- Email: [email protected]
2 mice
Neurobiology of Disease 36 (2009) 413–420
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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i
Y-27632 improves rotarod performance and reduces huntingtin levels in R6/2 mice Mei Li a,b, Yong Huang c, Aye Aye K. Ma a,b, Emil Lin c, Marc I. Diamond a,b,⁎ a b c
Department of Neurology, University of California, San Francisco, CA 94143, USA Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA
a r t i c l e
i n f o
Article history: Received 1 May 2009 Revised 24 June 2009 Accepted 28 June 2009 Available online 8 July 2009
a b s t r a c t Huntington disease (HD) is a devastating, untreatable, dominantly inherited neurodegenerative disease. It is caused by an expanded CAG codon repeat that leads to an elongated polyglutamine tract in the N-terminus of the huntingtin (Htt) protein. Few mechanism-based therapeutic leads have been developed. Y-27632, an inhibitor of the Rho-associated kinase ROCK, reduces Htt aggregation in cultured cells and Htt-induced neurodegeneration in Drosophila, but its effect in mice is unknown. We determined that Y-27632 is bioavailable in brain, with a half-life of 60–90 min. We then initiated a trial in R6/2 mice, which express Htt exon 1, administering 100 mg/kg/day of Y-27632 in drinking water. We did not observe a significant effect on brain weight, inclusion number or size, striatal medium spiny neuron number, clasping behavior, or lifespan. However, Y-27632 treatment improved rotarod performance significantly, and also reduced soluble brain Htt levels. The ROCK signaling pathway thus remains a promising therapeutic target for HD, and more potent inhibitors may prove useful. © 2009 Published by Elsevier Inc.
Introduction Huntington disease (HD) is an untreatable, devastating autosomal dominant neurodegenerative disease characterized by psychiatric disturbance, motor impairment, and dementia. CAG codon expansion in the huntingtin gene past 37 repeats leads to an expanded, pathogenic polyglutamine tract within the huntingtin (Htt) protein (1993). This expansion destabilizes Htt, leading to misfolding, aggregation, inclusion formation, and toxicity (Shao and Diamond, 2007). Emerging evidence suggests that small oligomeric aggregates may constitute the pathologic species in polyglutamine diseases (Li et al., 2007; Takahashi et al., 2007a; Takahashi et al., 2007b; Pattison and Robbins, 2008). Multiple studies have identified aggregation inhibitors, and several of these compounds have been tested in mouse models with modest effects on motor performance (Apostol et al., 2003; Chopra et al., 2007; Kazantsev et al., 2002; Tanaka et al., 2004; Zhang et al., 2005). We previously established a cellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET), which was used to identify Y-27632, an inhibitor of the Rhoassociated kinase ROCK. Y-27632 reduced Htt aggregation ∼15% in cells, and inhibited photoreceptor degeneration in a Drosophila model of HD based on neuronal expression of Htt exon 1 (Pollitt et al., 2003).
⁎ Corresponding author. GH-S572B, 600 16th Street, San Francisco, CA 94143-2280, USA. Fax: +1 415 514 4112. E-mail address: [email protected] (M.I. Diamond). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.nbd.2009.06.011
We have observed that both ROCK and a related kinase PRK-2 are inhibited by Y-27632 to reduce polyglutamine aggregation, and these kinases each contribute to the effects of Y-27632 in cells (Shao et al., 2008a). In studies of the downstream molecular mechanism by which Y-27632 reduces intracellular aggregation, we have identified profilin, an Htt interacting protein (Goehler et al., 2004), as a novel target of ROCK (Shao et al., 2008b). Profilin potently inhibits Htt aggregation in cells (Shao et al., 2008b), and a Drosophila homologue has been observed to suppress polyglutamine-mediated toxicity (Burnett et al., 2008). Profilin is phosphorylated at Ser-137 by ROCK, which blocks its ability to directly bind the Htt peptide. Thus, ROCK inhibition promotes dephosphorylation at Ser-137 and enables profilin's protective effects (Shao et al., 2008b). There is no evidence that PRK-2 directly phosphorylates profilin. Importantly, knockdown of profilin completely blocks the anti-aggregation effects of Y-27632 in cells, suggesting that ROCK signaling through profilin is a key signaling pathway in the regulation of aggregation (Shao et al., 2008b). Given our emerging knowledge about its molecular mechanisms in cells, and its effects in Drosophila, there is a strong rationale to test Y-27632 in a mouse model of HD. R6/2 mice express the Htt exon 1 peptide from the huntingtin promoter (Mangiarini et al., 1996), and have been widely used in preclinical trials for HD (Chen et al., 2000; Ferrante et al., 2002; Hockly et al., 2003a; Ferrante et al., 2003; Fox et al., 2004; Ferrante et al., 2004; Chopra et al., 2007; Bates and Hockly, 2003). These mice exhibit a progressive neurologic phenotype that leads to early death, generally by 12–16 weeks. This is associated with early and progressive accumulation of the Htt peptide in intracellular inclusions
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(Davies et al., 1997; Scherzinger et al., 1997). We used this model to test the efficacy of orally administered Y-27632.
have spontaneously died, which frequently occurred without the first two criteria being met.
Materials and methods
Rotarod testing
Y-27632 was a generous gift from Mitsubishi Pharma, Japan. Antibodies Mouse anti-human huntingtin N-terminal antibody clone EM48, mouse anti-expanded polyglutamine antibody clone 1C2, mouse antihuman and mouse full length huntingtin antibody MAB2166 and rabbit anti-dopamine and cAMP regulated phosphoprotein of 32 kD (DARPP32) were purchased from Chemicon International Inc.; mouse anti-glial fibrillary acidic protein (GFAP) was kindly shared by Zhaolin Hua, M.D., Ph.D. and Robert Edwards, M.D. at UCSF. Rabbit antiphospho-profilin 1 at serine 137 was developed by our laboratory (Shao et al., 2008b). Y-27632 detection in vivo Frozen brain specimens were pulverized, and then homogenized in H2O. Acetonitrile was added to precipitate protein with chloroquine (100 ng/ml) as an internal standard (IS), and Y-27632 concentration in the supernatant was measured by HPLC-tandem mass spectrometry (LC-MS-MS). To create reference standards, 14 known concentrations of Y-27632 (1.7–1700 ng/ml) were split into vehicle-treated mouse blood and brain specimens and carried through the extraction/ purification procedure. The LC/MS/MS system consisted of Shimadzu LC-10 AD pumps, a Waters intelligent Sample Processor 717 Plus autosampler, and a Micromass Quattro LC Ultima triple quadrupole tandem mass spectrometer. The mass spectrometer was set for electrospray ionization in positive ion mode. Y-27632 and IS were detected with multiple reaction monitoring (MRM) at 248.3 N 93.3 m/z and 320.9 N 247.6 m/z, respectively. The sample cone voltage and collision energy for both Y-27632 and IS were set at 30 eV and 25 eV, respectively. Y-27632 and IS were separated by silica column (50 mm × 4.6 mm) with mobile phase composed of 90% acetonitrile, 0.06% TFA and 2.5 mM ammonium formate. The flow rate was 1.8 ml/ min and 1/10 was split into the mass detector. Mouse husbandry R6/2 mice in the B6/CBA background were backcrossed with C57B/6 females (Jackson Laboratory, Bar Harbor, ME) for more than seven generations. Genotyping and CAG repeat numbers were detected from tail DNA by PCR. Mice were maintained in a barrier animal facility at the University of California San Francisco, housed five per cage with food and water available ad libitum, and kept on a 12 h light/dark cycle. All studies and procedures were done under the approval of the Institutional Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Therapeutic trials For analysis of rotarod performance, 16 female R6/2 animals were used. These consisted of 7 littermate pairs, and two additional animals. Littermates were evenly divided between the two groups. For the analysis of Y-27632 on lifespan, 17 female R6/2 animals were used. These consisted of 8 littermates evenly divided between treatment and control groups, and an additional animal was studied in the treatment group. For lifespan studies, animals were followed in accordance with IACUC approval until they lost N30% of body weight, or exhibited a moribund appearance (based on poor movement or grooming) at which point they were euthanized, or were found to
We performed rotarod evaluations using a rotarod apparatus (Columbus Instruments. Columbus, OH) as described previously (Li et al., 2007). We carried out four rotarod tests per session, allowing at least 15 min of rest for the mice between each test. The rotarod accelerated from 4 to 40 rpm over 15 s and was maintained at 40 rpm thereafter. The lowest value from all four tests was discarded. The remaining three values were averaged, and the data were analyzed by the student's t-test, assuming equal variance between the two groups. Histology and cell counts At age 13.5 weeks, mice were sacrificed and brains were cut in sagittal midline sections. The left side of the brain was freshly frozen by ethanol-dry ice for biochemical studies and right side of the brain was embedded in OCT compound and freshly frozen by ethanol-dry ice for histological studies. 12 μm serial coronal sections cut through the whole striatum were collected and fixed with 4% paraformaldehyde. Every 8th slice was immunostained with DARPP32 and GFAP antibodies and DAPI. Adjacent slices were stained with hematoxylin/ eosin. Cells in the striatum were counted using a previously published method (Chopra et al., 2007), with minor modifications. 36–60 serial adjacent images were taken from one large image of a whole slice using a Nikon 6 dimensional microscope (Nikon) at the Nikon Imaging Center at UCSF (http://nic.ucsf.edu/6D.html). A 10× objective and automated program NIS-Elements Advanced Research 3.0 (http:// www.nis-elements.com/) were used. DARPP32 positive cells, GFAP positive cells or DAPI positive cells were counted by analyzing the large images with Image J (NIH, http://rsbweb.nih.gov/ij/). The area of the striatum at the level of crossing of the anterior commissure was also measured by Image J. Statistical analysis was performed using a student's t-test. Immunohistochemistry Brain, pancreas and muscle embedded in OCT compound were cut on a cryostat (Leica 1850). 12 μm sagittal brain sections cut from the midline, and coronal sections cut from half of the brain were air-dried for 20 min. Dried sections were fixed with 4% paraformaldehyde for 10 min at 4 °C, washed with 0.05% Tween-20 in phosphate-buffered saline, and then incubated with 5% goat serum and 0.1% Triton X-100 in phosphate-buffered saline for 30 min at room temperature followed by overnight primary antibody incubation at 4 °C (mouse anti-huntingtin N-terminal antibody EM48 (1:400) and rabbit antiphospho profilin 1 at serine137(1:1000; n = 12 each in Y-27632 and vehicle-treated groups), or rabbit anti-DARPP32(1:500) and mouse anti-GFAP(1:250; n = 6 each in Y-27632 and vehicle-treated groups)), and washing with 0.05% Tween-20 in phosphate-buffered saline. Goat anti-mouse IgG conjugated with Alexa 546 and goat anti-rabbit IgG conjugated with Alexa 488 (1:4000; Molecular Probes) were applied to the sections for 2 h at room temperature in the dark. The sections were then washed for 1 h and counterstained with 4′6′ diamidino-2phenylindole (DAPI) (50 ng/ml) for 30 s, washed, and mounted with ProLong Gold anti-fade reagent (Molecular Probes). Fluorescence images were collected with a Nikon spectral laser confocal microscope C1s1 with excitation wavelength at 405 nm, 488 nm and 561 nm and detection at 435–450 nm, 515–530 nm, and 605–675 nm. Immunoprecipitation 50 mg of brain powder was dounce-homogenized with 25 strokes on ice in 500 μl RIPA buffer and centrifuged 15,000 ×g for 30 min at
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Fig. 1. Evaluation of Y-27632 clearance in serum and brain. Wild-type mice were given an intraperitoneal dose of 10 mg/kg Y-27632. Two animals were sacrificed at the indicated time intervals, and serum (A) and brain tissue (B) was harvested for mass spectrometry to determine compound levels. Error bars represent the range. Absolute concentrations were determined by using control tissue extracts spiked with known amounts of compound.
4 °C. 2 μl of 1C2 antibody (Chemicon) was covalently conjugated to a 20 μl bead volume of anti-mouse IgG beads (Sigma) by DSS (Pierce) as previously described (Li et al., 2007). Beads were washed thoroughly with RIPA buffer. 200 μl supernatant extracted with the above RIPA buffer was added with 1C2-mouse IgG beads overnight at 4°C followed by at least four washes in RIPA buffer. Samples were boiled for 5 min in 1% SDS sample buffer prior to SDS-PAGE and Western blot analyses. Blots were then probed with 1C2 (1:2500 dilution), washed with TBS and 0.5% Tween-20, and then incubated with horseradish peroxidase-conjugated mouse True Blot (1:5000 dilution, eBioscience) that does not recognize denatured rabbit IgG heavy or light chain. Horseradish peroxidase signal was detected by ECL-Plus (Amersham Biosciences). Quantitative PCR RNA was prepared from whole brain by using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Real time PCR was performed as described previously (Wang et al., 2006), except the internal control was actin instead of Rpl19. R6/2 transgene primers were: TCA GGT TCT GCT TTT ACC TGC (forward) and CTT CAT CAG CTT TTC CAG GG (reverse). Relative Htt exon 1 transcript was measured by [sample(Ct Htt exon1 − Ct actin) − minimum sample(Ct Htt exon1 − Ct actin)]2. Results Y-27632 bioavailability We first determined whether Y-27632 would penetrate the blood brain barrier. We administered 10 mg/kg as a single intraperitoneal dose to a group of 14 mice and sacrificed two of them at 15 min, 30 min, 1.5 h, 6 h, 8 h, 12 h and 24 h after injection. We then measured Y-27632 serum and brain concentrations by HPLC-tandem mass spectrometry. Peak serum concentration was 3–3.5 μM and brain concentration was 0.3–0.65 μM 15 min after injection (Fig. 1). Half-life in each tissue was ∼60–90 min and the brain/serum ratio was ∼1/6 Table 1 Concentrations in serum and brain after oral administration of Y-27632. Sample no.
Serum conc. (nM)
Brain conc. (nM)
Serum/brain ratio
1 2 3 4 5
59 91 298 393 51
6.9 17.6 47.5 20.2 10.3
8.7 5.2 6.3 19.5 4.9
Five animals were administered 50 mg/kg/day of Y-27632 orally for one week. Animals were then sacrificed, and serum and brain levels were determined by liquid chromatography–mass spectrometry.
(Fig. 1). These studies indicated that at this dose Y-27632 readily penetrates the blood brain barrier, although with a fairly short halflife. Peak CNS concentrations were slightly less than desired to achieve optimal ROCK inhibition. Next, we tested for oral availability, first confirming the stability of Y-27632 in drinking water (Supp. Fig. 1). We treated a cohort of 5 wild-type mice with 50 mg/kg/day Y-27632 in drinking water for 7 days and determined concentrations in serum and brain (Table 1). Finally, we used dose escalation to determine the highest amount tolerated. We administered Y-27632 in drinking water to a cohort of 5 of wild-type mice beginning at 75 mg/kg/day and gradually increasing by 25 mg/kg/day every 3–4 days. The mice tolerated up to 250 mg/kg/day, however we were concerned about using 250 mg/kg/day for an extended period due to limiting compound and possible toxicity. Thus, we decided to use 100 mg/ kg/day administered orally for an efficacy trial. Y-27632 improves rotarod function R6/2 mice are very well characterized, and determinants of experimental variability include littermate status, gender, weight, and CAG repeat length (Hockly et al., 2002; Hockly et al., 2003b). To reduce variation we only used female mice, controlling for initial body weight and littermate status, and controlling for CAG repeat number by PCR (all animals contained 110 to 120 CAG repeats within the Htt exon 1 transgene). For the rotarod trial, 16 identically aged R6/2 females, including 7 littermate pairs, were divided into two groups. 8 R6/2 females received 100 mg/kg/day Y-27632 in drinking water. The second group received vehicle control (water). We administered Y27632 beginning at age 4 weeks. After an initial drop in the weight of the treated animals, we observed no significant differences between the two groups over the course of the study (Fig. 2A). No significant changes were observed in clasping responses, a pathological limb reflex observed in these animals (Fig. 2B). 3 weeks after beginning treatment we initiated rotarod testing once per week, as soon as it was possible (rotarod testing in very young animals is not feasible). An accelerating rotarod (Columbus Instruments) was set to increase gradually from 4–40 rpm over 15 s, and maintained at 40 rpm thereafter. Four trials were carried out at each time point, and the shortest trial was discarded; the remaining three trials were averaged. Both groups exhibited a decline in performance over time. At age 7.5, 8.5 and 9.5 weeks, there were no significant differences between treatment and control groups. At age 10.5 weeks there was a significant improvement in performance in the treated vs. control animals: 55.8 ± 9.0 s vs. 24.4 ± 16.2 s, (p b 0.001, t-test); at 12.5 weeks 33.8 ± 16.0 vs. 13.7 ± 12.1 s, (p b 0.05, t-test) (Fig. 2C). A direct comparison of the rotarod performance of littermates at 12.5 weeks illustrates a relatively consistent benefit of the compound (Fig. 2D). An individual comparison of performance for each animal indicated an improvement in median function in the treated animals after
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Fig. 2. Analysis of Y-27632 on behavior in R6/2 mice. Cohorts of female R6/2 mice were randomized according to littermate status, weight, and CAG repeat length into two groups of 8 animals. Y-27632 at 100 mg/kg/day was administered by putting the compound in drinking water, controlling for daily intake to ensure accurate dosing. Dosing began at age 4 weeks. Rotarod testing began at age 7.5 weeks. (A) Y-27632 treatment had no significant effect on weight. (B) Y-27632 treatment had no significant effect on development of the clasping reflex. (C) Y-27632 treatment significantly improved rotarod performance beginning at age 10.5 weeks. ⁎p b 0.05; ⁎⁎p b 0.01 (t-test). (D) Comparison of littermates tested shows a consistent improvement in rotarod performance in the treated animals at 12.5 weeks. (E) Plots of individual animal's performance at each time point indicate an improvement in median performance on the rotarod in the treated group.
10 weeks (Fig. 2E), ruling out a large effect on a small number of animals. At 13.5 weeks, three animals in the treatment group died due to handling-induced seizures, but no animals in the control group.
Y-27632 has no effect on lifespan Because we observed handling-induced seizures, especially in the treated animals, we were concerned that despite its benefit on rotarod performance that Y-27632 might somehow lower the seizure threshold. Thus, we decided to test its effects on lifespan in the absence of the extensive handling required for rotarod testing. We repeated the trial with an additional 17 female R6/2 littermate mice, sorted by the same criteria used previously, with 9 animals in the Y-27632 treatment group and 8 animals in control group. Y-27632 was given from age 4 weeks. The median lifespan of Y-27632 treated animals was 126 days and control animals was 120 days, which matched the lifespan of other cohorts of R6/2 mice maintained in our colony (Supp. Fig. 2). There was no significant effect of treatment on lifespan (Fig. 3A), and no effect on body weight in this group (Fig. 3B).
Y-27632 does not change medium spiny cell density in the striatum Striatal medium spiny neuron loss is a principal feature of Huntington disease pathology. Thus, we tested whether Y-27632 treatment would change the medium spiny neuron density in R6/2 mice. We sacrificed 6 pairs of treated and control mice at 13.5 weeks. Brains were weighed and cut in a sagittal midline section. Half of the brain was embedded in OCT compound, and frozen in ethanol-dry ice for pathological studies; the other half was snap-frozen for biochemical studies. The brains embedded in OCT compound were cut on a coronal plane, with 12 μm serial sections cut from the anterior through the posterior striatum. Every 8th section was collected and double stained with DARPP32 (dopamine and cAMP regulated phosphoprotein of 32 kD), a marker for medium spiny neurons, and GFAP (glial fibrillary acidic protein), a marker for astrocytes. Adjacent images were collected by an automated fluorescence microscope (Nikon 6D). The images were then recombined to encompass a large image of the entire slice section. By analyzing the data using Image J (n = 6 samples per condition), we found that Y-27632 treatment had no statistically significant effect on the area of the striatum, medium spiny neuron
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Fig. 3. Analysis of Y-27632 on lifespan in R6/2 mice. Cohorts of female R6/2 mice were randomized according to littermate status, weight, and CAG repeat length into a treatment group of 9 animals, and a control group of 8 animals. The treatment group received 100 mg/kg/day of Y-27632 in drinking water, controlling for intake. Animals were weighed 2×/ week. (A) No effect of treatment was observed on survival. (B) No significant effect of treatment was observed on weight.
density, or astrocyte density. We noted an apparent increase in total cell density, and diminished astrocyte density in the striatum (Fig. 4; Supp. Fig. 3), indicating that other types of glial cell density might be slightly increased by Y-27632 treatment, but this did not rise to statistical significance.
27632 selectively reduces the levels of soluble Htt exon 1 protein, suggesting that it alters Htt exon 1 biochemistry, either promoting incorporation into a form not detected by immunoprecipitation, or by increasing its degradation. Discussion
Y-27632 reduces soluble Htt exon 1 levels in R6/2 mouse brain To confirm an effect of Y-27632 on Htt Exon 1 in the brain, we directly tested whether treatment altered inclusion formation or soluble protein levels. Various sections of brain were evaluated by immunohistochemistry using the EM48 antibody, which recognizes aggregated forms of Htt exon 1. We noted no significant differences in inclusion size or density in striatum, forebrain cortex, hippocampus or cerebellum (Fig. 5, Supp. Fig. 4). In R6/2 brain, much of the Htt exon 1 peptide is in an aggregated state, making accurate assessments of total protein levels difficult. We used formic acid to solubilize the aggregated protein to attempt to estimate total mutant Htt exon 1 levels. However the Htt exon 1 protein rapidly re-aggregated in solution, making this impossible (data not shown). Thus, we homogenized brains in RIPA buffer, and immunoprecipitated soluble Htt using 1C2 antibody (Chemicon International), which does not react with aggregated species. We probed the immunoprecipitated protein by western blot with 1C2, comparing treated littermates to their controls. 7/8 R6/2 mice treated with Y-27632 had reduced levels of soluble Htt relative to actin controls (Figs. 6A, B). qPCR indicated that this was not associated with a corresponding change in Htt exon 1 transgene expression (Fig. 6C). We also measured levels of endogenous mouse full length Htt (MAB2166, Chemicon International), but did not observe changes between Y-27632 treated and control R6/2 mice (Fig. 6D). Thus, Y-
In this study we evaluated oral administration of Y-27632 in the R6/2 mouse model of HD. We found that Y-27632 treatment significantly improves rotarod performance in these mice, and reduces soluble Htt peptide. Whereas vehicle-treated animals exhibited a fall in rotarod performance of ∼ 85% over the course of the trial period, the treated animals only experienced a drop of ∼50%. These effects were paralleled by a reduction in soluble Htt in 7/8 of the treated littermates. We did not observe statistically significant effects on brain weight, gross atrophy, lifespan, clasping, inclusion formation, or neuron counts in the striatum. We estimate that peak brain levels of Y-27632 were likely to have been ∼100 nM in this study. This is considerably less than levels ranging from 5–50 μM that have been studied in cultured cells, and which reduced aggregation of Htt by about 15–25% (Pollitt et al., 2003; Shao et al., 2008b). Given the modest effects of saturating Y-27632 treatment in cultured cells, it is perhaps not surprising that gross pathological analysis of tissue specimens did not reveal significant changes in inclusion formation. We also checked for effects on inclusion formation in other tissues that would likely have been exposed to higher drug levels, including pancreas and muscle, but did not observe effects on inclusion frequency. We stained with a phospho-profilin antibody developed in our laboratory (Shao et al., 2008b), but did not observe any changes in phospho-profilin in the brains of the treated mice. This could be due to a failure of ROCK to
Fig. 4. Analysis of Y-27632 on brain weight, striatum atrophy and cell densities was performed on 6 controls and 6 treated R6/2 mice. (A) Average of brain weight at 13.5 weeks was calculated. There was no significant difference between control and treated groups. (B) Average striatal area at the level of crossing of the anterior commissure. There was no significant difference between control and treated groups. (C–E) Half brain coronal sections ranging from anterior striatum to posterior striatum from 6 pairs of R6/2 mice in control and Y-27632 group were cut as 12 μm sections, and every 8th section was stained with DAPI, and DARPP32 and GFAP antibodies. Whole striatal images were taken by an automated fluorescence microscope (Nikon 6D). Cell numbers on each large image were counted by using Image J software. No significant effects of Y-27632 treatment were observed on (C) DAPI positive cell density (total cells); (D) DARPP32 positive cell density (medium spiny neurons); or (E) GFAP positive cell density (astrocytes). Error bars = S.E.M.
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Fig. 5. Analysis of Y-27632 on inclusion formation and phospho-profilin levels. Representative histopathology is shown. Various brain regions were stained with EM48 antibody (red), phospho-profilin antibody (green), and DAPI (blue). EM48 recognizes diffuse and aggregated forms of Htt exon 1. Phospho-profilin was distributed in the cytoplasm of forebrain cortical neurons, hippocampal CA3 neurons and cerebellar Purkinje neurons but not in striatal neurons. No effect of Y-27632 treatment was observed on cellular atrophy, phospho-profilin level or distribution, inclusion size or frequency. Scale bars are shown.
phosphorylate profilin in brain, but is more likely due to sub-saturating doses of Y-27632. Whether a more potent ROCK inhibitor, or higher brain concentrations, would produce more apparent effects on profilin phosphorylation or inclusion formation awaits further study. Y-27632 treatment selectively reduced brain Htt exon 1 levels, without affecting Htt exon 1 gene expression or full length mouse Htt. This suggests that the compound exerts some effect on the protein's metabolism in vivo, and is in keeping with a recent report which indicates an enhancing effect of Y-27632 treatment on Htt degradation via proteasome and autophagy pathways (Bauer et al., 2009). Our prior work suggests that Htt exon 1 is not cleared via the proteasome (Chandra et al., 2008), but autophagy may play an important role (Ravikumar and Rubinsztein, 2006; Ravikumar et al., 2004). As we have mentioned, it is very difficult to quantify precisely total Htt levels in vivo due to its rapid aggregation. However, our analysis of Htt Exon 1 transgene expression via qPCR and endogenous Htt levels effectively rules out a primary effect of Y-27632 on the Htt promoter, and suggests either that the total protein level is reduced, or that soluble protein is more rapidly cleared. In cultured cells we have not observed a consistent effect of Y-27632 treatment on Htt exon 1 levels, but this
may relate to the fact that it has been relatively over-expressed via transient transfection. As we learn more about mechanisms that govern Htt exon 1 solubility vs. aggregation, it will be interesting to determine whether these conformational states have a direct effect on protein degradation. Multiple parameters likely govern lifespan of R6/2 mice. These include peripheral effects such as weight loss and muscle atrophy, and central effects such as seizures. Consistent with this, two studies besides our own have noted a dissociation between lifespan and improved performance in behavior assays (Chopra et al., 2007; Chou et al., 2005). In this study, we did not observe significant weight loss in either group of animals, even at later points in life, or profound defects in glucose metabolism (data not shown), suggesting that muscle wasting or systemic illness did not play a major role. After the rotarod trials, we observed fatal handling-induced seizures in 3/8 animals treated with Y-27632, but in none of the controls. Thus, it is possible that despite a benefit in motor function, drug treatment may have lowered the seizure threshold. However, one report indicates that Y-27632 may reduce seizures in a mouse model (Inan and Buyukafsar, 2008).
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identifying therapeutic leads based on the inhibition of intracellular Htt aggregation. These results also provide a strong rationale for further study of molecular mechanisms by which Y-27632 functions. Finally, they suggest that development of more effective ROCK inhibitors with better CNS penetration might form the basis of an important new therapeutic strategy for HD. Acknowledgments We thank Mitsubishi Pharma for generously providing the Y-27632 used for this study. We thank Stanley Prusiner, M.D. for the help in obtaining Y-27632; Samuel Pleasure, M.D., Ph.D. for use of his cryostat; Clifford Bryant, Ph.D. for detecting Y-27632 stability; Kurt Thorn, Ph.D.; Amie Lee, M.D.; and the Nikon Imaging Center at UCSF for use of the Nikon C1s1 spectral confocal microscope and Nikon 6D microscope. We also thank Jieya Shao, Ph.D.; Zhaolin Hua, M.D., Ph.D.; Guangnan Li, Ph.D.; and Brian Feldman, M.D., Ph.D. for advice, reagents and technical support. ML was supported by a grant from the Muscular Dystrophy Association. MID was supported by grants from the Muscular Dystrophy Association, the NIH:NINDS, and the Taube Family Foundation Program in Huntington's Disease Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nbd.2009.06.011. Fig. 6. Y-27632 reduces soluble Htt exon 1 brain levels. (A) Brains from the animals studied in the behavior assay were homogenized and soluble Htt exon 1 levels were determined by immunoprecipitation and western blot with 1C2 antibody, which recognizes soluble forms of Htt exon 1. In 7/8 littermate pairs, there was a relative reduction in soluble Htt exon 1 levels. (B) Band intensity of the Htt exon 1 was compared to actin for each sample, and averaged. There was a significant decrease in the average Htt exon 1 band intensity relative to actin. ⁎p b 0.05; error bars = S.E.M. (C) qPCR was used to analyze Htt exon 1 gene expression in each of the samples. There was no effect of Y-27632 treatment on Htt exon 1 transcript. (D) Full length Htt expression was analyzed by western blot on whole brain lysates. No significant effect of Y-27632 treatment was observed.
We carefully searched for biochemical evidence of ROCK inhibition in the brain, but faced difficult hurdles. We have previously created a phospho-specific antibody to Ser-137 of profilin 1 that detects changes in profilin phosphorylation in a variety of cultured cells, including primary neurons (Shao et al., 2008b). We used this antibody to probe brain samples by immunohistochemistry and western blot, but did not detect effects of Y-27632 treatment on profilin phosphorylation. This is likely due to several factors. First, we were not able to achieve micromolar concentrations of Y-27632 in the brain for extended periods. We estimate that we achieved levels of ∼100 nM within the brain at peak levels, below the IC50 level of 140 nM previously reported for the kinase in cells (Ishizaki et al., 2000; Uehata et al., 1997). In this setting it could be very difficult to detect changes in phospho-profilin. However, partial ROCK inhibition may be the most therapeutically useful, given the importance of ROCK in a variety of cellular pathways. For example, another ROCK inhibitor HA-1077 (Fasudil) has been used at a lower dose (60 mg administered twice daily intravenous injection for 14 days) for ischemic stroke patients (Shibuya et al., 2005), indicating that partial inhibition of ROCK may still beneficial, even without the dramatic effects on protein phosphorylation required to document kinase inhibition biochemically. Development of effective therapies for HD will be hastened by the identification of factors that may be specifically targeted by drugs to reduce Htt toxicity. Our prior work has suggested that Y-27632 reduces Htt aggregation in cells through inhibition of ROCK signaling to profilin. Y-27632 also blocks Htt neurodegeneration in Drosophila. Taken together with this study, these data validate the approach of
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Ishizaki, T., et al., 2000. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol. Pharmacol. 57, 976–983. Kazantsev, A., et al., 2002. A bivalent Huntingtin binding peptide suppresses polyglutamine aggregation and pathogenesis in Drosophila. Nat. Genet. 30, 367–376. Li, M., et al., 2007. Soluble androgen receptor oligomers underlie pathology in a mouse model of spinobulbar muscular atrophy. J. Biol. Chem. 282, 3157–3164. Mangiarini, L., et al., 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493–506. Pattison, J.S., Robbins, J., 2008. Protein misfolding and cardiac disease: establishing cause and effect. Autophagy 4, 821–823. Pollitt, S.K., et al., 2003. A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor. Neuron 40, 685–694. Ravikumar, B., Rubinsztein, D.C., 2006. Role of autophagy in the clearance of mutant huntingtin: a step towards therapy? Mol. Aspects Med. 27, 520–527. Ravikumar, B., et al., 2004. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585–595. Scherzinger, E., et al., 1997. Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90, 549–558.
Shao, J., Diamond, M.I., 2007. Polyglutamine diseases: emerging concepts in pathogenesis and therapy. Hum. Mol. Genet. 16 (Spec No. 2), R115–R123. Shao, J., et al., 2008a. ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation. FEBS Lett. 582, 1637–1642. Shao, J., et al., 2008b. Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation. Mol. Cell. Biol. 28, 5196–5208. Shibuya, M., et al., 2005. Effects of fasudil in acute ischemic stroke: results of a prospective placebo-controlled double-blind trial. J. Neurol. Sci. 238, 31–39. Takahashi, T., et al., 2007a. Soluble polyglutamine oligomers formed prior to inclusion body formation are cytotoxic. Hum. Mol. Genet. Takahashi, Y., et al., 2007b. Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy. J. Biol. Chem. 282, 24039–24048. Tanaka, M., et al., 2004. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat. Med. 10, 148–154. Uehata, M., et al., 1997. Calcium sensitization of smooth muscle mediated by a Rhoassociated protein kinase in hypertension. Nature 389, 990–994. Wang, J.C., et al., 2006. Novel arylpyrazole compounds selectively modulate glucocorticoid receptor regulatory activity. Genes Dev. 20, 689–699. Zhang, X., et al., 2005. A potent small molecule inhibits polyglutamine aggregation in Huntington's disease neurons and suppresses neurodegeneration in vivo. Proc. Natl. Acad. Sci. U. S. A. 102, 892–897.
Contents lists available at ScienceDirect
Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i
Y-27632 improves rotarod performance and reduces huntingtin levels in R6/2 mice Mei Li a,b, Yong Huang c, Aye Aye K. Ma a,b, Emil Lin c, Marc I. Diamond a,b,⁎ a b c
Department of Neurology, University of California, San Francisco, CA 94143, USA Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA
a r t i c l e
i n f o
Article history: Received 1 May 2009 Revised 24 June 2009 Accepted 28 June 2009 Available online 8 July 2009
a b s t r a c t Huntington disease (HD) is a devastating, untreatable, dominantly inherited neurodegenerative disease. It is caused by an expanded CAG codon repeat that leads to an elongated polyglutamine tract in the N-terminus of the huntingtin (Htt) protein. Few mechanism-based therapeutic leads have been developed. Y-27632, an inhibitor of the Rho-associated kinase ROCK, reduces Htt aggregation in cultured cells and Htt-induced neurodegeneration in Drosophila, but its effect in mice is unknown. We determined that Y-27632 is bioavailable in brain, with a half-life of 60–90 min. We then initiated a trial in R6/2 mice, which express Htt exon 1, administering 100 mg/kg/day of Y-27632 in drinking water. We did not observe a significant effect on brain weight, inclusion number or size, striatal medium spiny neuron number, clasping behavior, or lifespan. However, Y-27632 treatment improved rotarod performance significantly, and also reduced soluble brain Htt levels. The ROCK signaling pathway thus remains a promising therapeutic target for HD, and more potent inhibitors may prove useful. © 2009 Published by Elsevier Inc.
Introduction Huntington disease (HD) is an untreatable, devastating autosomal dominant neurodegenerative disease characterized by psychiatric disturbance, motor impairment, and dementia. CAG codon expansion in the huntingtin gene past 37 repeats leads to an expanded, pathogenic polyglutamine tract within the huntingtin (Htt) protein (1993). This expansion destabilizes Htt, leading to misfolding, aggregation, inclusion formation, and toxicity (Shao and Diamond, 2007). Emerging evidence suggests that small oligomeric aggregates may constitute the pathologic species in polyglutamine diseases (Li et al., 2007; Takahashi et al., 2007a; Takahashi et al., 2007b; Pattison and Robbins, 2008). Multiple studies have identified aggregation inhibitors, and several of these compounds have been tested in mouse models with modest effects on motor performance (Apostol et al., 2003; Chopra et al., 2007; Kazantsev et al., 2002; Tanaka et al., 2004; Zhang et al., 2005). We previously established a cellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET), which was used to identify Y-27632, an inhibitor of the Rhoassociated kinase ROCK. Y-27632 reduced Htt aggregation ∼15% in cells, and inhibited photoreceptor degeneration in a Drosophila model of HD based on neuronal expression of Htt exon 1 (Pollitt et al., 2003).
⁎ Corresponding author. GH-S572B, 600 16th Street, San Francisco, CA 94143-2280, USA. Fax: +1 415 514 4112. E-mail address: [email protected] (M.I. Diamond). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.nbd.2009.06.011
We have observed that both ROCK and a related kinase PRK-2 are inhibited by Y-27632 to reduce polyglutamine aggregation, and these kinases each contribute to the effects of Y-27632 in cells (Shao et al., 2008a). In studies of the downstream molecular mechanism by which Y-27632 reduces intracellular aggregation, we have identified profilin, an Htt interacting protein (Goehler et al., 2004), as a novel target of ROCK (Shao et al., 2008b). Profilin potently inhibits Htt aggregation in cells (Shao et al., 2008b), and a Drosophila homologue has been observed to suppress polyglutamine-mediated toxicity (Burnett et al., 2008). Profilin is phosphorylated at Ser-137 by ROCK, which blocks its ability to directly bind the Htt peptide. Thus, ROCK inhibition promotes dephosphorylation at Ser-137 and enables profilin's protective effects (Shao et al., 2008b). There is no evidence that PRK-2 directly phosphorylates profilin. Importantly, knockdown of profilin completely blocks the anti-aggregation effects of Y-27632 in cells, suggesting that ROCK signaling through profilin is a key signaling pathway in the regulation of aggregation (Shao et al., 2008b). Given our emerging knowledge about its molecular mechanisms in cells, and its effects in Drosophila, there is a strong rationale to test Y-27632 in a mouse model of HD. R6/2 mice express the Htt exon 1 peptide from the huntingtin promoter (Mangiarini et al., 1996), and have been widely used in preclinical trials for HD (Chen et al., 2000; Ferrante et al., 2002; Hockly et al., 2003a; Ferrante et al., 2003; Fox et al., 2004; Ferrante et al., 2004; Chopra et al., 2007; Bates and Hockly, 2003). These mice exhibit a progressive neurologic phenotype that leads to early death, generally by 12–16 weeks. This is associated with early and progressive accumulation of the Htt peptide in intracellular inclusions
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(Davies et al., 1997; Scherzinger et al., 1997). We used this model to test the efficacy of orally administered Y-27632.
have spontaneously died, which frequently occurred without the first two criteria being met.
Materials and methods
Rotarod testing
Y-27632 was a generous gift from Mitsubishi Pharma, Japan. Antibodies Mouse anti-human huntingtin N-terminal antibody clone EM48, mouse anti-expanded polyglutamine antibody clone 1C2, mouse antihuman and mouse full length huntingtin antibody MAB2166 and rabbit anti-dopamine and cAMP regulated phosphoprotein of 32 kD (DARPP32) were purchased from Chemicon International Inc.; mouse anti-glial fibrillary acidic protein (GFAP) was kindly shared by Zhaolin Hua, M.D., Ph.D. and Robert Edwards, M.D. at UCSF. Rabbit antiphospho-profilin 1 at serine 137 was developed by our laboratory (Shao et al., 2008b). Y-27632 detection in vivo Frozen brain specimens were pulverized, and then homogenized in H2O. Acetonitrile was added to precipitate protein with chloroquine (100 ng/ml) as an internal standard (IS), and Y-27632 concentration in the supernatant was measured by HPLC-tandem mass spectrometry (LC-MS-MS). To create reference standards, 14 known concentrations of Y-27632 (1.7–1700 ng/ml) were split into vehicle-treated mouse blood and brain specimens and carried through the extraction/ purification procedure. The LC/MS/MS system consisted of Shimadzu LC-10 AD pumps, a Waters intelligent Sample Processor 717 Plus autosampler, and a Micromass Quattro LC Ultima triple quadrupole tandem mass spectrometer. The mass spectrometer was set for electrospray ionization in positive ion mode. Y-27632 and IS were detected with multiple reaction monitoring (MRM) at 248.3 N 93.3 m/z and 320.9 N 247.6 m/z, respectively. The sample cone voltage and collision energy for both Y-27632 and IS were set at 30 eV and 25 eV, respectively. Y-27632 and IS were separated by silica column (50 mm × 4.6 mm) with mobile phase composed of 90% acetonitrile, 0.06% TFA and 2.5 mM ammonium formate. The flow rate was 1.8 ml/ min and 1/10 was split into the mass detector. Mouse husbandry R6/2 mice in the B6/CBA background were backcrossed with C57B/6 females (Jackson Laboratory, Bar Harbor, ME) for more than seven generations. Genotyping and CAG repeat numbers were detected from tail DNA by PCR. Mice were maintained in a barrier animal facility at the University of California San Francisco, housed five per cage with food and water available ad libitum, and kept on a 12 h light/dark cycle. All studies and procedures were done under the approval of the Institutional Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Therapeutic trials For analysis of rotarod performance, 16 female R6/2 animals were used. These consisted of 7 littermate pairs, and two additional animals. Littermates were evenly divided between the two groups. For the analysis of Y-27632 on lifespan, 17 female R6/2 animals were used. These consisted of 8 littermates evenly divided between treatment and control groups, and an additional animal was studied in the treatment group. For lifespan studies, animals were followed in accordance with IACUC approval until they lost N30% of body weight, or exhibited a moribund appearance (based on poor movement or grooming) at which point they were euthanized, or were found to
We performed rotarod evaluations using a rotarod apparatus (Columbus Instruments. Columbus, OH) as described previously (Li et al., 2007). We carried out four rotarod tests per session, allowing at least 15 min of rest for the mice between each test. The rotarod accelerated from 4 to 40 rpm over 15 s and was maintained at 40 rpm thereafter. The lowest value from all four tests was discarded. The remaining three values were averaged, and the data were analyzed by the student's t-test, assuming equal variance between the two groups. Histology and cell counts At age 13.5 weeks, mice were sacrificed and brains were cut in sagittal midline sections. The left side of the brain was freshly frozen by ethanol-dry ice for biochemical studies and right side of the brain was embedded in OCT compound and freshly frozen by ethanol-dry ice for histological studies. 12 μm serial coronal sections cut through the whole striatum were collected and fixed with 4% paraformaldehyde. Every 8th slice was immunostained with DARPP32 and GFAP antibodies and DAPI. Adjacent slices were stained with hematoxylin/ eosin. Cells in the striatum were counted using a previously published method (Chopra et al., 2007), with minor modifications. 36–60 serial adjacent images were taken from one large image of a whole slice using a Nikon 6 dimensional microscope (Nikon) at the Nikon Imaging Center at UCSF (http://nic.ucsf.edu/6D.html). A 10× objective and automated program NIS-Elements Advanced Research 3.0 (http:// www.nis-elements.com/) were used. DARPP32 positive cells, GFAP positive cells or DAPI positive cells were counted by analyzing the large images with Image J (NIH, http://rsbweb.nih.gov/ij/). The area of the striatum at the level of crossing of the anterior commissure was also measured by Image J. Statistical analysis was performed using a student's t-test. Immunohistochemistry Brain, pancreas and muscle embedded in OCT compound were cut on a cryostat (Leica 1850). 12 μm sagittal brain sections cut from the midline, and coronal sections cut from half of the brain were air-dried for 20 min. Dried sections were fixed with 4% paraformaldehyde for 10 min at 4 °C, washed with 0.05% Tween-20 in phosphate-buffered saline, and then incubated with 5% goat serum and 0.1% Triton X-100 in phosphate-buffered saline for 30 min at room temperature followed by overnight primary antibody incubation at 4 °C (mouse anti-huntingtin N-terminal antibody EM48 (1:400) and rabbit antiphospho profilin 1 at serine137(1:1000; n = 12 each in Y-27632 and vehicle-treated groups), or rabbit anti-DARPP32(1:500) and mouse anti-GFAP(1:250; n = 6 each in Y-27632 and vehicle-treated groups)), and washing with 0.05% Tween-20 in phosphate-buffered saline. Goat anti-mouse IgG conjugated with Alexa 546 and goat anti-rabbit IgG conjugated with Alexa 488 (1:4000; Molecular Probes) were applied to the sections for 2 h at room temperature in the dark. The sections were then washed for 1 h and counterstained with 4′6′ diamidino-2phenylindole (DAPI) (50 ng/ml) for 30 s, washed, and mounted with ProLong Gold anti-fade reagent (Molecular Probes). Fluorescence images were collected with a Nikon spectral laser confocal microscope C1s1 with excitation wavelength at 405 nm, 488 nm and 561 nm and detection at 435–450 nm, 515–530 nm, and 605–675 nm. Immunoprecipitation 50 mg of brain powder was dounce-homogenized with 25 strokes on ice in 500 μl RIPA buffer and centrifuged 15,000 ×g for 30 min at
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Fig. 1. Evaluation of Y-27632 clearance in serum and brain. Wild-type mice were given an intraperitoneal dose of 10 mg/kg Y-27632. Two animals were sacrificed at the indicated time intervals, and serum (A) and brain tissue (B) was harvested for mass spectrometry to determine compound levels. Error bars represent the range. Absolute concentrations were determined by using control tissue extracts spiked with known amounts of compound.
4 °C. 2 μl of 1C2 antibody (Chemicon) was covalently conjugated to a 20 μl bead volume of anti-mouse IgG beads (Sigma) by DSS (Pierce) as previously described (Li et al., 2007). Beads were washed thoroughly with RIPA buffer. 200 μl supernatant extracted with the above RIPA buffer was added with 1C2-mouse IgG beads overnight at 4°C followed by at least four washes in RIPA buffer. Samples were boiled for 5 min in 1% SDS sample buffer prior to SDS-PAGE and Western blot analyses. Blots were then probed with 1C2 (1:2500 dilution), washed with TBS and 0.5% Tween-20, and then incubated with horseradish peroxidase-conjugated mouse True Blot (1:5000 dilution, eBioscience) that does not recognize denatured rabbit IgG heavy or light chain. Horseradish peroxidase signal was detected by ECL-Plus (Amersham Biosciences). Quantitative PCR RNA was prepared from whole brain by using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Real time PCR was performed as described previously (Wang et al., 2006), except the internal control was actin instead of Rpl19. R6/2 transgene primers were: TCA GGT TCT GCT TTT ACC TGC (forward) and CTT CAT CAG CTT TTC CAG GG (reverse). Relative Htt exon 1 transcript was measured by [sample(Ct Htt exon1 − Ct actin) − minimum sample(Ct Htt exon1 − Ct actin)]2. Results Y-27632 bioavailability We first determined whether Y-27632 would penetrate the blood brain barrier. We administered 10 mg/kg as a single intraperitoneal dose to a group of 14 mice and sacrificed two of them at 15 min, 30 min, 1.5 h, 6 h, 8 h, 12 h and 24 h after injection. We then measured Y-27632 serum and brain concentrations by HPLC-tandem mass spectrometry. Peak serum concentration was 3–3.5 μM and brain concentration was 0.3–0.65 μM 15 min after injection (Fig. 1). Half-life in each tissue was ∼60–90 min and the brain/serum ratio was ∼1/6 Table 1 Concentrations in serum and brain after oral administration of Y-27632. Sample no.
Serum conc. (nM)
Brain conc. (nM)
Serum/brain ratio
1 2 3 4 5
59 91 298 393 51
6.9 17.6 47.5 20.2 10.3
8.7 5.2 6.3 19.5 4.9
Five animals were administered 50 mg/kg/day of Y-27632 orally for one week. Animals were then sacrificed, and serum and brain levels were determined by liquid chromatography–mass spectrometry.
(Fig. 1). These studies indicated that at this dose Y-27632 readily penetrates the blood brain barrier, although with a fairly short halflife. Peak CNS concentrations were slightly less than desired to achieve optimal ROCK inhibition. Next, we tested for oral availability, first confirming the stability of Y-27632 in drinking water (Supp. Fig. 1). We treated a cohort of 5 wild-type mice with 50 mg/kg/day Y-27632 in drinking water for 7 days and determined concentrations in serum and brain (Table 1). Finally, we used dose escalation to determine the highest amount tolerated. We administered Y-27632 in drinking water to a cohort of 5 of wild-type mice beginning at 75 mg/kg/day and gradually increasing by 25 mg/kg/day every 3–4 days. The mice tolerated up to 250 mg/kg/day, however we were concerned about using 250 mg/kg/day for an extended period due to limiting compound and possible toxicity. Thus, we decided to use 100 mg/ kg/day administered orally for an efficacy trial. Y-27632 improves rotarod function R6/2 mice are very well characterized, and determinants of experimental variability include littermate status, gender, weight, and CAG repeat length (Hockly et al., 2002; Hockly et al., 2003b). To reduce variation we only used female mice, controlling for initial body weight and littermate status, and controlling for CAG repeat number by PCR (all animals contained 110 to 120 CAG repeats within the Htt exon 1 transgene). For the rotarod trial, 16 identically aged R6/2 females, including 7 littermate pairs, were divided into two groups. 8 R6/2 females received 100 mg/kg/day Y-27632 in drinking water. The second group received vehicle control (water). We administered Y27632 beginning at age 4 weeks. After an initial drop in the weight of the treated animals, we observed no significant differences between the two groups over the course of the study (Fig. 2A). No significant changes were observed in clasping responses, a pathological limb reflex observed in these animals (Fig. 2B). 3 weeks after beginning treatment we initiated rotarod testing once per week, as soon as it was possible (rotarod testing in very young animals is not feasible). An accelerating rotarod (Columbus Instruments) was set to increase gradually from 4–40 rpm over 15 s, and maintained at 40 rpm thereafter. Four trials were carried out at each time point, and the shortest trial was discarded; the remaining three trials were averaged. Both groups exhibited a decline in performance over time. At age 7.5, 8.5 and 9.5 weeks, there were no significant differences between treatment and control groups. At age 10.5 weeks there was a significant improvement in performance in the treated vs. control animals: 55.8 ± 9.0 s vs. 24.4 ± 16.2 s, (p b 0.001, t-test); at 12.5 weeks 33.8 ± 16.0 vs. 13.7 ± 12.1 s, (p b 0.05, t-test) (Fig. 2C). A direct comparison of the rotarod performance of littermates at 12.5 weeks illustrates a relatively consistent benefit of the compound (Fig. 2D). An individual comparison of performance for each animal indicated an improvement in median function in the treated animals after
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Fig. 2. Analysis of Y-27632 on behavior in R6/2 mice. Cohorts of female R6/2 mice were randomized according to littermate status, weight, and CAG repeat length into two groups of 8 animals. Y-27632 at 100 mg/kg/day was administered by putting the compound in drinking water, controlling for daily intake to ensure accurate dosing. Dosing began at age 4 weeks. Rotarod testing began at age 7.5 weeks. (A) Y-27632 treatment had no significant effect on weight. (B) Y-27632 treatment had no significant effect on development of the clasping reflex. (C) Y-27632 treatment significantly improved rotarod performance beginning at age 10.5 weeks. ⁎p b 0.05; ⁎⁎p b 0.01 (t-test). (D) Comparison of littermates tested shows a consistent improvement in rotarod performance in the treated animals at 12.5 weeks. (E) Plots of individual animal's performance at each time point indicate an improvement in median performance on the rotarod in the treated group.
10 weeks (Fig. 2E), ruling out a large effect on a small number of animals. At 13.5 weeks, three animals in the treatment group died due to handling-induced seizures, but no animals in the control group.
Y-27632 has no effect on lifespan Because we observed handling-induced seizures, especially in the treated animals, we were concerned that despite its benefit on rotarod performance that Y-27632 might somehow lower the seizure threshold. Thus, we decided to test its effects on lifespan in the absence of the extensive handling required for rotarod testing. We repeated the trial with an additional 17 female R6/2 littermate mice, sorted by the same criteria used previously, with 9 animals in the Y-27632 treatment group and 8 animals in control group. Y-27632 was given from age 4 weeks. The median lifespan of Y-27632 treated animals was 126 days and control animals was 120 days, which matched the lifespan of other cohorts of R6/2 mice maintained in our colony (Supp. Fig. 2). There was no significant effect of treatment on lifespan (Fig. 3A), and no effect on body weight in this group (Fig. 3B).
Y-27632 does not change medium spiny cell density in the striatum Striatal medium spiny neuron loss is a principal feature of Huntington disease pathology. Thus, we tested whether Y-27632 treatment would change the medium spiny neuron density in R6/2 mice. We sacrificed 6 pairs of treated and control mice at 13.5 weeks. Brains were weighed and cut in a sagittal midline section. Half of the brain was embedded in OCT compound, and frozen in ethanol-dry ice for pathological studies; the other half was snap-frozen for biochemical studies. The brains embedded in OCT compound were cut on a coronal plane, with 12 μm serial sections cut from the anterior through the posterior striatum. Every 8th section was collected and double stained with DARPP32 (dopamine and cAMP regulated phosphoprotein of 32 kD), a marker for medium spiny neurons, and GFAP (glial fibrillary acidic protein), a marker for astrocytes. Adjacent images were collected by an automated fluorescence microscope (Nikon 6D). The images were then recombined to encompass a large image of the entire slice section. By analyzing the data using Image J (n = 6 samples per condition), we found that Y-27632 treatment had no statistically significant effect on the area of the striatum, medium spiny neuron
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Fig. 3. Analysis of Y-27632 on lifespan in R6/2 mice. Cohorts of female R6/2 mice were randomized according to littermate status, weight, and CAG repeat length into a treatment group of 9 animals, and a control group of 8 animals. The treatment group received 100 mg/kg/day of Y-27632 in drinking water, controlling for intake. Animals were weighed 2×/ week. (A) No effect of treatment was observed on survival. (B) No significant effect of treatment was observed on weight.
density, or astrocyte density. We noted an apparent increase in total cell density, and diminished astrocyte density in the striatum (Fig. 4; Supp. Fig. 3), indicating that other types of glial cell density might be slightly increased by Y-27632 treatment, but this did not rise to statistical significance.
27632 selectively reduces the levels of soluble Htt exon 1 protein, suggesting that it alters Htt exon 1 biochemistry, either promoting incorporation into a form not detected by immunoprecipitation, or by increasing its degradation. Discussion
Y-27632 reduces soluble Htt exon 1 levels in R6/2 mouse brain To confirm an effect of Y-27632 on Htt Exon 1 in the brain, we directly tested whether treatment altered inclusion formation or soluble protein levels. Various sections of brain were evaluated by immunohistochemistry using the EM48 antibody, which recognizes aggregated forms of Htt exon 1. We noted no significant differences in inclusion size or density in striatum, forebrain cortex, hippocampus or cerebellum (Fig. 5, Supp. Fig. 4). In R6/2 brain, much of the Htt exon 1 peptide is in an aggregated state, making accurate assessments of total protein levels difficult. We used formic acid to solubilize the aggregated protein to attempt to estimate total mutant Htt exon 1 levels. However the Htt exon 1 protein rapidly re-aggregated in solution, making this impossible (data not shown). Thus, we homogenized brains in RIPA buffer, and immunoprecipitated soluble Htt using 1C2 antibody (Chemicon International), which does not react with aggregated species. We probed the immunoprecipitated protein by western blot with 1C2, comparing treated littermates to their controls. 7/8 R6/2 mice treated with Y-27632 had reduced levels of soluble Htt relative to actin controls (Figs. 6A, B). qPCR indicated that this was not associated with a corresponding change in Htt exon 1 transgene expression (Fig. 6C). We also measured levels of endogenous mouse full length Htt (MAB2166, Chemicon International), but did not observe changes between Y-27632 treated and control R6/2 mice (Fig. 6D). Thus, Y-
In this study we evaluated oral administration of Y-27632 in the R6/2 mouse model of HD. We found that Y-27632 treatment significantly improves rotarod performance in these mice, and reduces soluble Htt peptide. Whereas vehicle-treated animals exhibited a fall in rotarod performance of ∼ 85% over the course of the trial period, the treated animals only experienced a drop of ∼50%. These effects were paralleled by a reduction in soluble Htt in 7/8 of the treated littermates. We did not observe statistically significant effects on brain weight, gross atrophy, lifespan, clasping, inclusion formation, or neuron counts in the striatum. We estimate that peak brain levels of Y-27632 were likely to have been ∼100 nM in this study. This is considerably less than levels ranging from 5–50 μM that have been studied in cultured cells, and which reduced aggregation of Htt by about 15–25% (Pollitt et al., 2003; Shao et al., 2008b). Given the modest effects of saturating Y-27632 treatment in cultured cells, it is perhaps not surprising that gross pathological analysis of tissue specimens did not reveal significant changes in inclusion formation. We also checked for effects on inclusion formation in other tissues that would likely have been exposed to higher drug levels, including pancreas and muscle, but did not observe effects on inclusion frequency. We stained with a phospho-profilin antibody developed in our laboratory (Shao et al., 2008b), but did not observe any changes in phospho-profilin in the brains of the treated mice. This could be due to a failure of ROCK to
Fig. 4. Analysis of Y-27632 on brain weight, striatum atrophy and cell densities was performed on 6 controls and 6 treated R6/2 mice. (A) Average of brain weight at 13.5 weeks was calculated. There was no significant difference between control and treated groups. (B) Average striatal area at the level of crossing of the anterior commissure. There was no significant difference between control and treated groups. (C–E) Half brain coronal sections ranging from anterior striatum to posterior striatum from 6 pairs of R6/2 mice in control and Y-27632 group were cut as 12 μm sections, and every 8th section was stained with DAPI, and DARPP32 and GFAP antibodies. Whole striatal images were taken by an automated fluorescence microscope (Nikon 6D). Cell numbers on each large image were counted by using Image J software. No significant effects of Y-27632 treatment were observed on (C) DAPI positive cell density (total cells); (D) DARPP32 positive cell density (medium spiny neurons); or (E) GFAP positive cell density (astrocytes). Error bars = S.E.M.
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Fig. 5. Analysis of Y-27632 on inclusion formation and phospho-profilin levels. Representative histopathology is shown. Various brain regions were stained with EM48 antibody (red), phospho-profilin antibody (green), and DAPI (blue). EM48 recognizes diffuse and aggregated forms of Htt exon 1. Phospho-profilin was distributed in the cytoplasm of forebrain cortical neurons, hippocampal CA3 neurons and cerebellar Purkinje neurons but not in striatal neurons. No effect of Y-27632 treatment was observed on cellular atrophy, phospho-profilin level or distribution, inclusion size or frequency. Scale bars are shown.
phosphorylate profilin in brain, but is more likely due to sub-saturating doses of Y-27632. Whether a more potent ROCK inhibitor, or higher brain concentrations, would produce more apparent effects on profilin phosphorylation or inclusion formation awaits further study. Y-27632 treatment selectively reduced brain Htt exon 1 levels, without affecting Htt exon 1 gene expression or full length mouse Htt. This suggests that the compound exerts some effect on the protein's metabolism in vivo, and is in keeping with a recent report which indicates an enhancing effect of Y-27632 treatment on Htt degradation via proteasome and autophagy pathways (Bauer et al., 2009). Our prior work suggests that Htt exon 1 is not cleared via the proteasome (Chandra et al., 2008), but autophagy may play an important role (Ravikumar and Rubinsztein, 2006; Ravikumar et al., 2004). As we have mentioned, it is very difficult to quantify precisely total Htt levels in vivo due to its rapid aggregation. However, our analysis of Htt Exon 1 transgene expression via qPCR and endogenous Htt levels effectively rules out a primary effect of Y-27632 on the Htt promoter, and suggests either that the total protein level is reduced, or that soluble protein is more rapidly cleared. In cultured cells we have not observed a consistent effect of Y-27632 treatment on Htt exon 1 levels, but this
may relate to the fact that it has been relatively over-expressed via transient transfection. As we learn more about mechanisms that govern Htt exon 1 solubility vs. aggregation, it will be interesting to determine whether these conformational states have a direct effect on protein degradation. Multiple parameters likely govern lifespan of R6/2 mice. These include peripheral effects such as weight loss and muscle atrophy, and central effects such as seizures. Consistent with this, two studies besides our own have noted a dissociation between lifespan and improved performance in behavior assays (Chopra et al., 2007; Chou et al., 2005). In this study, we did not observe significant weight loss in either group of animals, even at later points in life, or profound defects in glucose metabolism (data not shown), suggesting that muscle wasting or systemic illness did not play a major role. After the rotarod trials, we observed fatal handling-induced seizures in 3/8 animals treated with Y-27632, but in none of the controls. Thus, it is possible that despite a benefit in motor function, drug treatment may have lowered the seizure threshold. However, one report indicates that Y-27632 may reduce seizures in a mouse model (Inan and Buyukafsar, 2008).
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identifying therapeutic leads based on the inhibition of intracellular Htt aggregation. These results also provide a strong rationale for further study of molecular mechanisms by which Y-27632 functions. Finally, they suggest that development of more effective ROCK inhibitors with better CNS penetration might form the basis of an important new therapeutic strategy for HD. Acknowledgments We thank Mitsubishi Pharma for generously providing the Y-27632 used for this study. We thank Stanley Prusiner, M.D. for the help in obtaining Y-27632; Samuel Pleasure, M.D., Ph.D. for use of his cryostat; Clifford Bryant, Ph.D. for detecting Y-27632 stability; Kurt Thorn, Ph.D.; Amie Lee, M.D.; and the Nikon Imaging Center at UCSF for use of the Nikon C1s1 spectral confocal microscope and Nikon 6D microscope. We also thank Jieya Shao, Ph.D.; Zhaolin Hua, M.D., Ph.D.; Guangnan Li, Ph.D.; and Brian Feldman, M.D., Ph.D. for advice, reagents and technical support. ML was supported by a grant from the Muscular Dystrophy Association. MID was supported by grants from the Muscular Dystrophy Association, the NIH:NINDS, and the Taube Family Foundation Program in Huntington's Disease Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nbd.2009.06.011. Fig. 6. Y-27632 reduces soluble Htt exon 1 brain levels. (A) Brains from the animals studied in the behavior assay were homogenized and soluble Htt exon 1 levels were determined by immunoprecipitation and western blot with 1C2 antibody, which recognizes soluble forms of Htt exon 1. In 7/8 littermate pairs, there was a relative reduction in soluble Htt exon 1 levels. (B) Band intensity of the Htt exon 1 was compared to actin for each sample, and averaged. There was a significant decrease in the average Htt exon 1 band intensity relative to actin. ⁎p b 0.05; error bars = S.E.M. (C) qPCR was used to analyze Htt exon 1 gene expression in each of the samples. There was no effect of Y-27632 treatment on Htt exon 1 transcript. (D) Full length Htt expression was analyzed by western blot on whole brain lysates. No significant effect of Y-27632 treatment was observed.
We carefully searched for biochemical evidence of ROCK inhibition in the brain, but faced difficult hurdles. We have previously created a phospho-specific antibody to Ser-137 of profilin 1 that detects changes in profilin phosphorylation in a variety of cultured cells, including primary neurons (Shao et al., 2008b). We used this antibody to probe brain samples by immunohistochemistry and western blot, but did not detect effects of Y-27632 treatment on profilin phosphorylation. This is likely due to several factors. First, we were not able to achieve micromolar concentrations of Y-27632 in the brain for extended periods. We estimate that we achieved levels of ∼100 nM within the brain at peak levels, below the IC50 level of 140 nM previously reported for the kinase in cells (Ishizaki et al., 2000; Uehata et al., 1997). In this setting it could be very difficult to detect changes in phospho-profilin. However, partial ROCK inhibition may be the most therapeutically useful, given the importance of ROCK in a variety of cellular pathways. For example, another ROCK inhibitor HA-1077 (Fasudil) has been used at a lower dose (60 mg administered twice daily intravenous injection for 14 days) for ischemic stroke patients (Shibuya et al., 2005), indicating that partial inhibition of ROCK may still beneficial, even without the dramatic effects on protein phosphorylation required to document kinase inhibition biochemically. Development of effective therapies for HD will be hastened by the identification of factors that may be specifically targeted by drugs to reduce Htt toxicity. Our prior work has suggested that Y-27632 reduces Htt aggregation in cells through inhibition of ROCK signaling to profilin. Y-27632 also blocks Htt neurodegeneration in Drosophila. Taken together with this study, these data validate the approach of
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