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VGF and striatal cell damage in in vitro and in vivo models of Huntington’s disease

Huntington’s disease (HD) is an inherited genetic disorder, characterized by cognitive dysfunction and abnormal body movements, and at present there is no effective treatment for HD. Therapeutic options for HD are limited to symptomatic treatment approaches and there is no cure for this devastating disease. Here, we examined whether SUN N8075, (2S)-1-(4-amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl)phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate, which exerts neuroprotective effects by antioxidant effects and induction of VGF nerve growth factor inducible (VGF), has beneficial effects in STHdh cells derived from striatum of knock-in HD mice and R6/2 HD mice. In an in vitro study, SUN N8075 inhibited the cell death caused by mutant huntingtin (mHtt) and upregulated the VGF mRNA level via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, 30 amino acid of VGF C-terminal peptide, AQEE-30 inhibited the cell death and the aggregation of mHtt. In an in vivo study, SUN N8075 improved the survival and the clasping response in the R6/2 mice. Furthermore, SUN N8075 increased the number of surviving neurons in the striatum of the R6/2 mice. These findings suggest that SUN N8075 may be an effective candidate for HD treatments.


Introduction
Huntington's disease (HD) is a hereditary neurodegenerative disease that is caused by a mutation in the gene coding for a ubiquitously expressed protein, huntingtin (Htt) (Rubinsztein 2002). Mutant Htt (mHtt) causes the death of neurons via its aggregation, particularly in medium spiny neurons (MSNs), the phenotype that accounts for 90% of striatal neurons. The demise of MSNs causes dysfunctional motor, cognitive and behavioral symptoms (Reiner et al. 1988). One possible mechanism of neuronal degeneration in HD is a reduction in neurotrophic factor activity (Zuccato et al. 2001). However, the underlying mechanisms of neurodegeneration and mHtt aggregation are not well understood, and at present, there are no effective treatments for HD. Therefore, therapeutic options for HD are limited to symptomatic approaches and there is no cure yet in sight for this devastating disease. In our recent studies, we examined a novel agent against neuronal degeneration, SUN N8075 [(2S)-1-(4-amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl) phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate], which was synthesized as an inhibitor for Na + and T-type Ca 2+ channels with a potent antioxidative effect (Annoura et al. 2000). Our previous studies revealed potent neuroprotective activities of this agent in an in vivo transient middle cerebral artery occlusion model (Kotani et al. 2007), Parkinson's disease (Oyagi et al. 2008), retinal damage induced by intravitreous injection of N-methyl-Daspartate or high-intraocular pressure (Akane et al. 2011), and amyotrophic lateral sclerosis (ALS) (Shimazawa et al. 2010). These neuroprotective effects were the result of multiple mechanisms such as the compound's antioxidant activity (Kotani et al. 2007;Oyagi et al. 2008;Akane et al. 2011) and the upregulation of VGF nerve growth factor inducible (VGF) (Shimazawa et al. 2010). However, the neuroprotective effects of SUN N8075 have not yet been studied in HD. In the present study, we examined whether SUN N8075 inhibited the cell death of STHdhQ111 cells, a model of HD by aggregation of mHtt. Furthermore, we investigated the effects of SUN N8075 on the survival, motor performance, and suppression of striatal neuronal degeneration in R6/2 model mice.

Cell lines
The striatally derived cell lines, STHdhQ7 and STHdhQ111, were purchased from Coriell Institute for Medical Research (Camden, NJ). These cell lines were established from striatal precursors obtained from E14 mouse embryos that expressed either Htt with 7 polyQ or mHtt with 111 polyQ. Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS; Valeant, Costa Mesa, CA) at 33°C with 5% CO 2 . Because various stresses such as ER-stress and oxidative stress contribute to the pathology of HD (Browne et al. 1997;Reijonen et al. 2008), we performed the in vitro experiments under conditions of starvation stress.

Cell death assay
To evaluate the effects of SUN N8075 on cell death induced by aggregation of mHtt, both STHdhQ7 and Q111 cells were seeded at a density of 5 9 10 3 cells per well into 96-well plates, and then incubated in a humidified atmosphere of 95% air and 5% CO 2 at 33°C for 24 h. SUN N8075 was dissolved in dimethylsulfoxide (DMSO; Nacalai Tesque Inc., Kyoto, Japan) and diluted with phosphate-buffered saline (PBS) containing 1% DMSO, after which it was added to the wells at final concentrations of 0.1, 0.3, 1, and 3 lmol/L upon replacing the medium with DMEM or DMEM containing 3% FBS. We also evaluated the effects of VGF peptide on STHdhQ111 cells, by adding 30 amino acids of the rat VGF C-terminal peptide AQEE-30 (Medical & Biological Laboratories Co., Ltd, Aichi, Japan) and 21 amino acids of the rat VGF peptide TLQP-21 (R&D Systems, Inc., MN, USA) after dissolving in PBS. In the experiment using MEK inhibitors, U0126 (Promega Corporation, WI, USA) and PD184352 (Sigma), SUN N8075 and MEK inhibitor were added together. U0126 and PD184352 were dissolved in DMSO and diluted with 1% DMSO PBS (at final concentration of 10 lmol/L). Cell viability was measured 12 h after treatment with SUN N8075. At the end of cultivation, Hoechst 33342 (Molecular Probes, OR) and propidium iodide (PI) (Zuccato et al. 2001) (Molecular Probes, OR, USA) were added to the culture medium for 15 min. Hoechst 33342 freely enters living cells and cells that have suffered apoptosis or necrosis. PI is a membrane-impermeant dye that is generally excluded from viable cells. Images were collected using a digital camera (COOLPIX 4500; Nikon, Tokyo, Japan). We used an image-processing software (Image-J ver. 1.33f; National Institutes of Health, Bethesda, MD) for cell counting.

Fluorescence immunostaining
STHdh cells were incubated in the Lab-Tek II Chamber Slide system (Thermo) for 24 h, and then treated with SUN N8075 and AQEE-30. After 12 h of treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated in 3% normal goat serum for 30 min and then incubated in Can Get Signal â Immunostain solution A (Toyobo Co., Ltd., Osaka, Japan) containing anti-huntingtin monoclonal antibody, clone 4A4.2 (EMD Millipore Corporation) at 4°C overnight. Next, the cells were washed with PBS and incubated at room temperature with Alexa Fluor 488 rabbit anti-mouse antibody (1:1000 dilution; Molecular Probes) for 1 h, and then incubated with Hoechst 33342 (1:1000) in PBS for 5 min. Huntingtin aggregates were viewed with a confocal microscope (FV10i, Olympus, Tokyo, Japan). The percentage of cells that contained aggregates within each group was determined.

Study animals
Transgenic male and female R6/2 mice (B6CBA-TgN [HDexon1]62Gpb) were purchased from the Jackson Laboratory (Bar Harbor, ME). The R6/2 line is characterized by a rapidly progressive HD phenotype that leads to death in 13-14 weeks (Mangiarini et al. 1996). We carefully observed the development of HD symptoms and identified symptomatic disease onset as when the mice showed clasping behavior. All mice were housed in a room under lighting conditions of 12-h light and 12-h dark.

Animal welfare and ethical statements
All experiments were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University.

Drugs
We have previously reported that SUN N8075 exerted the beneficial effects on the model mice of neurodegenerative disease such as Parkinson's disease and ALS (Oyagi et al. 2008). Therefore, in this study, we performed the experiment in equivalent doses. SUN N8075 was dissolved in 6% Captisol solution, and subcutaneously administered at doses of 30 mg/kg, once daily from 4 weeks of age until the end of the animals' lives. In the control group, vehicle (6% Captisol solution) was subcutaneously administered at 10 mL/kg.

Survival
Mortality was scored as the day when the mouse was unable to right itself within 30 s after being placed on its back.

Rotarod test
Mice were tested for their ability to maintain balance on a rod (3 cm diameter) rotating at 5 rpm on a rotarod apparatus (Bio-Medica Ltd, Osaka, Japan). To adapt the mice to the apparatus, they were allowed to adjust to balancing on the rod as it rotated (5 rpm) for 7 days from when they were 21 days old. After this period of adaptation, locomotor performance was evaluated as the rod rotated at 5 rpm starting at 28 days of age. Testing was conducted two times per week until the animals became moribund. Each session consisted of three trials (10 min/trial). Performance time for each trial was recorded as the longest time the mice could stay on the rod without falling.

Clasping performance
An analysis of clasping was performed by suspending the mice from their tails at least 1 foot above a surface for 1 min, as previously described by Baquet et al. (2004).

Tissue processing
At 10 weeks of age, mice were anesthetized with sodium pentobarbital (80 mg/kg; i.p.; Nacalai Tesque Inc.) and perfused with 2% paraformaldehyde solution, after which their brains were immersed in the same fixative solution for 24 h. After fixation, the brains were soaked in 25% (w/v) sucrose at 4°C for 1 day, and then frozen in embedding compound (Tissue-Tek; Sakura Finetechnical Co. Ltd., Tokyo, Japan). Coronal sections (Bregma + 0.14 mm) were stained with cresyl violet (Sigma).

Data analysis and statistical procedures
Data were presented as means AE SEM. Statistical comparisons were made by way of a Tukey's test, or Dunnett's test, or Student's t-test. Statistical analysis of the mean age of clasping and survival was performed with a log-rank test. P < 0.05 was considered to indicate statistical significance.

SUN N8075 inhibited cell death in STHdh cells
We investigated the effects of SUN N8075 on cell death caused by the aggregation of mHtt in STHdhQ111 cells. STHdhQ7 cells, which express normal Htt, were used as a control.
Representative fluorescence images of Hoechst 33342 and PI staining are shown in Figure 1A observed in both STHdhQ7 and Q111 cells was significantly increased under the starvation stress condition compared with each control group (Fig. 1B). Furthermore, cell death in vehicle-treated STHdhQ111 cells was significantly increased compared with vehicle-treated STHdhQ7 cells (Fig. 1B). Notably, SUN N8075 significantly inhibited cell death in STHdhQ111 cells at concentrations of 0.3-3 lmol/L, with the most prominent effects observed at 3 lmol/L (Fig. 1B).

SUN N8075 upregulated the phosphorylation of ERK1/2
To confirm the mechanism by which SUN N8075 mediates its neuroprotective effects, we examined the effects of SUN N8075 treatment on levels of phospho-ERK1/2 (p-ERK1/2), a molecule known to be involved in signaling pathways that modulate cell survival. Western blot analysis results showed that SUN N8075 upregulated p-ERK1/2 levels in STHdhQ111 cells as early as 1 h after treatment (Fig. 2B). Furthermore, p-ERK1/2 levels were significantly decreased in STHdhQ111 cells under the starvation stress condition, and SUN N8075 significantly suppressed the downregulation of p-ERK1/2 1 h after treatment ( Fig. 2C and D).

SUN N8075 upregulation of Vgf mRNA and VGF peptide expression exerted protective effects in STHdhQ111 cells
We have previously reported that SUN N8075 increased VGF, a neuropeptide with neuroprotective effects against endoplasmic reticulum stress (ER-stress) (Shimazawa et al. 2010). To confirm whether SUN N8075 induced VGF expression in STHdh cells, we performed quantitative RT-PCR. In STHdhQ111 cells, Vgf mRNA levels were significantly decreased in the serum-deprived condition; however, treatment with SUN N8075 significantly suppressed the downregulation of Vgf mRNA level 6 h after treatment (Fig. 3A). Furthermore, we performed a cell death assay to confirm whether VGF has neuroprotective effects in STHdhQ111 cells. A schematic diagram of the rat VGF precursor protein and processed peptides is shown in Figure 3B. The VGF peptide AQEE-30 significantly inhibited STHdhQ111 cell death at concentrations from 0.3-3 lmol/L (Fig. 3C); however, no effects  were observed with the second VGF peptide tested, TLQP-21 (Fig. 3D).

SUN N8075 and AQEE-30 inhibited the aggregation of mHtt
To evaluate the effects of SUN N8075 and VGF peptides on the aggregation of mHtt, we performed fluorescence immunostaining. Representative fluorescence images are shown in Figure 4A, and the percentage of cells that included the aggregates was determined (Fig. 4B). In the nontreated group, there were some aggregates in STHdhQ111 cells (Fig. 4A). On the other hand, in the groups which treated with SUN N8075 and AQEE-30, aggregates of mHtt were significantly decreased (Fig. 4B).

MEK inhibitor suppressed the upregulation of Vgf mRNA and neuroprotective effects induced by SUN N8075
VGF is upregulated through mitogen-activated protein kinase kinase (MAPKK) and its target ERK (MEK/ERK) signaling pathway (Monteggia et al. 2004;Duman and Monteggia 2006;Adachi et al. 2008). Based on the results depicted in Figures 2, 3 of the present reports, we proposed the hypothesis that the upregulation of Vgf mRNA and improved cell viability caused by SUN N8075 treatment may be suppressed by inhibition of ERK activation.
To confirm this hypothesis, we performed RT-PCR and cell death assay using the MEK inhibitors U0126 and PD184352. Treatment of STHdhQ111 cells with U0126 significantly suppressed the upregulation of Vgf mRNA induced by SUN N8075 6 h after treatment (Fig. 5A). Importantly, the expression level of Vgf mRNA expression level was not changed by U0126 treatment alone. Thus, the protective effect of SUN N8075 in STHdhQ111 cells was significantly decreased by treatment with U0126 and PD184352 ( Fig. 5B and C).

SUN N8075 prolonged survival in R6/2 HD mice
In an in vitro study, it was confirmed that SUN N8075 inhibited cell death. Next, we evaluated the effects of SUN N8075 in vivo using the HD model animal (R6/2 mouse) for the 5 0 -end of the human huntingtin gene carrying (CAG)115-(CAG)150 repeat expansions. The treatment with SUN N8075 commenced when the R6/2 mice were 4 weeks of age. The effects of SUN N8075 on survival in R6/2 mice are shown in Figure 5. Treatment with SUN N8075 (30 mg/kg per day; s.c.) significantly prolonged the mean lifespan by 25.0% (P = 0.014) in the R6/2 mice compared to vehicle-treated R6/2 mice (Fig. 6A). There were no significant differences in body weight between SUN N8075-treated and vehicle-treated R6/2 mice at any age (Fig. 6B).

Motor dysfunction in SUN N8075-treated R6/2 HD mice
Hindlimb and forelimb clasping have been observed in transgenic lines in which there is motor dysfunction or degeneration, including HD mouse models expressing mutant Htt (Auerbach et al. 2001;Guidetti et al. 2001). Additionally, HD model mice show a progressive decrease in retention times in the rotarod compared to agematched wild-type littermate mice (Rodriguez-Lebron et al. 2005). We investigated the effects of SUN N8075 on the progression of these phenotypes by using the tail suspension and rotarod tests in R6/2 mice. Before disease onset, R6/2 mice often splay their legs out when suspended; however, the clasping response was observed at onset (Fig. 6C). Treatment with SUN N8075 (30 mg/kg per day; s.c.) significantly delayed onset of the clasping response. Kaplan-Meier life curves suggested that  treatment with SUN N8075 significantly increased survival (P = 0.039) in R6/2 mice (Fig. 6D). On the other hand, no significant differences were observed in motor performance in the rotarod test between SUN N8075-treated and vehicle-treated R6/2 mice (Fig. 6E).
SUN N8075 protected against neuronal cell death in R6/2 mice We evaluated the effect of SUN N8075 on neuronal loss in the brains of R6/2 mice at 10 weeks of age. Representative photomicrographs of neuronal cells in the striatum are shown in Figure 7A-F. The high-magnification images of cresyl violet-stained brain sections ( 7G).

Discussion
In the present study, we demonstrated that SUN N8075 exerted neuroprotective effects in both in vitro and in vivo HD models. The efficacy of SUN N8075 was clearly indicated by the suppression of neuronal loss of striatum in the R6/2 mice. Furthermore, SUN N8075 upregulated Vgf mRNA expression via the phosphorylation of ERK1/2, resulting in suppression of cell death. VGF is a secreted polypeptide that is expressed throughout the brain, especially in neurons, as well as in several endocrine and neuroendocrine that include the pancreas and pituitary and it regulates food intake and/or energy balance (Salton et al. 2000;Levi et al. 2004). Furthermore, the neuroprotective effects of VGF have been suggested to improve depression by enhancing hippocampal synaptic plasticity as well as neurogenesis in the dentate gyrus (Thakker-Varia et al. 2007). In the present study, SUN N8075 suppressed cell death (Fig. 1) and upregulated Vgf mRNA expression (Fig. 3A). Furthermore, AQEE-30, which is well known as a VGF peptide, suppressed cell death, and the aggregation of mHtt in STHdhQ111 cells (Figs. 3C, 4); however, TLQP-21, another VGF peptide, did not exert a suppressive effect on STHdhQ111 cell death (Fig. 3D). In this study we have chosen two VGF peptides, AQEE-30 and TLQP-21. It is known that bioactive peptides are generated mainly by cleavage of VGF C-terminal. Because the sequence of AQEE-30 and TLQP-21 do not overlap, investigating these peptides may contribute to the greater understanding of the effects of VGF. These results indicate that SUN N8075 suppresses cell death via the upregulation of VGF expression and it inhibited the mHtt aggregation. In this study, we evaluated the mHtt aggregation as previously reported (Wang et al. 2011). It would have been useful to discriminate between the nuclear and cytosolic aggregate since nuclear aggregates have been reported as being more toxic to the cells (Davies et al. 1997), however, in STHdhQ111 cells, we were not able to determine the localization of mHtt because the expression of mHtt was not at a high enough level. VGF is encoded by a gene that is responsive to brainderived neurotrophic factor (BDNF) (Thakker-Varia et al. 2007) and is upregulated by BDNF through the MEK/ ERK signaling pathway. We previously reported that SUN N8075 inhibited ER-stress-induced neuronal cell death via an increase in VGF expression and activation of the MEK/ERK pathway (Shimazawa et al. 2010). MEK/ERK signaling has been implicated in the hyperphosphorylation of Elk-1 and CREB, which are transcription factors induced by ERK activation; importantly, earlier studies have observed Elk-1 and CREB hyperphosphorylation in the striatum of R6/2 mice (Lievens et al. 2002;Obrietan and Hoyt 2004). Furthermore, several protein tyrosine phosphatases, which increase the dephosphorylation/inactivation of ERK1/2, were found to result in increased cell death in an HD model using PC12 cells such that the downregulation of p-ERK1/2 phosphorylation was correlated with the aggregation of mHtt (Wu et al. 2002). It has also been reported that the polyphenols fisetin and resveratrol provide neuroprotective effects via ERK activation in R6/2 mice (Maher et al. 2011). In the present study, ERK1/2 phosphorylation was induced 1 h after treatment with SUN N8075 (Fig. 2B-D). Furthermore, SUN N8075 increased the expression of Vgf mRNA 6 h after treatment, and this upregulation of Vgf mRNA was inhibited by an MEK inhibitor, thus attenuating the beneficial effects of SUN N8075 on cell death (Figs. 3, 5A-C). These results suggest that SUN N8075 exerts neuroprotective effects via activation of the MEK/ERK pathway.
In our in vivo study, SUN N8075 prolonged the mean lifespan of R6/2 mice by 25.0% compared with that of vehicle-treated R6/2 mice (Fig. 6A). In a previous study, various medicinal agents applied to R6/2 mice, such as riluzole, minocycline, and cystamine, prolonged the mean lifespan by only 10.2%, 14.0%, and 19.5%, respectively (Chen et al. 2000;Dedeoglu et al. 2002;Schiefer et al. 2002). These findings indicate that the therapeutic effect of SUN N8075 on survival is more potent than that of these previously tested compounds. Furthermore, treatment with SUN N8075 delayed the onset of clasping behavior (Fig. 6D) and inhibited neuronal cell death in the striatum of R6/2 mice (Fig. 7). We can speculate that SUN N8075 may have delayed onset of the clasping phenotype via the inhibition of neuronal cell death; however, further study will be required to elucidate the mechanisms of SUN N8075's effects in R6/2 mice.
In conclusion, we demonstrated that SUN N8075 inhibited cell death, via the upregulation of VGF expression and ERK 1/2 phosphorylation in STHdhQ111 cells. Additionally, treatment with SUN N8075 prolonged survival, delayed symptom onset, and inhibited the striatal neuronal cell death in the R6/2 mouse model of HD mice. SUN N8075 may be an important candidate compound for the treatment of HD and other neurodegenerative disorders. Disclosure S. T. and T. I. are employees of Asubio Pharma.