3. Material and methods
3.6 Measuring reactive oxygen species (ROS) activity in vitro by
The effects of NH043-1 on the ROS generations by glutamate-induced excitotoxicity in SH-SY5Y cells were measured by using CL analysis system. SH-SY5Y cells were plated in 6 cm dish (1 x 106 cells/dish) for 24 h, and the cells were pre-treated with 15 uM NH043-1 or 10 uM MK-801 for 1 h, then challenged with 100 mM MSG for 24 h. Then the cells were washed with PBS and lysed with 60 uL RIPA on ice for 5 min, and sonicated and centrifuged at 13,000 rpm for 20 min at 4oC. The 200 uL protein samples (20 ug) were mixed with 0.5 mL of 0.2 mM luminal (5-amino-2, 3-dihydro-1, 4-phthalazinedione, Sigma). After 5 min, CL was measured by the amounts of chemiluminescence with a CL analysis system (CLD-110, Tohoku Electronic Inc. Co., Sendai, Japan).
3.7 Measuring mitochondrial membrane potential in vitro by flow cytometery
The effects of NH043-1 on the mitochondrial membrane potential by glutamate-induced excitotoxicity in SH-SY5Y cells
were measured by using flow cytometry with JC-1 dye staining.
SH-SY5Y cells were plated in 6 cm dish (1 x 106 cells/dish) for 24 h, and then the cells were pre-treated with 15 uM NH043-1 or 10 uM MK-801 for 1 h, followed by being challenged with 100 mM MSG for 12 h. The cells were washed and collected by centrifugation at 2000 rpm for 3 min, and after washing with PBS, the cells were stained using JC-1 dye for 30 min. For positive control group, 5 uM carbonyl cyanide 3-chlorophenyl hydrazine (CCCP) was added to induce mitochondrial membrane depolarization. According to the kit’s protocol, the cells were washed and re-suspended in PBS, and analyzed with a flow cytometer by two parameter dot-plots. A total of 10,000 cells were recorded in each case.
3.8 Dot-blot filter retardation assay
The effects of NH043-1 on the nTBP/Q36-EGFP and nTBP/Q79-EGFP aggregation in nTBP/Q36-EGFP and nTBP/Q79-EGFP SH-SY5Y cells, respectively, induced by doxycycline were measured by using BRL dot-blot instrument (Bio-Rad). The nTBP/Q36 and nTBP/Q79 cells were plated in 6 cm dish (1 x 106 cells/dish) for 24 h, and the cells were
pre-treated with 15 uM NH043-1 for 1 h, then treated with 10 uM doxycycline and 10 uM RA for 5 d. The cells were washed and collected by centrifugation at 2,000 rpm for 3 min at 4 oC, and then removed PBS. The cells were incubated on ice for 30 min in the buffer (100 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl pH 8.8, 0.5% NP-40, 100 mM EDTA, and cocktail).
After centrifugation at 14,000 rpm for 5 min at 4 oC, the precipitates were dissolved with the buffer (15 mM MgCl2, 20 mM Tris-HCl pH 8.0, and 0.5 mg/mL DNase I) at 37 oC for 60 min. The protein concentrations were determined with bicinchoninic acid (BCA) protein assay kit (Thermo scientific).
Twenty mg protein was mixed with 2% SDS solution to 0.2 mL and loaded to dot-blot machine through a cellulose acetate membrane. Membrane was washed with 0.1% SDS solution, blocked with 5% non-fat milk in TBST solution at room temperature for 1 h, and washed thrice with TBST for 5 min, then, incubated with primary TBP (N-12) antibody (1:1000) in dilute buffer overnight at 4oC. After three quick washes in TBST buffer for 5 min, the membranes were treated with HRP-conjugated secondary anti-rabbit antibodies for 1 h, and the proteins were visualized by using an enhanced chemiluminesence (ECL) detection reagent (Millipore), and
detected with an ImageQuestTM LAS-4000 (Fujifilm Co., Tokyo, Japan). The expressions of protein were quantified by ImageJ (National Institute of Health, USA)
3.9 Animal model
SCA17 transgenic mice bearing expanded TBP (Q109) were kindly supplied by Dr. Hsiu-Mei Hsieh, NTNU. Mice were housed in individually ventilated cages with a 12 h light/dark cycle. All mice were bred and maintained in the animal facility at NTNU under specific pathogen-free conditions in accordance with institutional guidelines of The Animal Care and Use Committee at NTNU. The animals were used for motor behavioral assessments, footprint test, and western blotting analysis of aggregated TBP and cleaved-caspase-3 of the mouse cerebellum. The body weights of mice for rotarod test, and footprint test were measured twice a month.
3.9.1 Motor behavioral assessments
The mice were trained on the rotarod before drug treatment to establish the baseline of behavior activity
by using a linear acceleration from 2 rpm to 20 rpm over 5 min, and then maintained at 20 rpm for another 5 min. For experimental period, mice were placed with a linear acceleration from 4 to 30 rpm with over 5 min.
The rotarod analysis was performed every two-week until 20th week. On every test, mice were subjected to three trials, each with a maximum duration of 600 sec and latency of fall was recorded. Mice were tested between 12:00 and 18:00.
3.9.2 Footprint patterns analysis
The test is widely used to determinate motor skill, coordination and balance. Hind- and forefeet of mice were coated with red and blue nontoxic paint, and the animals were allowed to walk along a runway over a fresh sheet of white paper. The distances between the center of the hind and preceding front footprint, length of steps and distance of parallel fore-paw/hind-paw were recorded over a sequence of six consecutive steps, excluding footprints made at the beginning and end of the run. The footprint patterns were determined
quantitatively by the measurements of stride length and fore-paw/hind-paw overlap. The tests were measured by every half of month.
3.9.3 Western blot analysis of aggregated TBP and cleaved-caspase-3 protein in the cerebellum of tested mice
All mice were anesthetized using urethane (1.5 mg/kg, i.p), and after 10 min, cerebella of mice (n=6, for each group) were collected in cold RIPA buffer, homogenized by 22G and 26G (32 mm and 13 mm) needles (TERUMO® Needle, NEOLUS) on ice, and stored at -80oC. The protein concentrations of homogenates of cerebella were measured with bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Then, 40 mg protein was resolved on 12.5%
SDS-PAGE, and after electrophoresis and semi-dry blotting, the PVDF membrane was blocked with 5%
non-fat milk solution in TBST at room temperature for 1 h or overnight at 4oC. The PVDF was washed thrice with TBST for 5 min, then, incubated with primary
antibody in dilute buffer overnight at 4oC. After three quick washes in TBST buffer for 5 min, the membrane was treated with HRP-conjugated secondary antibodies for 1 h, and the proteins were visualized by using an enhanced chemiluminesence (ECL) detection reagent (Millipore) and detected with an ImageQuestTM LAS-4000 (Fujifilm Co., Tokyo, Japan). The expression of proteins was quantified by ImageJ (National Institute of Health, USA).
4 Results
4.1 NH043-1 effectively protects SH-SY5Y cells against MSG-induced excitotoxicity.
MTT assay, a colorimetric assay, was performed for measuring the activity of cellular enzymes representing the cell viability. MSG was used to induce excitotoxicity in neuroblastoma SH-SY5Y cell. First, cells were treated with 20, 40, 60, 80, and 100 mM MSG for 24 h to determine the IC50
toxicity of MSG. The IC50 toxicity of MSG was determined to be 100 mM for 24 h (Figure 1A), and 100 mM MSG was used with following experiments. Then, the IC50 of active compounds of CHMs on SH-SY5Y cells were measured, and half or one-fifth of compounds IC50 (1/2X or 1/5X IC50) were used to screen the most effective compound against MSG. As shown in Table 1, NH043-1 exhibited 85% increased of cell viability, compared to MSG. Therefore, NH043-1 was chosen for further studies.
The treatment of SH-SY5Y cells with 5, 10, 15, 20, and 25 uM of NH043-1 against MSG for 24 h showed that 15 uM of NH043-1 significantly protected the cells against 100 mM MSG-induced excitotoxicity, exhibiting 85% cell viability, as 10
uM MK801 did. MK801 is an antagonist of NMDA receptor and was used as positive control in this study (Figure 1B).
Collectively, our results indicate that NH043-1 showed a remarkably inhibitory effect on MSG-induced excitotoxicity.
4.2 NH043-1 attenuates the apoptosis of SH-SY5Y cells induced by MSG.
To examine whether NH043-1 inhibits apoptosis of SH-SY5Y cells mediated by MSG, flow cytometry analysis using Annexin-V staining, which is an apoptosis marker, was carried out. The results demonstrated that the treatment of 100 mM MSG for 24 h induced 54.13% apoptotic cells, however, NH043-1 at 15 uM and MK801 at 10 uM caused 9.04% and 9.73% apoptosis in the presence of MSG, respectively (Figure 2).
It indicates that NH043-1 effectively reduced apoptosis of SH-SY5Y cells induced by MSG.
4.3 NH043-1 decreases the expressions of calpain-2 and SBDPs in SH-SY5Y cells treated with MSG.
To study whether NH043-1 protects SH-SY5Y cell apoptosis against MSG-induced excitotoxicity via calcium-dependent apoptosis, Western blot analysis was performed. Calpain-2, a thiol proteinase, is activated by the increases of intracellular free calcium ions and the reduction of Bcl-2 level, and SBDPs was elevated by calcium-induced calpain-2 in glutamate-induced cell death (Anderton et al. 2011, Miao et al. 2012). The calpain-2 and SBDPs were investigated.
The 20% increments of both calpain-2 and SBDPs respectively were found by treating the cells with 100 mM MSG for 6 h. By pretreatment with 15 uM NH043-1 and MSG for 6 h, the expression of calpain-2 and SBDPs were decreased 200% and 250% (Figure 3). It indicates that NH043-1 significantly protected cell apoptotic death through calcium-induced apoptosis in MSG-treated SH-SY5Y cells.
4.4 NH043-1 decreases the expressions of Bax, but increases that of Bcl-2 in SH-SY5Y cells treated with MSG.
To study whether NH043-1 protects SH-SY5Y cell apoptosis against MSG-induced excitotoxicity via mitochondria-dependent apoptosis, Western blot analysis was performed. The increases of intracellular free calcium ions were related to the reduction of Bcl-2 level and the increment of Bax level (Anderton et al. 2011, Miao et al. 2012). The cell-survival protein, Bcl-2, and pro-apoptotic protein, Bax, which both mediate the release of cytochrome C from mitochondria, were investigated. The 50% increments of Bax, and the 40%
decrement of Bcl-2, respectively were found by treating the cells with 100 mM MSG for 6 h. By pretreatment with 15 uM NH043-1 and MSG for 6 h, the expression of Bax were decreased 100%, while that of Bcl-2 was increased 150%
(Figure 4). It indicates that NH043-1 significantly protected cell apoptotic death through mitochondria-dependent apoptosis in MSG-treated SH-SY5Y cells.
4.5 NH043-1 inhibits the expressions of caspase family proteins mediated by MSG.
Caspases, a family of cysteine proteases implicated in classical apoptotic death, were initially shown to play a role in delayed excitotoxic injury, which is associated with caspase-9, caspase-3 and nuclear enzyme PARP activation. To study whether NH043-1 protects SH-SY5Y cells death by MSG-induced excitotoxicity through caspase-dependent apoptosis, Western blot analysis was carried out. Treating the cells with 100 mM MSG for 24 h resulted in the 40%, 60%, and 190% increases of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP respectively. By treating 15 uM NH043-1 against MSG for 24 h, the expressions of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, were significantly decreased 125%, 87%, and 52% inhibition, respectively, that of MSG treated cells, suggesting that NH043-1 increased cell viability by inhibiting the expressions of caspase family proteins in MSG-treated SH-SY5Y cells (Figure 5).
4.6 NH043-1 inhibits the intracellular ROS induced by MSG.
The glutamate-induced cytotoxicity was associated with mitochondrial dysfunction and increased ROS production in neuronal cells (Votyakova and Reynolds 2005). To investigate that NH043-1 would inhibit the accumulation of ROS implicated by MSG-induced excitotoxicity, the ROS level was measured by CL assay. Luminol is activated with an oxidant to exhibit its chemiluminescence. The emission spectrum is blue glow and can be detected with a chemiluminescence detector. We found that the intracellular ROS was 80% increment by 100 mM MSG-induced excitoxtoicity, but extracellular ROS was not affected. By treating with 10 uM NH043-1 or 10 uM MK801 for 24 h after MSG treatment, the intracellular ROS was 80%
inhibition and the extracellular ROS did not alter (Figure 6). The results suggested that NH043-1 reduced the intracellular ROS production induced by MSG.
4.7 NH043-1 blocks the decrease of mitochondrial membrane potential (MMP) mediated by MSG.
To study whether NH043-1 blocks the decrease of MMP in SH-SY5Y cells mediated by MSG, flow cytometry analysis was performed. JC-1 dye could be used as an indicator of mitochondrial membrane potential in a variety of cell types. The results demonstrated that the treatment of 100 mM MSG exhibited 62±10% MMP in relative to that of the control after 24 h MSG treatment. However, NH043-1 at 15 uM showed 85±13% MMP by MSG and the treatment of 10 uM MK801 showed 90±5% MMP. Five uM CCCP, the disruptor of electron transport chain, showed 18±10% MMP (Figure 7). It suggested that NH043-1 effectively reduced the loss of MMP in SH-SY5Y cells arose by MSG.
4.8 NH043-1 effectively increases cell viability against Dox-induced nTBP/Q
79-EGFP cells.
To further study the effect of NH043-1 on SCA17, inducible nTBP/Q36-EGFP and nTBP/Q79-EGFP cells were used.
The cells were induced with 10 ug/mL Dox to express
nTBP/Q36-EGFP and nTBP/Q79-EGFP with or without 15 uM of NH043-1 for 5 d. The cell viability was not changed at each group in nTBP/Q36-EGFP. The treatment of 15 uM NH043-1 protected the cells against nTBP/Q79-EGFP-induced cytotoxicity for 5 d, and the increase of 20% cell viability was observed. It indicates that NH043-1 showed a protective inhibitory effect on nTBP/Q79-EGFP-induced cytotoxicity (Figure 8A).
4.9 NH043-1 inhibits the expression of caspase family proteins mediated by Dox-induced nTBP/Q
79-EGFP cells.
To study whether NH043-1 protects cell death induced by nTBP/Q79-EGFP expression through caspase-dependent apoptosis, Western blot analysis was carried out. The 190%, 80%, and 90% up-regulation of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, respectively, were observed after inducing nTBP/Q79-EGFP expression with 10 uM Dox for 5 d. The treatment of 15 uM NH043-1 significantly decreased the expressions of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, resulting in 88%, 100%, and 44% inhibition, respectively (Figure 8B and 8C). It suggests
that NH043-1 increased cell viability by inhibiting the expressions of caspase family proteins induced by nTBP/Q79-EGFP expression through caspase-dependent apoptosis.
4.10 Effects of NH043-1 on polyQ protein aggregation by Dox-induced nTBP/Q
36-EGFP and nTBP/Q
79-EGFP cells.
To investigate whether NH043-1 decreases protein aggregation by Dox-induced in nTBP/Q79-EGFPSH-SY5Y cells, dot-blot assay and Western blot were performed.
nTBP/Q79-EGFP protein aggregates were increased higher than that of nTBP/Q36-EGFP by dot-blot and western blot analysis, after inducing with 10 uM Dox for 5 d. However, 15 uM NH043-1 significantly decreased the amounts of nTBP/Q79-EGFP protein aggregates, 67% and 90% inhibition examined by dot-blot and Western blot analysis, respectively (Figure 9). It suggests that NH043-1 increased cell viability by inhibiting the protein aggregation of nTBP/Q79-EGFP proteins.
4.11 NH043-1 ameliorates the neurological behavior of SCA17 transgenic mice.
To investigate the therapeutic effect of NH043-1 for SCA17 in vivo, a SCA17 transgenic mouse model was used (Ref). From
10- to 20-week-old mice, the body weights of SCA17 transgenic mice were not much different from those of control mice (Figure 10A). The experiments were carried out by i.p. administration of 4.5mg/kg of NH043-1 in saline to SCA17 transgenic mice once every two days from 8-week to 20-week, whereas control mice were given saline. First, motor coordination was examined by rotarod performance, and the results showed that saline-treated SCA17 mice, 269±36 seconds in the average of falling time was shorten than that of control mice group, 558±33 seconds, but it was improved in NH043-1-treated SCA17 mice, 393±36 seconds comparing with that of saline-treated SCA17 mice at 20 weeks (Figure 10B). Furthermore, the footprint patterns were measured to estimate left and right paw overlaps. We found that left and right paw overlaps of saline-treated SCA17 mice were 1.5±0.2 cm and 1.6±0.2 cm, respectively, longer than that of WT mice, 0.6±0.1 cm for left paw and right paw. However, the paw overlaps of SCA17 mice were shorter in NH043-1-treated
SCA17 mice to 0.8±0.1 cm and 1.1±0.1 cm for left and right paw, respectively, comparing with that of the saline-treated SCA17 mice (Figure 11). The results indicated that NH043-1-treated rescued the motor coordination deficits in SCA17 mice.
4.12 NH043-1 attenuates TBP/polyQ aggregation and cleaved-casapase-3 protein in cerebella of SCA17 mice.
To examine whether the NH043-1 treatment decreases the TBP/polyQ proteins aggregation and cleaved-caspase-3 proteins expression in SCA17 mice cerebella, we measured TBP/polyQ proteins aggregation and the level of cleaved-caspase-3 protein in cerebella of mice brain, and the results indicates that the up-regulation of aggregated TBP/polyQ proteins and cleaved-caspase-3, was both 100% in the cerebella of 20-week saline-treated SCA17 mice, comparing with that of control mice.
Interestingly, 4.5mg/kg NH043-1-treated SCA17 mice showed the decrease of the expressions of aggregated TBP/polyQ and cleaved-caspase-3, 100% and 25%, respectively, indicating that NH043-1 significantly inhibited the expression of aggregated
TBP/polyQ and cleaved-caspase-3 in cerebella of SCA17 mice (Figure 12).
5 Discussion
Recently, CHMs have been used for the treatment of neurodegenerative disorders through the alterations in different receptor-mediated pathways, such as oxymatrine showing partial protection in the cortical neurons via down-regulation of NR2B containing NMDA receptors and up-regulation of Bcl-2 family (Zhang et al. 2013). Glutamate is the predominant excitatory neurotransmitter in the CNS, and excessive cellular glutamate release has been connected to excitotoxic events in neurodegenerative disease, where excessive glutamate binding to specific receptor, such as NMDA receptor, results in cell death (Mattson 2008, Lau and Tymianski 2010, Yang et al. 2011). In the present study, we observed MSG-mediated excitotoxicity in a dose-dependent manner in SH-SY5Y cells (Figure 1A), and screened many kinds of compounds from CHMs against MSG. NH043-1 was found to increase cell viability of MSG-induced in SH-SY5Y cells (Figure 1B). In addition, NH043-1 pretreatment protected the cells from apoptosis induced by MSG in SH-SY5Y cells, demonstrated by Annexin-V/PI staining followed by analysis with flow cytometry (Figure 2). Conclusively, NH043-1 exhibits protective effects on MSG-induced excitotoxicity
Exploration of mechanism underlying implies that glutamate excitotoxicity is related to the loss of mitochondrial membrane potential and calcium imbalance (Abramov and Duchen 2008, Pivovarova et al. 2013). The increase of cytosolic calcium in glutamate-treated hippocampal neurons triggered calpain activation (Brustovetsky et al. 2010). Besides, glutamate-mediated excitotoxicity induced apoptosis through mitochondrial apoptotic pathway (Pfeiffer et al. 2013, Zhang et al. 2014). The mitochondrial pathway plays an important role in neurodegenerative disorders.
Cytochrome C releasing from the inter-membrane of mitochondria into cytoplasm binds Apaf-1, leading to the activation of caspase-9 and caspase-3. In addition, mitochondrial apoptotic pathway is mediated by members of the Bcl-2 family, such as Bax and Bcl-2 (Rana et al. 2007), that may have particularly important roles in mediating or protecting against calcium-mediated neuronal death by modifying mitochondrial membrane permeability (Chen et al. 2012, Duchen 2012). In this study, several pieces of evidence demonstrated that NH043-1 inhibited SH-SY5Y cells apoptosis by MSG through mitochondrial pathway. NH043-1 inhibited the MSG-induced calcium-dependent protein, calpain-2, SDBPs, and pro-apoptotic protein, Bax, but increased anti-apoptotic protein, Bcl-2 in SH-SY5Y cells (Figure 3 and Figure 4), as well as decreased the
cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP in SH-SY5Y cells (Figure 5), suggesting that NH043-1 could prevent mitochondria-mediated apoptosis.
Over-loading of calcium in neuron cells initiates the loss of mitochondrial membrane potential and produces ROS, leading to apoptosis (Lin and Beal 2006, Duchen 2012, Pfeiffer et al. 2013). In this study, we observed that NH043-1 inhibited the loss of mitochondrial membrane potential and the production of ROS (Figure 6 and Figure 7).
The neuroprotective effects of NH043-1 on AD were explored in recent years, and NH043-1 was first showed to prevent memory impairment and oxidative damage induced by Aβ25-35, suggesting that NH043-1 may be a potential therapeutic agent against AD Furthermore, NH043-1 was also demonstrated to inhibit Aβ aggregation. In addition to AD model, the applications of NH043-1 on other neurodegenerative diseases are rarely defined. This study was designed to investigate the effect of NH043-1 on SCA17 cells and transgenic mice model. We demonstrated that NH043-1 increased cell viability and decreased the expression of cleaved-caspase-9,
cleaved-caspase-3, and cleaved-PARP in nTBP/Q79-EGFP SCA17 cells (Figure 8).
Protein misfolding and aggregation in the brain have been implicated as a common molecular pathogenesis of various neurodegenerative diseases (Proctor et al. 2010, Chang et al. 2011, Burke et al. 2013). SCA17 is characterized by protein aggregates in N-terminal region of TBP and selective loss of cerebellar neurons, particularly Purkinje cells (Koide et al. 1999, Nakamura et al. 2001, Orr and Zoghbi 2007). Therefore, SCA17 could share a common molecular mechanism leading to neuronal cell death mediated by protein aggregation (Chang et al. 2011, Huang et al. 2011, Roshan et al. 2012). We demonstrated that NH043-1 effectively suppressed aggregate formation by using Western blot and dot-blotting assay in nTBP/Q79-EGFP SCA17 cells (Figure 9).
The pathogenesis of the SCA17 transgenic mice recapitulates the aggregation of protein, the loss of Purkinji cells, the motor coordination deficits, and cerebellum atrophy observed in SCA17 patients (Friedman et al. 2008, Chang et al. 2011). We demonstrated that NH043-1 improved the motor performance on an accelerating rotarod (Figure 10B) and ameliorated gait abnormalities, including