Transcriptional therapy with the histone deacetylase inhibitor trichostatin A ameliorates experimental autoimmune encephalomyelitis

Journal of Neuroimmunology 164 (2005) 10 – 21 www.elsevier.com/locate/jneuroim

Transcriptional therapy with the histone deacetylase inhibitor trichostatin A ameliorates experimental autoimmune encephalomyelitis Sandra Cameloa,1, Antonio H. Iglesiasa,1, Daehee Hwangb,1, Brice Duec, Hoon Ryuf, Karen Smithf, Steven G. Graya, Jaime Imitolad, German Durana, Basel Assaf a, Brett Langleye, Samia J. Khouryd, George Stephanopoulosb, Umberto De Girolamic, Rajiv R. Ratane, Robert J. Ferrantef, Fernando Dangonda,* a

Laboratory of Transcriptional and Immune Regulation, Brigham and Women’s Hospital, Department of Neurology, Harvard Medical School, Boston, MA, United States b Bioinformatics and Metabolic Engineering Laboratory, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States c Division of Neuropathology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States d Center for Neurologic Diseases, Brigham and Women’s Hospital, Department of Neurology, Harvard Medical School, Boston, MA, United States e Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA, United States f Geriatric Research and Education and Clinical Center, Bedford VA Medical Center, Bedford, MA and Neurology, Pathology, and Psychiatry Departments, Boston University School of Medicine, Boston, MA, United States Received 5 November 2004; received in revised form 11 February 2005; accepted 17 February 2005

Abstract We demonstrate that the histone deacetylase (HDAC) inhibitor drug trichostatin A (TSA) reduces spinal cord inflammation, demyelination, neuronal and axonal loss and ameliorates disability in the relapsing phase of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). TSA up-regulates antioxidant, anti-excitotoxicity and pro-neuronal growth and differentiation mRNAs. TSA also inhibits caspase activation and down-regulates gene targets of the pro-apoptotic E2F transcription factor pathway. In splenocytes, TSA reduces chemotactic, pro-Th1 and pro-proliferative mRNAs. A transcriptional imbalance in MS may contribute to immune dysregulation and neurodegeneration, and we identify HDAC inhibition as a transcriptional intervention to ameliorate this imbalance. D 2005 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Histone deacetylase; Trichostatin A; Microarrays; Experimental autoimmune encephalomyelitis

1. Introduction Multiple sclerosis (MS) is a Th1 cytokine-driven inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) (Peterson et al., 2001). Neuronal or axonal injury may account for

* Corresponding author. Laboratory of Transcriptional and Immune Regulation, Brigham and Women’s Hospital Laboratories, Room 312, 65 Landsdowne Street, Cambridge MA 02139, United States. Tel.: +1 617 768 8597; fax: +1 617 768 8595. E-mail address: [email protected] (F. Dangond). 1 These authors contributed equally to the work. 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.02.022

disability in MS and can occur in the absence of overt inflammation (Trapp et al., 1999). Mechanisms that have been implicated in MS pathogenesis include oxidative stress, excitotoxicity, autoimmunity and hormonal imbalance. Therapies that target these distinct pathophysiologic events have been shown to be effective in experimental autoimmune encephalomyelitis (EAE), a Th1 cytokinedriven model of MS. Based on these findings, some are being tested in humans (Youssef et al., 2002). However, drugs with preferential effects on the chronic relapsing (i.e., post-inflammation peak) phase of EAE, in which axonal loss correlates with disability (Wujek et al., 2002), have yet to be identified (Steinman, 1999).

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Histone deacetylase (HDAC) enzymes repress genes via condensation of the nucleosome and interactions with transcription factors. For instance, HDACs bind repressor element 1 silencing transcription factor (REST), which is known to counteract neuronal differentiation traits (Ballas et al., 2001), and Sp1, another transcription factor that mediates neuronal antioxidant pathways (Ryu et al., 2003a,b). Compounds that inhibit HDAC enzymes range from nonspecific agents such as the short-chain fatty acid sodium phenylbutyrate (SPB), to highly specific hydroxamic acids such as trichostatin A (TSA). These structurally diverse agents induce histone hyperacetylation with concomitant up-regulation of neuronal maturation and antioxidant gene expression, indicating REST (Ballas et al., 2001; Hakimi et al., 2002) and Sp1 (Ryu et al., 2003a,b) transcription factor derepression. E2F1 is a transcription factor that promotes immune cell proliferation (Wu et al., 2001). E2F1 activity is triggered by activation of T cells via the IL-2 receptor (IL-2R), and thus holds special relevance for MS and EAE, diseases driven by activated Th1 T cells. In fact, we have shown that peripheral blood mononuclear cells (PBMCs) from MS patients exhibit an activated E2F pathway signature (Iglesias et al., 2004), suggesting E2F as a potential therapeutic target in this disease. Accordingly, E2f1deficient mice have less severe EAE and their splenocytes have decreased g-IFN and elevated IL-4 responses to antigenic stimulation in vitro (Iglesias et al., 2004), suggesting a contribution by E2F1 to Th1 differentiation. E2F1 overexpression, in addition, contributes to neuronal apoptosis (Hou et al., 2000), further supporting our rationale for targeting the E2F pathway in MS. We have previously shown that HDAC mRNAs are elevated in activated immune cells (Dangond et al., 1998), suggesting that they may serve as markers of activation and potential targets of treatment. We have also shown that TSA blocks immune cell proliferation and suppresses the pro-Th1 factor g-IFN (Dangond and Gullans, 1998), in accordance with a growing literature suggesting that HDAC inhibitors cause a shift to a Th2 phenotype (Saemann et al., 2000). For instance, HDAC inhibitors down-regulate the CD28 costimulatory molecule (Moreira et al., 2003), the IL-2 receptor (Moreira et al., 2003) and IL-12 (p35 and p40) (Saemann et al., 2000) and up-regulate IFN-a and -h (Shuttleworth et al., 1983). Since MS is associated with transcriptional dysregulation (Iglesias et al., 2004), we tested whether the transcriptional drug TSA is clinically beneficial for EAE and whether it has neuroimmunoprotective effects.

2. Materials and Methods 2.1. EAE induction For disease induction, we injected 6- to 8-week-old C57BL/6 female mice (Jackson Laboratory, Ann Harbor,

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ME) subcutaneously with 150 Ag myelin oligodendrocyte glycoprotein (MOG)35 – 55 peptide in PBS and CFA containing 0.4 mg of Mycobacterium tuberculosis (H37Ra, Difco, Detroit, MI), and i.p. on days 1 and 3 with 200 ng Pertussis (List Biological, Campbell, CA), as described (Iglesias et al., 2004). 2.2. HDAC inhibitor dose TSA (Biomol, Plymouth Meeting, PA and Wako, Richmond, VA) in PBS (9):DMSO(1) vehicle was administered from day 4 until day 40. Exp. 1 (7.5 mg/kg/dose/day i.p.) required 10 mice on vehicle and 9 on TSA; exp. 2 (same dose) utilized 15 per group. Mice were sacrificed on day 40 after immunization and tissues were collected for histology and molecular studies. 2.3. EAE Score Mice were assessed clinically as described (Milicevic et al., 2003; Suen et al., 1997), using the following score system: 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

No clinical signs Incomplete flaccid tail Flaccid tail Score of one but not two with righting reflex > 5 s Flaccid tail and wobbly gait Paralysis of one limb Complete bilateral hind limb paralysis Complete hind limb paralysis and partial fore limb weakness 4.0 Complete paralysis of hind and fore limbs 5.0 Death. The following definitions were also applied: (1) remission: clinical improvement characterized by at least a half point decrease in the disability score for
2 consecutive days, after the peak score of the acute phase, (2) mean peak of the remission phase: mean of the maximal score reached during remission, and (3) index of disease: the sum of the daily average disease score divided by the average score at disease onset and multiplied by 100 (Suen et al., 1997). The index of disease was determined at days 30 and 40 following EAE induction. 2.4. Histopathology After intracardiac fixative perfusion, cords were isolated, cut into 2-mm lengths and snap-frozen from 4 vehicle and 3 TSA samples that were representative of the mean score of each group throughout the post-inflammatory peak phase. Cords were then fixed in formalin, embedded in paraffin, and serially sectioned in parallel at 20 Am, yielding 7– 10 (200 Am) lumbo-sacral levels per cord, for sequential comparison. H and E, Oil Red-O,

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Sudan Black B and Bielchowsky-stained slides were graded by half-steps from 0.0 (best) to 3.0 (worst) by a blinded pathologist. Unbiased stereological counts of Nissl-stained ventral horn neurons, using Neurolucida Stereo Investigator software optical dissector method (Microbrightfield, Colchester, VT), were taken from serial-cut 50-Am sections of the midthoracic (T4 –T6) spinal cord (4 samples from each group). The total areas were defined in 20 serial-step sections in which counting frames were randomly sampled. An arbitrary line was traced from the central canal horizontally to delineate the ventral horn region to be analyzed. Neuron areas were measured by microscopic videocapture using an automatic Windows-based image analysis system (Optimas, Bioscan Incorporated, Edmonds, WA). All computer-identified cell profiles were differentiated from glial cells and manually verified as neurons based on morphological characteristics, and exported to Microsoft Excel. Crosssectional areas and volume assessment were analyzed using Statview (SAS Institute, Cary, NC). 2.5. Immunohistochemistry We used Abs for activated caspase 3 (casp3) (BD Biosciences, San Jose, CA) and 9 (casp9) (Cell Signaling Technology, Beverly, MA), and ac-histones H3 and H4 (Upstate Biotechnology, Lake Placid, NY), followed by a conjugated second Ab method (Ferrante et al., 2003). Tissue sections (50 Am each) were preincubated in absolute methanol 0.3% hydrogen peroxide solution for 30 min, washed 3 in phosphate-buffered saline (PBS) for 10 min each, placed in 10% normal goat serum (Gibco Labs, Grand Island, NY) for 1 h, incubated free floating in primary antiserum at room temperature for 12 –18 h and washed 3 in PBS. Sections were then placed in periodate-conjugated goat anti-rabbit IgG at 1:1000 in PBS or goat anti-mouse IgG at 1:1000 in PBS (Boehringer-Mannheim, Indianapolis, IN), washed 3 in PBS, and reacted with 3,3V diaminobenzidine HCl (1 mg/ml) in Tris – HCl buffer with 0.005% hydrogen peroxide. Specificity for the antisera used in this study was examined in each immunochemical experiment by using cell lysate samples (BD Biosciences, San Jose, CA, and Cell Signaling Technology, Beverly, MA) as positive controls and by omission of the primary and secondary Abs to determine the amount of background generated from the detection assay. 2.6. Protein Analysis Abs recognizing E2F1 (Santa Cruz Biotechnologies, Santa Cruz, CA), tubulin-a (Sigma-Aldrich, St. Louis, MO) or Abs that recognize both pro-caspase 9 and active caspase 9 (Stressgen Biotechnology Corp., San Diego, CA) were used for western blots. Briefly, tissues or cells were homogenized and suspended in Laemmli buffer, sonicated and incubated on ice. Proteins were recovered after a 20-

min high-speed spin, resolved by 4– 20% gradient Tris – Glycine gel (Novex, San Diego, CA) electrophoresis, and transferred to nitrocellulose (Millipore, Bedford, MA). Blots were 5% nonfat milk-blocked and probed with primary Ab at 1:500 – 1:1000, then with 1:10,000 HRP-conjugated Ab (Affinity Bioreagents, Golden, CO), and exposed to the ECL system (NEN Life Science Products, Boston, MA) and then to X-ray film. RayBioi mouse arrays for 32 cytokines (RayBiotech, Atlanta, GA) were used as recommended by the manufacturer, by incubating with 50 Ag of pooled protein from MOG-stimulated and unstimulated splenocytes cultured in the presence or absence of TSA (50 nM). Chemiluminescence buffers provided with the RayBioi kit were used as recommended before exposure to X-ray film. 2.7. Microarrays Murine U74A chips (Affymetrix, Santa Clara, CA), containing ¨12,426 probe sets, were used for mRNA expression analysis. Three groups of pooled spinal cord RNA were used: control mice injected only with pertussis (no EAE group), EAE mice on vehicle and EAE mice on TSA (n = 3 mice per group). RNA isolation and microarray hybridization were performed as described (Dangond et al., 2004). 2.8. Statistical analysis of microarray data The average intensity of each microarray was first scaled to a 1500 target intensity using Affymetrix MAS5 software. Before statistical analysis, all average difference intensity values of less than 300 or negative values were assigned an arbitrary value of 300. Using these data, we performed a hypothesis test to compute P values of observing particular expression levels for genes by chance, when they are not differentially expressed (null hypothesis). In most hypothesis tests (e.g., t-test), we assume that a statistical measure follows a certain distribution: for example, the difference in expression levels between two conditions follows a t-distribution in t-test. However, the actual (i.e., real) distribution can deviate seriously from the assumed (i.e., theoretical) one, depending on how significantly the data violate underlying assumptions for such tests (e.g., normality and equal variance in t-test). This deviation results in inaccurate estimation of P values, thus increasing a false positive rate in selection of differentially expressed genes. To resolve this problem, we estimated a non-parametric empirical distribution directly from the data using Gaussian kernel density estimation (Wand and Jones, 1995), and used the estimated distribution in our hypothesis test, instead of using an assumed distribution. First, as a statistical measure, for each gene i we computed a log ratio (r i ) between mean expression levels of the pooled 3 EAE (untreated) spinal cord samples ( gEAE ) and of 3 noni

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EAE (i.e., pertussis only) control samples ( gCi ) (Lock et al., 2002): r i = log10(( gEAE ) / ( gCi )). Second, we obtained an i empirical probability density function (PDF) of those ratios using a kernel density estimator. The density for a certain ratio (r)
is defined by n ratios (r i ) in: fˆ ðr;hÞ ¼ i ðnhÞ1 ~ni¼1 K rr where K (Gaussian kernel in this h study) is a unimodal probability density function satisfying XK(x)dx = 1. The bandwidth (h) is a positive number that determines the kernel width. We determined the optimal h to minimize the mean integrated squared error (MISE) between the estimated density (fˆ) 1=2and the target density RðK Þ 1=5 ( f ) for the normal kernel: hˆ ¼ 8p s. We defined 2 3l ðK Þ n 2

R(K) and l 2(K) by XK ðrÞ2 dr and Xr2 K ðrÞdr, respectively, and s is the estimated (sampled) standard deviation of log ratios. Since this bandwidth to minimize MISE tends to be large, we tried hˆ /2, hˆ /4, and hˆ /8 to choose an optimal bandwidth, and in most of cases hˆ /2 produced the optimal distribution structure. We assume that the estimated PDF represents the distribution of non-differentially expressed genes because a majority of genes are not differentially expressed. As most two-tailed tests, the left and right tails of the distribution represent down-regulated and upregulated genes. Finally, we used our empirically estimated distribution to perform a typical two-tailed hypothesis, as in performing a t-test using a t-distribution. The null hypothesis (H 0) is that means are equal (i.e., a ratio is zero), and the alternative hypothesis (H 1) is that means are not equal (i.e., a ratio is non-zero). For a log ratio of each gene, we calculated the area (a i ) under the PDF between the negative infinity and the log ratio. Then, the probability of observing the log ratio of gene i when the gene is not differentially expressed is calculated as P i = 2 min (a i ,1  a i ): if P i is less than a significance level (0.05 in this study), the test concludes that the gene is differentially expressed. The same method was used to compare cord RNA samples of TSA-treated vs. untreated EAE mice. 2.9. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) We performed one-step QRT-PCR in triplicate for each sample (n = 3 per group), with SYBR Green and an ABI PRISM 7700 System (PE Applied Biosystems, Foster City, CA). One-step QRT-PCR was performed following strict guidelines from the ABI PRISM manufacturer, including implementation of the recommended primer design parameters, optimization using standard curves with primers at various dilutions, assessment of ideal temperature conditions for primers to prevent melting and use of 100 ng RNA per experiment. A visual analysis was also performed to verify that amplification curves for h-actin and the target RNAs were comparable. In addition, we ran the PCR products in electrophoresis gels to confirm the presence of a single band and verify the absence of

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primer – dimer formation. The following custom-made primers (Integrated DNA Technologies, Coralville, IA) were used: Neurobiology panel Gpx1 FWD Gpx1 RVS NaV1.2 FWD NaV1.2 RVS Dbh FWD Dbh RVS CD18 (Mac-1) FWD CD18 (Mac-1) RVS Bak FWD Bak RVS Bid FWD Bid RVS Caspase 2 FWD Caspase 2 RVS Aif FWD Aif RVS

5V-CGACATTGCCTGGAACTTTGA-3V 5V-CGATGTCGATGGTACGAAAGC-3V FWD 5V-CCCATGTGGCCAATGTCA-3V 5V-GGCAAATCTTCCTCCATCCA-3V 5V-CCTATCTCCATGCATTGCAACA-3V 5V-TGGAGGTGATCTTAGGCAAAGG-3V 5V-CCACCGATGTGTGAGGATTG-3V 5V-GGCACAAGAGGTGTGGTTGTC-3V 5V-TCACCCCCACCTCATCgT-3V 5V-CCAAGGCAAAAATGGATGGAT-3V 5V-CGTCTTGTGCTGAACTTTGCTT-3V 5V-CATGGCTGGGATGAGTTCAGA-3V 5V-TGCCTACTCGCTCAGACATGAT-3V 5V-GAGTGAGGGCCTCAATGTACCA-3V 5V-AGGACTCCTTCCACCACAATGT-3V 5V-TTGAAGGTCGCAGAGTACGAAA-3V

Immunology panel Ifn-a FWD Ifn-a RVS IL-2Ra FWD IL-2Ra RVS IL-8R FWD IL-8R RVS IL-12p35 FWD IL-12p35 RVS Cd28 FWD Cd28 RVS

5V-AGGTGGATAACCAGCAGATCCA-3V 5V-GCAGATGAAGCCTTTGATGTGA-3V 5V-GCTGCCTCTTCCTGCTCATC-3V 5V-TTCCTCCATCTGTGTTGCCA-3V 5V-TGAGAACCAAGCTGATCAAGGA-3V 5V-AGCCAAGAATCTCCGTAGCATT-3V 5V-TCCCCACCAGTATCACAACTTG-3V 5V-TTTGGAAATGGGAATCAACACA-3V 5V-CCCTGCCAGAGACTTTGCA-3V 5V-TGAGGCTGTCCTTTCCTATCCA-3V

All fold changes were normalized to baseline actin levels, using the DCT method as recommended by the ABI PRISM 7700 System manufacturer (PE Applied Biosystems, Foster City, CA), with the following PE Applied Biosystems actin primers: h-Actin FWD h-Actin RVS

5V-TCCTCCTGAGCGCAAGTACTC-3V 5V-CTCCTGCTTGCTGATCCACAT-3V

2.10. Proliferation assays We plated splenocytes from EAE mice sacrificed on day 20 at 2  106 cells/ml with 0, 1, 10, and 100 Ag/ml of MOG peptide. After 48 h, 1 ACi 3H-dT (NEN, Boston, MA) was added for 16 h prior to harvesting and counting. 2.11. In vitro neuronal cultures and treatments Rat cortical neuronal cells were obtained from the cerebral cortex of fetal Sprague Dawley rats (day 17 of gestation) as described (Murphy et al., 1990). Cultures are approximately 85 – 90% neuronal. The remaining cells

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are glia or undifferentiated neuronal precursors. Cells were cultured in Minimum Essential Medium (MEM) (Invitrogen, Carlsbad, CA) with 10% FCS and penicillin/ streptomycin. Cells were seeded at a density of 1 106 cells/ml on poly-d-lysine-coated 8-well chamber slides (BD Biosciences, San Jose, CA) for a Live/Dead assay or poly-d-lysine-coated 96-well microtitre plates (Corning, New York, NY) for an MTT assay (both described below). Cells were treated with either 5 mM homocysteate (HCA – glutamate analog) (Sigma, St. Louis, MO), 125 ng/ ml Trichostatin A (TSA) (Biomol, Plymouth Meeting, PA) or both. The exposure of immature cortical neurons to HCA results in depletion of the antioxidant glutathione and in oxidative stress-induced cell death with the morphological and biochemical characteristics of apoptosis (Ratan et al., 1994). HCA was diluted from a 0.1 M stock in MEM, pH adjusted to 7.5. TSA was diluted from a 200 Ag/ml stock dissolved in DMSO. Control and HCA alone-treated cells received an equivalent volume of DMSO (0.625 Al DMSO per ml). 2.11.1. Live/Dead assay Neuronal cells were treated for 24 h and examined for cell survival using the Live/Dead assay kit (Molecular Probes, Eugene, OR), according to the manufacturer’s protocol. Briefly, the media from the cells were removed

and the cells were incubated with a 2 AM calcein AM, 4 AM ethidium homodimer (EthD-1) solution for 30 min at room temperature and examined under a Nikon Eclipse E600 microscope. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the non-fluorescent cell-permeable calcein AM to the fluorescent calcein (green fluorescence at ex/em ¨495 nm / ¨515 nm). Dead cells are distinguished by EthD-1 entering the cells with damaged membranes and undergoing a 40-fold enhancement of fluorescence upon binding to nucleic acids (red fluorescence at ex/em ¨495 nm / ¨635 nm). The images were captured using SPOT 3.2.4 software (Diagnostic Instruments, Sterling Heights, MI). 2.11.2. MTT assays Neuronal cells were treated for 24 h and survival was quantified using the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). Five microliters of MTT assay solution was added to the cell media of each well, mixed gently and incubated at 37 -C, 5% CO2 for 1 – 2 h. During this time, living cells convert the MTT tetrazolium component of the dye solution into a formazan product. Solubilization/stop solution was then added to the culture to solubilize the formazan product, and the absorbance was measured at 570 nm using a SpectraMAX 250 96well plate reader (Molecular Devices, Sunnyvale, CA).

Fig. 1. Clinical and immunological effects of TSA in EAE. (a) Mean clinical scores reflect a modest but statistically significant reduction in clinical disability by TSA in EAE (? TSA-treated and > vehicle-treated) (*P < 0.05 by Fisher’s protected least significant difference (PLSD) test). Representative exp. 1 is shown. Exp. 2 yielded similar results. (b) Clinical findings on data pooled from the two TSA exp. reveals that TSA-treated mice had a significant drop in the mean peak of the remission phase (*P < 0.04 by Fisher’s PLSD test) and a > 25% decrease in the index of disease measured at both days 30 and 40. (c) QRTPCR shows modulation of inflammatory genes in spleen tissues from TSA-treated EAE mice, as compared to vehicle-treated EAE mice. Note modulation of immune genes previously reported to be altered by HDAC inhibitors: up-regulated IFN-a and down-regulated IL-2 receptor, CD28 and IL12p35. The IL-8 chemokine receptor was also down-regulated in spleen tissues by in vivo TSA treatment. (d) Protein arrays show suppression of chemokine MIP-2 expression by TSA in MOG-stimulated and non-stimulated splenocytes in vitro. (e) TSA-treated EAE mice splenocytes have a decreased proliferative response to MOG35 – 55 re-stimulation in vitro. Splenocytes were obtained and plated on day 20 from TSA-treated (h) and vehicle-treated (g) EAE mice from exp. 1. The stimulation index, based on 3H-dT incorporation values, is shown TSD. (*= P < 0.01 by Student’s t-test).

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3. Results 3.1. Clinical effects of TSA Treatment of mice with TSA (7.5 mg/kg/day i.p.) on days 4– 40 post-MOG injection resulted in a decrease in EAE disability measures during the chronic relapsing phase (Fig. 1a – b). The mean peak of the remission phase had a statistical significant reduction ( P < 0.04) in TSAtreated mice. The disease index measured on day 30 and even on day 40, at the end-stage of the chronic relapsing phase, was also lower in TSA-treated mice than in vehicletreated controls. TSA had no effects on the time of disease onset.

Fig. 3. Nissl stain and stereologic counts of spinal cord size and ventral horn neurons. Representative sections performed on day 40. The midthoracic cords were serially sectioned in parallel, to obtain comparable levels between vehicle-treated and TSA-treated animals: (a – b) Nissl stain reveals that spinal cord volume is smaller in vehicle-treated mice than in TSA-treated mice; (c – d) Nissl stain at higher magnification reveals fewer ventral horn neurons (darkly stained cells) in vehicle-treated mice than in TSA-treated mice. The results reveal that TSA treatment of EAE mice is associated with significantly greater spinal cord volume and neuronal counts as compared to the vehicle-treated EAE group.

3.2. Immune effects of TSA QRT-PCR of spleen tissues showed TSA up-regulation of IFN-a and down-regulation of IL-2R-a, the chemokine IL-8R, the pro-Th1 IL-12p35 and the costimulatory CD28 (Fig. 1c). Protein arrays showed that in vitro TSA suppressed expression of the macrophage inflammatory protein 2 (MIP-2) chemokine in both MOG-stimulated and unstimulated splenocytes (Fig. 1d) after 24-h culture. In splenocytes derived from TSA-treated mice, we found decreased in vitro proliferation to MOG (Fig. 1e), Con A and PHA (all with P < 0.01, latter two not shown). Thus, TSA counteracts factors that promote a Th1 shift, chemotaxis and proliferation. 3.3. Histopathology Fig. 2. CNS tissue effects of TSA. Representative histopathologic comparison of spinal cords from 4 vehicle- and 3 TSA-treated EAE mice performed on day 40. The lumbosacral cords were serially sectioned in parallel, to obtain comparable levels between vehicle-treated and TSAtreated animals: (a – b) H and E stains show large inflammatory infiltrates only in the untreated mice; (c – d) Oil Red-O reveals decreased lipid breakdown products by TSA; (e – f) Sudan Black shows a demyelinating lesion (arrow) in the spinal cord of a vehicle-treated animal but not in the spinal cord of a TSA-treated animal (same level as panels [a – d]); (g – h) Bielchowsky, which darkly stains axons (shown by arrows in cross section and at very high magnification), reveals higher staining intensity in the spinal cord of a TSA-treated animal.

Spinal cord H and E stains revealed that TSA led to decreased inflammation, with the scattered distribution of few cells (Fig. 2a –b), occasional small-sized inflammatory foci throughout the tissues and an absence of large, distinct foci. Oil red-O stains showed a decrease in lipid breakdown products (Fig. 2c – d) and, at higher magnification, in lipidladen macrophages/microglia (not shown). Sudan Black B stains (Fig. 2e –f) showed less demyelination. Focal axonal loss areas as revealed by Bielchowsky stains (Fig. 2g– h)

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were only found in association with large inflammatory infiltrates, and therefore were only seen near or within lesions in vehicle-treated but not TSA-treated mice. In addition, 20 consecutive thoracic cord Nissl-stained serial sections were computed by stereologic analysis of cord volume and neuronal counts. The cord volume in untreated vs. TSA-treated mice was 2.015 T 0.017  109 vs. 3.034 T 0.012  109 Am3, P < 0.001 (Fig. 3a– b), and the ventral horn neuronal count, respectively, was 6100 T 190 vs. 7850 T 140, P < 0.01 (Fig. 3c – d). Histopathological analysis thus confirmed both neuronal preservation and anti-inflammatory roles for TSA.

3.4. Spinal cord microarrays reveal specific effects of TSA For gene expression analysis, we sacrificed all mice on day 40 purposely so that microarrays would give a snapshot of the late phase of EAE, in which neuronal loss may better reflect disability. TSA treatment led to changes in expression of 491 spinal cord genes. A number of representative genes from various pathways is shown after average linkage clustering using TreeView software (http://rana.lbl.gov) (Fig. 4a). There was up-regulation of the known TSA-responsive gene (Yang et al., 2001) estrogen receptor alpha (ER-a), whose product mediates

Fig. 4. TSA modulates genes known for their neuroprotective role. (a) Cluster analysis microarray representation of selected genes, from a total of 491 genes altered by TSA in cord tissues. Genes that depart with a > 2-fold change from vehicle-treated EAE mice are shown in green if exhibiting lower expression or in red if exhibiting higher expression (black—normalization assigned to all vehicle-treated EAE). Note up-regulation of known HDAC inhibitor-responsive genes, such as the Sp1 transcription factor target gene Mac-1, or genes responsible for neuronal growth, integrity or protection. Note up-regulation of other Sp1-dependent genes, such as estrogen receptor alpha (ER-a) and phenylethanolamine N-methyltransferase (Pnmt). Notably, several E2F1 pathway-dependent genes, such as CDC2, are down-regulated. Regulatory sequence analysis tools (http://rsat.ulb.ac.be/rsat/) were used to confirm the presence of either Sp1 or E2f binding sites within 5-kb upstream of the coding region of each gene. (b) Bar graph (QRT-PCR) representation of key genes altered by TSA in brain tissues. Besides induction of Gpx1 and Mac-1, there was up-regulation by TSA of the REST/HDAC complex-repressed genes dopamine beta hydroxylase (Dbh) and the sodium channel Nav1.2. An anti-apoptotic gene signature promoted by TSA was also shown by QRT-PCR. Note down-regulation by TSA of the proapoptotic genes Bcl2-antagonist/killer 1(Bak), BH3 interacting domain death agonist (Bid), caspase 2 (Casp2), and apoptosis inducing factor (Aif). Bars show genes with > 1.5-fold change in three separate QRT-PCR experiments.

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the protective effects of estrogen in EAE (Polanczyk et al., 2003) and elevation of the neuroprotective insulin-like growth factor 2 (Igf2), the anti-excitotoxicity glutamate transporter EAAT2, the neuronal integrity trait gene synaptojanin 2, and the antioxidant glutathione peroxidase (Gpx1). Importantly, the known TSA-responsive gene markers Mac-1 (Gore and Carducci, 2000) and hemoglobin beta (Ferrante et al., 2003) were up-regulated. As shown, several of these genes have Sp1 transcription factorbinding sites in their promoters. Most strikingly, a signature of down-regulation of known E2F1 target genes (Ma et al., 2002; Ren et al., 2002), such as CDC2, DNA polymerase alpha and others was evident in the spinal cords of TSA-treated mice. Our findings show that the beneficial effects of TSA on the chronic relapsing phase of EAE correlate with modulation of transcription factor pathways that have been implicated in inflammatory and neuronal injury mechanisms. 3.5. QRT-PCR in brain tissues from TSA-treated EAE mice We also examined gene expression of frontal lobe brain tissue from TSA-treated EAE mice and controls with a set of neurobiology primers using QRT-PCR. We confirmed TSAinduced up-regulation of glutathione peroxidase 1 (Gpx1) and Mac-1, and modulation of several genes whose expression changes were not evident by microarrays of spinal cords (Fig. 4b). These included up-regulation of the REST/HDAC-repressed neuronal trait genes sodium channel Nav1.2 and dopamine beta hydroxylase and downregulation of brain mRNAs for pro-apoptotic Bak, Bid and caspase 2, and the caspase-independent apoptosis-inducing factor. The results are consistent with modulation by TSA of neuronal integrity and survival pathways. 3.6. TSA counteracts caspase protein activation and down-regulates the E2F pathway Since oxidant stress has been implicated in MS and could lead to neuronal apoptosis, we analyzed the protein expression of caspases in EAE spinal cords. While readily detectable immunostaining of activated caspase 9 and 3 was observed in EAE spinal cords, TSA significantly decreased these immunoreactivities (Fig. 5a), correlating with histone H3 and H4 hyperacetylation. An in vitro model uses the glutamate analog homocysteate (HCA) to deplete the antioxidant glutathione (Ratan et al., 1994) leading to oxidant stress-induced neuronal death. Cell death in this model can be prevented by treating with caspase inhibitors (Tan et al., 1998) or TSA (Ryu et al., 2003a). We therefore examined whether TSA could negatively regulate neuronal caspase expression associated with HCA-induced oxidative stress. We found that oxidative stress enhances the active form of caspase 9, and coexposure to TSA prevents this expression at 8 h (Fig. 5b). Notably, the effects of TSA on caspase 9 are more evident in

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oxidant-stressed cells than in cells exposed to TSA alone. At 24 h, the live/dead assay revealed that adding TSA to HCA indeed led to rescue from cell death (Fig. 5c). Finally, we show that TSA inhibited pro-apoptotic E2F1 protein expression in these neurons at 24 h (Fig. 5d), suggesting the participation of transcription factor pathways in oxidantinduced cell death mechanisms. Taken together, these in vitro results suggest that the TSA-promoted decrease in spinal cord E2f1 targets, as evidenced by DNA microarrays, could reflect drug inhibition of E2f protein in neuronal cells. Importantly, our data support a positive correlation in vivo and in vitro between E2f pathway inhibition and neuronal survival.

4. Discussion Herein, we show that the specific HDAC inhibitor TSA harnesses neuronal survival and anti-inflammatory pathways in EAE, resulting in clinical amelioration during the chronic relapsing phase. Several lines of evidence point to the potential use of HDAC inhibitors as transcriptional modulators in MS. We have previously shown that HDAC mRNAs are elevated in proliferating immune cells after stimulation with mitogens (Dangond et al., 1998), and HDAC inhibitors inhibit this proliferation (Dangond and Gullans, 1998). In vitro HDAC inhibitors down-regulate Th1 factors implicated in MS, including g-IFN (Dangond and Gullans, 1998) and IL-12p35 (Saemann et al., 2000). Accordingly, we showed decreased IL-12p35 mRNA in spleens of TSA-treated mice. Moreover, microarray studies of CD4+ T cells have revealed that TSA down-regulates CD28 and IL-2R mRNAs (Moreira et al., 2003), consistent with our findings in spleen cells of TSA-treated EAE mice. We previously identified activation of E2F targets in MS PBMCs (Iglesias et al., 2004). The E2F pathway mediates convergent signals in T cell activation (Brennan et al., 1997) and is essential for cell proliferation (Wu et al., 2001). Accordingly, we now show that in vivo administered TSA inhibits splenocyte proliferation. TSA also inhibited immune cell CNS infiltration and microglial uptake of lipid breakdown products, consistent with antimicroglial activation effects recently described for HDAC inhibitors (Kim et al., 2004). The improvement in EAE disability by TSA also correlated with reduced axonal and neuronal loss, and allowed us to postulate the harnessing of several neural repair pathways. MIP-2 is a chemokine elevated during chronic EAE attacks (Glabinski et al., 2003) that is downregulated by estrogen (Matejuk et al., 2002), a hormone also known to exert EAE protection. TSA’s ability to simultaneously inhibit splenocyte MIP-2 protein and spinal cord estrogen receptor alpha mRNA suggest beneficial mechanisms in EAE at multiple levels. TSA also led to elevated spinal cord mRNA for Igf2, a neurotrophic (Kaspar et al., 2003; Longo et al., 2002) factor that has the added benefit of

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S. Camelo et al. / Journal of Neuroimmunology 164 (2005) 10 – 21

promoting remyelination by oligodendrocyte progenitor cells (Mason et al., 2003). Finally, TSA enhanced CNS expression of the HDAC/REST complex-repressed neuronal integrity genes Nav1.2 sodium channel (Ballas et al., 2001) and dopamine beta hydroxylase (Atouf et al., 1997), suggesting possible acquisition of the neuronal phenotype by progenitor cells or maintenance of maturity traits by neurons. Thus, neuronal or oligodendrocyte growth, survival and differentiation pathways are modulated at several levels by TSA treatment. Nitric oxide (NO) reacts with superoxide to form the prooxidant peroxynitrite. Oxidative stress leads to protein nitrosylation (Beltran et al., 2000), axonal damage and demyelination (Touil et al., 2001). Antioxidant enzymes such as Gpx1 reduce H2O2 (Jain et al., 1991), detoxify peroxynitrite (Sies et al., 1997), and, in EAE, decrease blood brain barrier permeability (Guy et al., 1989). We show that TSA led to increased CNS levels of Gpx1 mRNA. Recently, treatment of adoptive EAE with sodium phenylacetate, an HDAC inhibitor metabolite, was shown to decrease nitrosylation of spinal cord tissues (Dasgupta et al., 2003). Taken together, these observations suggest that HDAC inhibitors may protect neurons from free radical attack in EAE, in part via Gpx1 up-regulation. Recent studies have shown that neuronal apoptosis occurs concomitantly with reduced histone acetylation (Rouaux et al., 2003) (i.e., unopposed HDACs) or elevated E2F1 protein (Hou et al., 2000). We showed that TSA increased histone acetylation and counteracted E2F-dependent transcripts in EAE spinal cords, suggesting that it exerts a primary neuroprotective effect in vivo. Among the E2F target genes down-regulated by TSA in spinal cords, CDC2 stands out since it is a key mediator of E2F1-induced neuronal apoptosis (Konishi et al., 2002). In addition, TSA reduced E2F1 protein levels in cortical neuronal cells in vitro. E2F1-induced apoptosis is executed via caspase 3 activation (Hou et al., 2000). Caspase 3 is activated in EAE CNS tissues (Ahmed et al., 2002), and correlates with optic neuritis-related disability (Meyer et al., 2001). In this study, TSA decreased the CNS mRNAs for the pro-apoptotic Bak, Bid, caspase 2, and the caspase-independent apoptosisinducing factor (Cregan et al., 2002), and inhibited caspase 9 and 3 proteins. TSA-induced inhibition of caspase 9 protein in vitro was more efficient in neurons already exposed to oxidants than in non-exposed, suggesting that an endogenous protective response is already in place prior to TSA effects. This explanation is in line with the demonstration that non-histone proteins, such as Sp1,

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become acetylated and activated in response to both oxidant stress and TSA (Ryu et al., 2003a). Thus, TSA could amplify the beneficial (albeit insufficient) role of acetylated Sp1, and our finding of induced neuroprotective or neuronal function genes whose promoters contain Sp1binding sites, such as Gpx1 (Ho and Howard, 1992), Igf2 (Rodenburg et al., 1997), EAAT2 (Su et al., 2003), estrogen receptor alpha (ER-a) (deGraffenried et al., 2002) and phenylethanolamine N-methyltransferase (Pnmt) (Her et al., 2003) in spinal cords of TSA-treated mice, stands in support of this hypothesis. In vitro studies have shown that only a small percentage (¨2%) of genes in the human genome become transcriptionally active in response to HDAC inhibition (Marks et al., 2000). Our results in vivo support these observations, with TSA affecting only 491 (¨3.9%) of the ¨12,426 probe sets in the mouse arrays, some of which have been reported as in vitro targets of HDAC inhibitors. As concerns naturally arise regarding potential side effects of these systemically acting drugs, this limited repertoire of transcriptionally responsive genes to various HDAC inhibitors may be beneficial. Other advantages of these drugs include their reversibility of action and their proven clinical safety and efficacy in human diseases, such as sickle cell anemia (Dover et al., 1994). Further, pharmacokinetic studies of HDAC inhibitors in humans with cancer have shown that they are tolerable at oral doses needed to achieve biological activity (Gore and Carducci, 2000). This is relevant for MS, a disease for which no effective oral therapy is available. HDAC inhibitors ameliorate mouse models of neurodegenerative diseases, such as Huntington disease (Ferrante et al., 2003; Hockly et al., 2003; Ryu et al., 2003a) and spinal muscular atrophy (Chang et al., 2001), and their broad effects are being elucidated via microarrays (Ferrante et al., 2003). In our study, the high sensitivity of microarrays was evident in their ability to detect changes within a narrow improvement window by TSA. We do not have a clear explanation for the discrepancy of such drastic amelioration of CNS inflammation, demyelination and neuronal loss at a time of sampling (i.e., day 40) when the clinical improvement had almost subsided. One possibility is that the disability is due to lack of drug effects on pathways such as complement cascades, or occurrence of known untoward effects of HDAC inhibitors (e.g., high MHC) at the molecular level. However, this is in line with our expectation that not all HDAC inhibitor-induced transcriptional events would be beneficial for EAE. We therefore acknowledge that the general effect of HDAC

Fig. 5. TSA decreases neuronal caspase activation and E2F protein expression. (a) TSA-treated mice had decreased spinal cord caspase expression associated with enhanced histone acetylation, by immunohistochemistry; (b) Western blots of rat cortical neuron lysates reveal up-regulation of caspase 9 by the oxidant stressor HCA, and down-regulation by co-treatment with TSA (T) in vitro after 8 h, as compared to vehicle. A densitometry image (ratio of inactive caspase 9 to a-tubulin and ratio of active caspase 9 to a-tubulin) is shown; (c) Fluorescence microscopy illustrates effects of TSA in rescuing viability (survival is provided in percent as quantified by an MTT assay) of HCA-treated neurons at 24 h (green—live, red—dead) as follows: no treatment (for comparison purposes, we assigned it a 100% survival value), TSA (75%); HCA (15%), and HCA + TSA which results in rescue (74%). The percentage drop by TSA alone is attributed to lower proliferation of a small subset of glia and neuroblasts present in these cultures, since we found no change in the percentage of dead neurons. (d) TSA decreases E2F1 protein expression in these cortical neurons, and does so more prominently in neurons co-exposed to HCA.

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inhibition is to perturb an already dysregulated homeostatic process, shifting the balance of histone modifications towards acetylation and harnessing more beneficial genes than it does pro-disease (i.e., chemokine, pro-Th1) genes. Despite its shortcomings, TSA likely achieves neuroprotection by promoting antioxidant, anti-excitotoxicity, hormonal and growth responses, counteracting pro-proliferative or pro-apoptotic E2F target genes and caspase-dependent and independent apoptotic signals, and derepressing neuronal integrity traits in vivo. The growing availability of HDAC inhibitors with a range of specificity, efficacy and tolerability should provide new transcriptional tools to probe and treat CNS inflammation and neurodegeneration in MS.

Acknowledgments This work was supported by NIH 1KO8-CA80084 and R21-NS41623 (F.D.) and NIH NS045242, NS045806 and the Veterans Administration (R.J.F.). Thanks to the BWH GATC Center for technical support.

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