight mice. #, P<0.05: significant difference from Control mice and *, P<0.05: significant difference from MPTP and MPTP + UC2288 mice.
28 Figure 2 Effect of UC2288 on ROS generation, lipid peroxidation and inflammatory cytokines level.
(A) MDA levels in the brain striatum site were measured using a TBARS assay kit. H2O2 levels in the brain striatum site were measured using an H2O2 assay kit. GSH/GSSG levels in the brain striatum site were measured using a GSH/GSSG Ratio Detection Assay Kit. (B) Serum levels of cytokines were measured by ELISA. Inflammatory cytokines (TNF-α, IL-6, and IL-1β) were also examined. Each value is presented as mean±SD from eight mice. #, P<0.05: significant difference from Control mice and *, P<0.05: significant difference from MPTP and MPTP + UC2288 mice.