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(The FASEB Journal. 2005;19:1407-1421.)
© 2005 FASEB

HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis

Ajaib S. Paintlia1, Manjeet K. Paintlia1, Mushfiquddin Khan, Timothy Vollmer{dagger}, Avtar K. Singh* and Inderjit Singh2

Department of Pediatrics, Children Research Institute, Medical University of South Carolina, and
* Department of Pathology and Laboratory Medicine, Medical University of South Carolina and Ralph H. Johnson V. A. Medical Center, Charleston, SC 29425, and
{dagger} Barrow Neurological Institute, Phoenix, Arizona, USA

2Correspondence: Pediatrics Development Neurogenetics, 173 Ashley Avenue, 509 CRI, MUSC, Pediatrics, P.O. Box 250515, Charleston, SC 29425, USA. E-mail: singhi{at}musc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Impaired remyelination due to degeneration of both postmitotic oligodendrocytes and oligodendrocyte progenitors (OPs) is the major hallmark of inflammatory demyelination in multiple sclerosis (MS) lesions and experimental autoimmune encephalomyelitis (EAE). Here, we have demonstrated the potential of lovastatin, a HMG-CoA reductase inhibitor, for the restoration of impaired remyelination mediated through enhanced survival and differentiation of OPs in the spinal cord of treated EAE animals. Lovastatin treatment significantly increased the level of myelin lipids in the spinal cord of treated EAE animals, coinciding with the attenuation of disease severity and inflammatory demyelination as compared with untreated EAE animals. The increased expression of myelin proteins and transcription factors associated with differentiating oligodendrocytes along with the increase in number of NG2+/BrdU– and NG2+/BrdU+ cells, and the expression of proliferating OP-specific proteins, demonstrated the restoration of remyelination in the spinal cord of lovastatin-treated EAE animals. Corresponding to this, in vitro studies further corroborated the increased survival and differentiation of OPs in lovastatin-treated activated mixed glial cells suggesting that lovastatin protects against the degeneration of OPs and enhances their differentiation through induction of a pro-remyelinating environment in the spinal cord of treated EAE animals. Together, these data demonstrate that lovastatin has the potential to augment remyelination in MS lesions and other neuroinflammatory diseases.— Paintlia, A. S., Paintlia, M. K., Khan, M., Vollmer, T., Singh, A. K., Singh, I. HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis.


Key Words: lovastatin • oligodendrocyte progenitors • spinal cord • encephalomyelitis • multiple sclerosis • remyelination


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MULTIPLE SCLEROSIS (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) white matter characterized by the morphological hallmarks of inflammation, gliosis, demyelination, and loss of neuronal axons and oligodendrocytes (1) . In recent studies, investigators have concluded that the developing lesions in the MS brain are heterogeneous in nature (2 , 3) . Generally, the infiltrating cells are distributed throughout the developing acute lesions, in contrast to being restricted to the edges in clinically silent lesions. These lesions also differ in deposition of immunoglobulin and activated complement molecules, degeneration of oligodendrocytes, followed by demyelination and loss of neuronal axons, which correlates with permanent functional deficits (2 , 3) . Overall, these studies suggest the existence of multiple mechanisms for both demyelination and remyelination processes in MS lesions, which are responsible for the heterogeneity observed in MS pathology.

The concept of remyelination is of interest since naturally occurring remyelination is impaired in MS lesions. Active MS lesions are usually remyelinated by existing postmitotic oligodendrocytes and oligodendrocyte progenitors (OPs) unless this process is impaired by recurring demyelinating episodes, whereas the remyelination process is completely disrupted in silent demyelinated lesions in MS due to the lack of postmitotic oligodendrocytes and OPs in the lesions except in the border region between the plaque and peri-plaque white matter (4 , 5) . Remyelination helps to preserve axons, restore conduction velocity, and clinically silence the MS lesions. This can be achieved either by promoting endogenous repair mechanisms or by providing an exogenous source of myelinating cells. Current therapies are essentially targeted at promoting CNS repair, which include application of growth factors (6 , 7) , intravenous administration of remyelinating immunoglobulin auto-antibodies (8) , and the transplantation of OPs or embryonic stem cells (9 , 10) . These therapies have potential to induce remyelination in animal models and clinical trials on MS patients are currently under way.

Recruitment of OPs to the demyelinating lesions is critical for the induction of remyelination, which requires their proliferation followed by recruitment and differentiation into myelinating mature oligodendrocytes (11 , 12) . Neurotrophic factors such as insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, glial-derived neurotrophic factor (GDNF), neuregulin (glial growth factor-2; GGF-2), and ciliary neurotrophic factor (CNTF) are reported to be important for the proliferation and recruitment of OPs to the demyelinating lesions and their subsequent differentiation into mature oligodendrocytes for the purpose of myelinating the demyelinated axons (6 , 7 , 13) . Recent studies using animal models of MS and a human clinical trial suggest clinical relevance of the HMG-CoA reductase inhibitors, statins, as potential therapeutic agent for the treatment of MS patients: statins attenuate the neuroinflammatory response in the CNS (14 15 16 17 18) . The mechanisms by which statins interfere with the neuroinflammatory response are well established but the effects on the restoration of remyelination and neurological function remain to be elucidated. Moreover, it is important to study the effect of these drugs on remyelination since cholesterol is an important component of the myelin sheath. Therefore, in this study we have evaluated the effects of lovastatin on remyelination in the rat with experimental autoimmune encephalomyelitis (EAE), an animal model of MS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and antibodies
Guinea pig myelin basic protein (MBP), complete Freund’s adjuvant (CFA), DAPI and Hoechst 33342 stains, and murine anti-2'3'-cyclic nucleotide phosphodiesterase (CNPase) (clone 11-5B) and anti-mouse myelin basic protein (MBP, clone 1: 129-138) antibodies were purchased from Sigma (St. Louis, MO, USA) and Serotec (Raleigh, NC, USA). DMEM (4.5 g/L glucose) and FBS were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant rat IFN-{gamma}, TNF-{alpha}, and IL-1ß proteins were purchased from R&D Systems (Minneapolis, MN, USA). Lovastatin was purchased from Calbiochem (San Diego, CA, USA). Anti-mouse PDGF-{alpha}R antibodies were purchased from Research Diagnostic, Inc. (Flanders, NJ, USA). Rabbit and mouse anti-NG2 chondroitin sulfate proteoglycan, mouse anti-A2B5, and rabbit anti-ß-actin antibodies were purchased from Chemicon International (Temecula, CA, USA). Anti-rabbit glial fibrillary acidic protein (GFAP) antibodies were purchased from DAKO (Carpenteria, CA, USA). Secondary antibodies: Texas red-X-conjugated goat anti-mouse IgG (for PDGF-{alpha}R, NG2, CNPase, MBP), and FITC-conjugated goat anti-rabbit IgG (for GFAP and NG2), and anti-isolectin B4 antibodies were purchased from Vector Lab Inc. (Burlingame, CA, USA).

Induction and clinical assessment of EAE
Female Lewis rats (Harlan Laboratory, Harlan, IN, USA) weighing 225–300 g were housed in the animal care facility at the Medical University of South Carolina (MUSC) throughout the experiment and were provided with food and water ad lib. All experimental protocols were reviewed and approved by MUSC’s Animal Ethics Committee. The procedures used for the induction of EAE and for lovastatin (LOV) treatment were the same as those described earlier (15) . Four experimental groups were used: EAE with placebo treatment (EAE), EAE plus LOV treatment (E+LOV), control with placebo treatment (control), and control with LOV treatment (C+LOV). In brief, EAE was induced via subcutaneous administration of 50 µg of MBP (dissolved in PBS; pH 7.4) emulsified in an equal volume of CFA (Sigma) into the hind limb footpads of the animals (on the 1st and 7th day). EAE animals received an injection of vehicle (i.p., placebo, 0.1% Triton X-100 in PBS), each day. E+LOV animals were immunized similarly with MBP antigen and received LOV (i.p., 2 mg/kg-body wt, dissolved in 0.1% Triton X-100/PBS) immediately before MBP administration. Thereafter, LOV was administered for the duration of the experiment based upon our past experiences and studies suggest that LOV pretreatment before the onset of EAE disease is required to exert its protective effects (16) . For control groups, an emulsion of CFA/PBS was injected in hind limb foot pads in animals. Control and C+LOV groups received daily injections (i.p.) of placebo and LOV, respectively. A daily injection of pertussis toxin (i.p., 200 ng) was given to each animal in each group on the 1st and 2nd day of immunization. Clinical signs in EAE animals manifested as ascending paralysis starting on the 9th day of postimmunization (dpi) onwards, resulting in death in most animals by the 13th dpi. The clinical signs of EAE animals were scored by an experimentally blinded investigator as 0 = normal; 1 = piloerection; 2 = loss in tail tonicity; 3 = hind leg paralysis; 4 = paraplegia, and 5 = moribund or dead. Five animals/group in each experiment were killed to collect their spinal cords (lumbar region) on peak clinical day (13th dpi) and on remission (20th dpi) because obvious and marked pathological changes related to the demyelination/remyelination process in the spinal cord are known to occur in diseased animals during these time points. Each experiment was repeated four times.

BrdU administration
Cellular proliferation was examined during the development of acute EAE and remission using the thymidine analog BrdU (50 mg/kg, i.p., Sigma). Animals (n=3) from each group received a daily injection of BrdU for 2 days before being killed; thus, the proliferation response was determined at the 13th dpi and 20th dpi.

Assessment of myelin breakdown and its restoration
Lumbar spinal cord tissues were dissected out carefully from each group of animals as most marked pathological changes are detected in this region of the spinal cord in EAE. Lipids were extracted from ~50 mg of frozen spinal cord tissue from LOV-treated and untreated EAE animals and controls as described earlier (19) . Sphingomyelin, free cholesterol, and cholesterol esters were quantitated by high performance thin-layer chromatography followed by densitometry as described (20) . Both nonhydroxy and hydroxy forms of cerebrosides and sulfatides were analyzed and quantitated as described by Ganser et al. (21) .

Histology, immunohistochemistry/immunocytochemistry assessments
For histological examination of the tissue from experimental animals, lumbar spinal cord tissues were fixed in 10% buffered formalin (Stephens Scientific, Riverdale, NJ, USA), followed by processing and sectioning of tissue. The sections were then stained with H&E and luxol fast blue using standard protocols. For single-label immunohistochemistry, standard methodology was used whereby sections were incubated with primary antibodies (1:100), followed by incubation with secondary antibodies (1:100). Mouse IgG and rabbit polyclonal IgG were used as control antibodies. The sections were incubated with Texas red-conjugated IgG and FITC-conjugated IgG without primary antibodies for negative controls. The sections were analyzed by immunofluorescence microscopy (Olympus BX-60) with an Olympus digital camera (Optronics; Goleta, CA, USA) using a dual-band pass filter. Images were captured and processed with Adobe PhotoShop 7.0 and adjusted using the brightness and contrast to enhance image clarity.

For double-labeling with anti-NG2/anti-BrdU antibodies, the sections were incubated in 1N HCl at 37°C for 30 min, followed by PBS washings. The sections were incubated with anti-NG2 antibodies (1:100) at 4°C overnight. After washing three times in PBS, the sections were incubated with secondary antibodies such as FITC-conjugated rabbit anti-IgGs antibodies for NG2 and mouse anti-BrdU Cy5-conjugated IgGs for BrdU followed by their analyses as described above. Total numbers of both NG2+/BrdU– and NG2+/BrdU+ cells/field were determined by manual counting at magnifications 400x and 600x, respectively in 10 fields/slide using tissue sections from 4–5 animals/group in a blinded fashion. All NG2+/BrdU– and NG2+/BrdU+ cells within the white and gray matter were counted. Mean numbers of NG2+/BrdU– and NG2+/BrdU+ cells/field were computed for statistical analysis among groups and plotted. Likewise, for immunocytochemistry analysis, live mixed glial cells obtained after treatment were immunostained with primary antibodies in slide chambers followed by secondary antibodies or were stained/treated for double labeling and analyzed as described above.

Preparation of total RNA, GeneChip hybridization, and data analysis
Total RNA from spinal cord tissues or cells was purified using "TRIZOL Reagent" (Invitrogen) and RNA cleaning kits (Qiagen; Valencia, CA, USA) as described earlier (22) . RNA from similarly treated animals (n=5) was pooled in equal proportions to increase the sample size and to reduce individual disease variability among animals. RNA was converted to double-stranded cDNA and then to biotinylated cRNA as described earlier (15) . After confirming the quality of labeled cRNA with Affymetrix Test2 arrays, it was hybridized to Affymetrix Rat U34A GeneChip© arrays. These arrays had 8800 gene transcripts including appropriate transcripts for housekeeping genes as well as negative and positive control genes. Hybridization, washing, staining with streptavidin-phycoerythrin, and scanning with a probe array scanner (Gene Array Scanner; Affymetrix) were performed at the UCI DNA Microarray facility (University of California, Irvine, CA, USA). GeneChip© data were analyzed with Affymetrix Microarray Suite MAS 5.0 software and a one-sided Wilcoxon’s signed rank test was used to generate a detection P value (<0.05) to determine statistical significance of transcript expression on the chip. The software generated, based on the P value, a present (P<0.04), marginal (P<0.04 to P<0.05), or absent (P>0.05) call for each transcript. For comparison analysis, each probe set on the experimental array was compared with its counterpart on the baseline array to calculate the change in P value that was used to generate the difference call of increase (I: P<0.04); marginal increase (MI: P<0.04 to P<0.06); decrease (D: P>0.997); marginal decrease (MD: P>0.992 to P>0.997); or no change (NC: P>0.06 to P<0.997). Comparison analysis generated a signal-log ratio algorithm for each probe-pair on the experimental array to the corresponding probe pair across the baseline array. This strategy cancels out differences due to different probe binding coefficients. Software computed the signal log ratio number by using a one-step Tukey’s Biweight method by taking a mean of the log-ratio of probe-pair intensities across the two arrays. Two GeneChips©/group were used to repeat microarray analyses in two independent experiments. Fold changes were computed compared with control for each expressed transcript using standard formulae as described earlier (15) . The changes were considered significant if the magnitude of change was at least 1.5-fold or greater, with a P value <0.05 between experimental groups.

cDNA synthesis and real-time PCR analyses
Single-stranded cDNA was synthesized from pooled RNA samples from similarly treated animals/cells using a superscript preamplification system for first-strand cDNA synthesis (Invitrogen) as described earlier (22) . Real-time PCR was performed for some of the genes listed in Table 1 using IQTM SYBR Green Supermix and BIO-RAD Laboratories iCycler iQ RT-PCR (Bio-Rad; Hercules, CA, USA). Thermal cycling conditions were as follows: activation of iTaqTM DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30 s and 55–58°C for 30–60 s. The normalized mRNA expressions were computed with GAPDH expression as described previously (22) .


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  		Table 1. List of primer sequences used for real-time PCR analysisa

Immunoblotting
Immunoblotting of spinal cord tissue and cells was carried out as described earlier (15) . Briefly, tissues were homogenized or cells were lysed in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, and protease inhibitors mixture). Twenty microgram of protein/lane was separated by 10% SDS-PAGE and blotted to nitrocellulose (Amersham, UK). Immunoblots were incubated with primary antibodies (1:1,000) followed by incubation with secondary peroxidase-conjugated antibodies (1:10,000; Sigma). Immunoreactivity was detected using the enhanced chemiluminescence detection method according to the manufacturer’s instructions with subsequent exposure of immunblot to X-Ray films (Amersham, UK), followed by autoradiography.

Cortical mixed glial cell cultures and treatment
Rat cortical mixed glial cell cultures were generated from P1-P2 SD rat brains (Charles River, Wilmington, MA, USA) and OPs were purified from mixed glial cultures as described earlier (23) . Purity of OPs was determined by FACS analysis using anti-A2B5 (OPs), anti-GFAP (astrocytes), and anti-isolectin B4 (microglia) antibodies using standard protocols, which showed ~95% purity. Mixed glial cells at a density of 1 x 104 cells/slide were plated on glass chamber slides precoated with poly-D-lysine. After 24 h, the fresh DMEM without FBS was changed and cells were pretreated with LOV (1 µM) for 24 h before addition of cytokine mixture (CM: TNF-{alpha}, IL-1ß, and IFN-{gamma}; each 10 ng/mL). The proliferation of OPs was determined by immunostaining for PDGF-{alpha}R and NG2 antigens expressed by dividing OPs at days in vitro (DIV) 2 and DIV3, respectively. The differentiation of oligodendrocytes was determined by immunostaining for O1 and MBP at DIV5 and DIV6, respectively. The number of positive cells was counted manually in 10 fields/slide as described above. Similarly, the double-immunostaining for GFAP and MBP was performed to quantify reactive gliosis and survival of differentiating oligodendrocytes at DIV6. For mRNA and protein expression analysis, cells were treated with CM and LOV in 100 mm plates, and harvested after 12 h (mRNA) or 96 h (protein) post-treatment. All experiments were repeated three or four times.

Quantification of immunofluorescence intensity
The fluorescence in the different areas of slides immunostained with anti-MBP, anti-CNPase, or anti-GFAP antibodies was measured by using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA). The fluorescence intensity signals were plotted directly or classified as "weak" if the maximum peak level was below 130, "intermediate" if peak level was between 130 and 200, "moderate" if levels of half of the peaks were >200 and "strong" if all peak levels were > 200. A distance scale of 20 µm was chosen for measurement in all groups.

Thymidine uptake analysis
The purified primary OPs (1x104 cells/mL) were preincubated with/without LOV (1 µM) for 24 h in 96-well plates followed by incubation in conditioned media, obtained from mixed glial cell cultures treated similarly with LOV and CM for 24 h as described above and then 0.5 µCi of methyl-[3H] thymidine was added into each well. Media was removed after 16–18 h and cells were detached with the addition of 100 µL of 0.25% trypsin in Ca2+/Mg2+-free PBS containing 5.5 mM glucose. Cells were harvested onto glass fiber filters using a cell harvester (InfoTech AG; Switzerland) and the filters were washed thoroughly with water and counted in a MicroBeta system (Wallac; Turku, Finland).

Statistical analysis
Using the Student’s unpaired t test and ANOVA (Student-Newman-Keuls: comparison of all pairs of columns), P values were determined from three independent experiments using GraphPad software (GraphPad Software Inc., San Diego, CA, USA) for respective experiments. A P<0.05 was used as the criterion for statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lovastatin treatment attenuates myelin breakdown and facilitates its restoration
LOV-treated EAE animals had lower clinical scores (3.5±0.47) compared with EAE (4.6±0.57) animals on peak clinical day (E+LOV) and recovered neurological functions by the 20th dpi (E+LOV/R) in addition to having a body weight similar to healthy controls by the 45th dpi (Supplementary Fig. A, B). However, a relapse with low clinical scores was also observed between the 21st to 26th dpi in 10% of LOV-treated recovering animals. Histological examinations of the spinal cord sections showed lesser demyelination and inflammatory cell infiltration in the white matter region of E+LOV animals when compared with EAE animals (Supplementary Fig. C–E). Consistent with our previous observations, E+LOV animals that were recovered showed no cellular infiltration or demyelination in the white matter region of spinal cord (15) . To correlate these histological changes with myelin breakdown and its repair, the ratio of myelin lipid: protein was determined in each group of animals. There was a significant (P<0.001) increase in cholesterol ester in EAE animals when compared with controls (Fig. 1 A). Cerebrosides, sulfatides, and sphingomyelin lipids were significantly decreased in EAE animals compared with controls (Fig. 1B-D ). Conversely, E+LOV animals showed no significant change in myelin lipids when compared with controls (Fig. 1A-D ). Notably, no significant change in the levels of cholesterol was observed in animals from each group (Fig. 1D ). The levels of myelin lipids were close to healthy controls in E+LOV animals that were recovered (data not shown). Together, these data demonstrate that LOV limits the breakdown of the myelin sheath and improves its restoration in treated EAE animals.



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  		Figure 1. Lovastatin treatment attenuates myelin breakdown and facilitates its restoration. Spinal cord homogenates were analyzed for myelin lipids associated with the demyelination/remyelination process. The % change in the ratio of lipid and protein/group was computed and plotted. The levels of cholesterol ester (A), cerebrosides (nonhydroxy and hydroxy) (B), sulfatides (nonhydroxy and hydroxy) (C), sphingomyelin (D), and cholesterol (E) are shown in EAE, LOV-treated EAE (E+LOV), LOV-treated control (C+LOV), and placebo-treated control (CON) groups. Results are expressed as mean ± SD for n= 3–5/group in 3 independent experiments. **P<0.01, ***P<0.001 and NS (nonsignificant) vs. control, and #P<0.05 and ###P<0.001 vs. EAE.

Lovastatin treatment enhances the survival and differentiation of oligodendrocytes
Axonal loss and demyelination together with mononuclear infiltration into the CNS are the major hallmarks of EAE/MS lesions responsible for the multiplicity of neurological deficits in the affected animals (1) . Because LOV attenuates the progression of disease and restores the levels of myelin lipids in treated EAE animals, we next determined the expression of myelin proteins associated with remyelination. First, we performed microarray GeneChip analysis, which revealed the differential expression of mRNA for myelin-proteins in LOV-treated and untreated EAE animals relative to controls (Table 2 ). These observations were further confirmed by immunoblotting, immunohistochemistry, and real-time PCR analyses. Corresponding to microarray analysis, there were low levels of MBP and CNPase proteins in EAE animals compared with controls (Fig. 2 A). The levels of these myelin proteins were similar to controls in LOV-treated EAE animals. Consistent with these findings, the immunohistochemistry analysis also showed greater demyelination (arrowheads) as indicated by weak immunofluorescence for both MBP (left panel) and CNPase (right panel) in the white matter of EAE animals compared with controls (Fig. 2B ). Relatively lesser demyelination was seen in the white matter of E+LOV animals when compared with recovered E+LOV animals or controls. Furthermore, quantification of the immunofluorescence intensity signal also showed a significant decrease in signal for MBP and CNPase in EAE animals when compared with controls (Fig. 2C ). Although the intensity of the signal for these myelin proteins was significantly lower in E+LOV animals compared with controls, it was significantly greater than that seen in EAE animals. These results correlated with the mRNA expression for MBP, PLP, MOG, and MAG proteins, which was significantly lower in EAE animals as compared with controls (Fig. 2D-G ). Parallel to immunohistochemistry studies, the mRNA expression for these proteins was significantly lower in E+LOV animals compared with controls and significantly higher than that in EAE animals (Fig. 2D-G ). No significant change was observed in the mRNA expression for MOG and MAG proteins between recovered E+LOV animals and controls with the exception of MBP and PLP expressions (Fig. 2D-G ). Complementing these studies, the expression of transcription factors such as MyT1-L, GTX, and PPAR-{delta}, associated with the differentiating oligodendrocytes was also determined. Microarray analysis demonstrated increased expression of MyT2 mRNA (a transcription factor similar to MyT1-L) in LOV-treated EAE animals as compared with that in EAE animals (Table 2) . Real-time PCR analysis further corroborated these data and demonstrated a significant increase in expression of MyT1-L in LOV-treated EAE animals compared with untreated EAE and control animals (Fig. 2H ). The expression of GTX mRNA was significantly elevated in LOV-treated EAE animals compared with EAE animals (Fig. 2I ). Likewise, the expression of PPAR-{delta} mRNA was also significantly elevated in E+LOV animals compared with EAE animals (Fig. 2J and Table 2 ). However, the expression for PPAR-{delta} mRNA was significantly higher in E+LOV and EAE animals when compared with controls. Collectively, these changes in expression for myelin-proteins and transcription factors are indicative of enhanced remyelination mediated through increased survival and differentiation of postmitotic oligodendrocytes in the spinal cord of LOV-treated EAE animals.


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  		Table 2. Changes in expression of message for myelin proteins associated with proliferation and differentiation of oligodendrocyte progenitors in CNS of lovastatin-treated/untreated EAE and control animalsa



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  		Figure 2. Lovastatin treatment enhanced the survival and differentiation of oligodendrocytes in the spinal cord of EAE animals. The expression of myelin proteins and mRNA associated with differentiating oligodendrocytes was observed in the spinal cord of animals in each group. A representative immunoblot demonstrates the expression of MBP and CNPase including ß-actin (A). Representative sections of the spinal cord were immunostained with anti-MBP (left panel) and anti-CNPase (right panel) antibodies as described under Materials and Methods (B). The weak immunofluorescence in demyelinated regions in the white matter is indicated (arrowheads) in EAE and E+LOV at a magnification of 400x. Quantification of immunofluorescence (IFL) for MBP and CNPase in immunostained sections was performed as described under Materials and Methods (C). No demyelination was observed in the spinal cord of animals from placebo-treated CON, LOV-treated control (C+LOV), or recovered E+LOV/R groups. Real-time PCR analysis demonstrated mRNA expression of myelin-protein genes, i.e., MBP (D), PLP (E), MOG (F), and MAG (G) in each group as described under Materials and Methods. Similarly, mRNA expression of transcription factors, i.e., MyT1-L (H), GTX (I), and PPAR-{delta} (J) was analyzed in each group by real-time PCR analyses. Data are expressed as mean ± SD of 3 independent experiments run in triplicate each time. *P< 0.05, **P< 0.01, ***P< 0.001 and NS (nonsignificant) vs. control, and #P< 0.05, ##P<0.01, and ###P <0.001 vs. EAE.

Lovastatin treatment enhances the proliferation and recruitment of OPS
To further understand the effect of LOV on proliferation and recruitment of OPs in the spinal cord of treated EAE animals, we performed immunohistochemical analysis using anti-NG2 and anti-BrdU antibodies. We observed the scattered distribution of NG2+/BrdU– cells (arrowheads) in both the white and gray matter of spinal cord in each group of animals (Fig. 3 A). Manual counting revealed a significant decrease in the number of NG2+/BrdU– cells/field in EAE animals when compared with controls (Fig. 2B ). LOV treatment significantly increased the number of NG2+/BrdU– cells/field in recovered E+LOV animals compared with controls. Likewise, NG2+/BrdU+ cells were also found to be scattered throughout the gray and white matter of spinal cord in each group of animals (Fig. 3C ). There was a significant decrease in NG2+/BrdU+ cells/field in EAE animals compared with controls (Fig. 3D ). Similar to the NG2+/BrdU– cell counts, NG2+/BrdU+ cells/field were also increased significantly in recovered E+LOV animals when compared with controls (Fig. 3D ).



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  		Figure 3. Lovastatin enhanced proliferation and recruitment of OPs in the spinal cord of treated-EAE animals. To determine the impaired recruitment and proliferation of OPs in the spinal cord, the sections were immunostained with anti-BrdU and anti-NG2 antibodies as described under Materials and Methods. Representative sections from each group show the distribution of NG2+/BrdU– cells (green; red arrowheads) in the white matter (WM) of spinal cord of each group of animals (400x) (A). Graph represents the average number of NG2+/BrDU-– cell counts in 10 fields/section from 4–5 animals/group (B). Representative section demonstrates the colocalization of NG2 and BrdU immunostaining in the spinal cord of recovered E+LOV/R animals (600x) (C). Red arrowheads indicate the colocalization of NG2+ (green), BrdU+ (red), and NG2+/BrdU+ (yellow) in proliferating OPs, whereas a yellow arrowhead represents the migrated or resident OP. Graph represents the number of NG2+/BrdU+ cell counts in 10 fields/section from 4–5 animals/group (D). The spinal cord tissue homogenates from each group were analyzed for the expression of OP proteins, i.e., A2B5 (70 kDa) and PDGF-{alpha}R (150 kDa) including ß-actin by immunoblotting as described under Material and Methods (E). Graphs demonstrate real-time PCR analysis of mRNA expression for PDGF-{alpha}R (F), SOX10 (G), and Shh (H) in the spinal cord of each group of animals. *P< 0.05, **P< 0.01, ***P< 0.001, and NS (nonsignificant) vs. CON, and #P<0.05, ##P< 0.01, and ###P< 0.001 vs. EAE.

The expression of NG2 is also reported in other cell types such as endothelial cell and smooth muscle cell progenitors (24) ; therefore, we next validated these data by immunoblotting of spinal cord tissue homogenates with anti-A2B5 and anti-PDGF-{alpha}R antibodies, which cross-react with gangliosides (25) or glycoprotein (26) , and PDGF-{alpha}R (27) proteins, respectively, expressed in dividing OPs. There was a relative decrease in both A2B5 glycoprotein (a major cross reacting band of 70 kDa was observed in addition to several minor bands of greater than 120 kDa and from 30 to 40 kDa) and PDGF-{alpha}R (150 kDa) proteins in EAE animals compared with LOV-treated EAE animals and controls (Fig. 3E ). The protein level of PDGF-{alpha}R was increased (~4-fold) in recovered E+LOV animals compared with controls. Lysates of purified OPs and matured oligodendrocytes (cultured OPs for 6 days in vitro) were included as positive and negative controls, respectively (data not shown). Corresponding with this, the expression of PDGF-{alpha}R mRNA was also significantly increased in recovered E+LOV animals compared with controls (Fig. 3F ). In contrast, the expression of PDGF-{alpha}R mRNA was significantly decreased in EAE animals (Fig. 3F ). Parallel to these findings, the expression of SOX10, a transcription factor expressed exclusively in dividing OPs was significantly decreased in EAE animals as compared with controls (Fig. 3G ). Although the expression of SOX10 mRNA was slightly lower in E+LOV animals compared with controls, it was significantly higher than that seen in EAE animals. Corresponding with PDGF-{alpha}R expression, the SOX10 mRNA expression was significantly higher in recovered E+LOV animals than controls. Furthermore, the mRNA expression of Shh, a pre-OP (oligodendrocyte progenitor) -specific transcription factor also followed the same trend in EAE animals vs. controls except in E+LOV animals in which no significant difference was observed when compared with controls (Fig. 3H ). Altogether, these data are suggestive of a significant increase in both proliferation and recruitment of OPs in the spinal cord of LOV-treated EAE animals, especially on remission.

Lovastatin enhances the survival and proliferation of OPs in cultures of activated mixed glial cells
The in vivo studies described above indicate that LOV improves the proliferation, recruitment, and differentiation of OPs in treated EAE animals. These findings were further evaluated by in vitro studies with primary rat mixed glial cell cultures. These cells were treated with a cytokine mixture (CM; IFN-{gamma}, TNF-{alpha}, and IL-1ß) in the presence or absence of LOV for different time points. Mixed glial cells were preferred for this study because cell-to-cell interactions are needed for OPs with astrocytes for their proliferation/differentiation, and treatment with CM mimics the inflammatory disease state akin to that observed in EAE/MS brain. Immunocytochemical analysis revealed that the majority of OPs were PDGF-{alpha}R+ as compared with differentiating CNPase+ or MBP+ oligodendrocytes at 0 h in mixed glial cell cultures (data not shown). There was a significant decrease in PDGF-{alpha}R+ cells/field in CM-treated cells after 24 h (DIV2) of stimulation when compared with control cells, whereas LOV pretreatment significantly increased the number of PDGF-{alpha}R+ cells in CM+LOV-treated cells as compared with CM-treated and control cells (Fig. 4 A). PDGF-{alpha}R+ cells were bipolar (arrowheads) and clustered in all groups except in the CM-treated group as demonstrated by immunocytochemistry (Fig. 4B ). A similar trend was observed when cells were immunostained with anti-NG2 antibodies followed by manual counting. NG2+ cells/field were significantly decreased in CM-treated cells after 48 h (DIV3) of stimulation when compared with controls, whereas LOV pretreatment significantly increased the number of NG2+ cells in CM+LOV-treated cells as compared with CM-treated and control cells (Fig. 4C ). Like PDGF-{alpha}R+ cells, NG2+ cells demonstrated an increase in the number of processes (arrowheads) in all groups except those in the CM-treated group (Fig. 4D ). Consistent with protein expression for PDGF-{alpha}R and NG2, PDGF-{alpha}R mRNA expression was also decreased significantly in CM-treated cells, but increased significantly in CM+LOV-treated cells when compared with both CM-treated and control cells (Fig. 4E ). Both of these qualitative and quantitative analyses revealed the attenuation of cytokine-mediated loss of OPs and in turn an increase in proliferation of OPs in CM+LOV-treated cells. To further confirm these findings, we examined OPs proliferation by using thymidine uptake analysis. We observed an increase in OP proliferation in mixed glial cells; therefore, we preferred to use conditioned media obtained from similarly treated mixed glial cells for thymidine uptake analysis. Thymidine uptake was increased significantly in primary OPs cultured in CM+LOV-treated cell culture conditioned media, but this was significantly lower in primary OPs cultured in CM-treated culture conditioned media when compared with control glial cell culture conditioned media (Fig. 4F ). No significant change was observed for thymidine uptake in OPs cultured in conditioned media obtained from LOV-treated mixed glial cell cultures or controls (Fig. 4F ). LOV pretreatment did not protect purified OPs against the cytotoxic effects of mediators released in cultures of CM-treated mixed glial cells. Altogether, these data demonstrate that LOV treatment attenuates the inflammatory cytokine induced release of cytotoxic mediators and augments the survival and proliferation of OPs in activated mixed glial cell cultures.



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  		Figure 4. Lovastatin enhanced the survival and proliferation of OPs in mixed glial cell cultures. Cortical mixed glial cell cultures were treated with LOV and CM as described under Materials and Methods. The cells were immunostained using anti-PDGF-{alpha}R and anti-NG2 antibodies after 24 h (DIV2) and 48 h (DIV3) poststimulation. Graph represents PDGF-{alpha}R+ cell counts/field in 10 fields/slide from 3 independent experiments (A). Representative slides demonstrate PDGF-{alpha}R+ cells (yellow arrowheads) present in all groups (B). Graph represents NG2+ cell counts/field in 10 fields/slide from 3 independent experiments (C). Representative slides demonstrate NG2+ cells (yellow arrowheads) in all groups (D). Real-time PCR analyses demonstrate PDGF-{alpha}R mRNA expression in LOV- and CM-treated mixed glial cells after 12 h of stimulation (E). [3H] Thymidine uptake analyses of purified OPs pretreated (PT) with/without LOV and incubated in conditioned media obtained from mixed glial cell cultures after stimulation with cytokine mixture (CM) and LOV as described under Materials and Methods. Results in graphs are expressed as mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001, and NS (nonsignificant) vs. control, and ###P< 0.001 vs. CM. PT indicates OPs were pretreated with LOV (1 µM) for 24 h before stimulation with conditioned media.

Lovastatin enhances the differentiation of maturing oligodendrocytes in primary culture of activated mixed glial cells
To determine the effect of LOV treatment on differentiation of oligodendrocytes in activated mixed glial cells, immunocytochemistry was performed using anti-O1 and anti-MBP antibodies at DIV5 (120 h) and DIV6 (144 h), respectively. There were significantly fewer O1+ and MBP+ cells in the CM-treated cells when compared with controls (Fig. 5 A). Immunostaining revealed O1+ cells, characteristically stained around the cell body (arrowheads) and weakly stained on the processes, whereas MBP+ cells stained all over the cell body including extended processes and branches (Fig. 5B ). Notably, there was no significant change in O1+ and MBP+ cell counts among Cont+LOV– and CM+LOV-treated cells and controls, but there was an increase in the length and number of processes in Cont+LOV-treated cells compared with controls. Similarly, the mRNA expression for MBP and PLP was also decreased in CM-treated cells compared with controls (Fig. 5C ). Conversely, the expression for these proteins was significantly increased in CM+LOV– and Cont+LOV-treated cells as compared with controls. Furthermore, immunoblotting also revealed a decrease in CNPase protein levels in CM-treated cells compared with controls and this was restored by LOV treatment in CM+LOV-treated cells (Fig. 5D ). Together, these data show the increase in differentiated oligodendrocytes by LOV in activated mixed glial cell cultures.



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  		Figure 5. Lovastatin enhanced the differentiation of maturing oligodendrocytes in mixed glial cell cultures. Cortical mixed glial cells were treated with LOV in the presence/absence of CM and immunostained with anti-O1 and anti-MBP antibodies as described under Materials and Methods. Graph depicts the count of O1+ and MBP+ cells/field in each slide (n=6) from 3 independent experiments (A). Representative slides demonstrate O1+ (red) and MBP+ (red) cells as indicated (arrowheads), present in all groups after 120 h (DIV5) and 144 h (DIV6) (B). Likewise, representative slides of O1+/DAPI+ (blue) and MBP+/DAPI+ (blue) of the same field demonstrate the cell nuclei and cell numbers. MBP+ oligodendrocytes indicated by arrowheads (green) are photographed at a higher magnification (1000x) to compare the length and branches of processes. Graph depicts mRNA expression for MBP and PLP in similarly treated cells as described under Materials and Methods (C). Immunoblot demonstrates CNPase and ß-actin levels in similarly treated mixed glial cells at DIV4 as described under Materials and Methods (D). Graphs results are expressed as mean ± SD. *P < 0.05, ** P< 0.01, and *** P< 0.001 vs. control, and ###P< 0.001 vs. CM.

Lovastatin treatment attenuates reactive gliosis and induces a pro-remyelinating environment in the CNS and activated mixed glial cell cultures
Microarray analysis demonstrated an increase in the expression of GFAP in EAE animals compared with controls and its attenuation with LOV treatment (Table 2) . To determine the effect of LOV treatment on cytokine-induced reactive gliosis, double-immunostaining was performed with anti-GFAP and anti-MBP antibodies. Immunocytochemistry analysis revealed a characteristically bushy appearance of reactive hypertrophic astrocytes in CM-treated cells compared with controls (Fig. 6 A, B). Small and poorly differentiated oligodendrocytes (arrowheads) were observed in CM-treated cells, whereas LOV treatment demonstrated fully differentiating mature oligodendrocytes in Cont+LOV– and CM+LOV-treated cells. Quantitative analysis of GFAP immunofluorescence demonstrated a strong intensity for GFAP, but weak intensity for MBP in CM-treated cells (Fig. 6B ). Conversely, CM+LOV-treated cells had moderate GFAP and strong MBP intensities similar to controls. No significant change in intensities for GFAP and MBP was observed between Cont+LOV-treated cells and controls. In accordance with these results, the expression of GFAP mRNA was also increased significantly in CM-treated cells compared with controls (Fig. 6C ). However, no significant change in GFAP mRNA was observed among Cont+LOV– and CM+LOV-treated cells and controls.



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  		Figure 6. Lovastatin treatment attenuates reactive gliosis in mixed glial cell cultures. Cortical mixed glial cells were treated with LOV in the presence/absence of CM and immunostained with anti-GFAP and anti-MBP antibodies as described under Materials and Methods. Representative slides show GFAP+ (green) and MBP+ (red) cells (arrowheads) present in all groups (upper panel) after 144 h (DIV6) of stimulation (A). Likewise, representative slides GFAP+/Hoechst (blue) slides of the same field (lower panel) demonstrate the cell nuclei and cell numbers (A). Representative graphs depict the immunofluorescence intensities for GFAP (green curve) and MBP (red curve) in immunostained slides at 20 µm distance scale as described under Materials and Methods (B). A plot demonstrates the mRNA expression for GFAP in similarly treated cells as described under Materials and Methods (C). Plot data are expressed as mean ± SD. Magnification was at 400x (GFAP and MBP). ***P< 0.001 and NS (nonsignificant) vs. control.

Neurotrophic factors are known as important pro-remyelinating growth factors for the induction of proliferation/differentiation of OPs (7) . Microarray analysis revealed the up-regulation of mRNA expression for various neurotrophic factor proteins (i.e., CNTF, GDNF, BDNF, IGF-1, FGF-9, and LIF) in LOV-treated EAE animals when compared with EAE animals and controls (Table 3 ). Corresponding to this, real-time PCR analysis also demonstrated an increase in expression of BDNF in E+LOV animals when compared with EAE animals and controls (Fig. 7 A). Although the expression of BDNF in E+LOV animals was significantly lower than that in controls but was significantly increased in recovered E+LOV animals. CNTF expression was not remarkably altered in EAE animals, but was significantly increased in LOV-treated EAE animals when compared with controls (Fig. 7B ). Furthermore, the expression for PDGF, GGF-2, and LIF was significantly decreased in EAE animals compared with controls (Fig. 6C-E ). The expression was significantly lower (PDGF), close to control (GGF-2) or elevated (LIF) in E+LOV animals, but was significantly elevated in recovered E+LOV animals when compared with controls with the exception of PDGF and GGF-2 expression in controls (Fig. 6C-E ). Next, we determined the expression of these neurotrophic factors in LOV– and CM-treated mixed glial cells. In agreement with the in vivo data, we observed a significant increase in mRNA expression of GGF-2, BDNF, and LIF in LOV+CM-treated cells compared with controls (Fig. 6F-H ). There was no expression of these neurotrophic factors in CM-treated cells. Notably, Cont+LOV-treated cells also demonstrated a significant increase in expression of BDNF and GGF-2 when compared with controls. Together, these data show that LOV treatment attenuates the reactive gliosis, which in turn helps to induce a pro-remyelinating environment in the CNS.


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  		Table 3. Changes in expression of message for neurotrophic factors required for proliferation and differentiation of oligodendrocyte progenitors in CNS of lovastatin-treated/untreated EAE and control animals



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  		Figure 7. Lovastatin treatment enhances the release of neurotrophic factors in the spinal cord of treated-EAE animals and in mixed Glial cell cultures. The mRNA expression of neurotrophic factors was determined by real-time PCR analysis in the spinal cord of LOV-treated/untreated EAE animals and controls as well as CM-treated mixed glial cell cultures as described under Materials and Methods. Graphs represent the mRNA expression of BDNF (A), CNTF (B), PDGF (C), GGF-2 (D), and LIF (E) in the spinal cord of animals from each group. Likewise, graphs represent the mRNA expression for BDNF (F), LIF (G), and GGF-2 (H) in LOV-and CM-treated mixed glial cells. The data in graphs are expressed as mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001, and NS (nonsignificant) vs. control, #P< 0.05, ##P< 0.01, and ###P< 0.001 vs. EAE, and $$$P< 0.001 vs. CM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, we and others have demonstrated the anti-inflammatory properties of HMG-CoA reductase inhibitors, i.e., statins in acute and remitting-relapsing EAE (14 15 16 , 18) . The present study reveals a correlation between attenuated mononuclear infiltration and decreased demyelination in the spinal cord on peak clinical day, and early restoration of neurological functions during remission (within a week) mediated through the increased survival and differentiation of OPs in LOV-treated EAE animals.

The myelin-lipid profiles associated with demyelination/remyelination processes correlate well with disease severity and the inflammatory response in LOV-treated/untreated EAE animals. Cholesterol ester accumulation in the brain is considered to be associated with demyelination during MS and other CNS demyelinating disease such as X-adrenoleukodystrophy (X-ALD) (22 , 28 , 29) . In our study, the levels of cholesterol ester were increased significantly in the spinal cord of untreated EAE animals compared with LOV-treated EAE animals and controls, which supports our hypothesis that LOV attenuates demyelination in treated EAE animals. Likewise, the increase in biosynthesis of both cerebrosides and galactolipids is considered to be an important determinant of myelin development, remyelination, and axo-glial organization in various CNS demyelination models (30 , 31) . Accordingly, the observed increase in expression of these myelin lipids and proteins, in parallel to mRNA expression of MyT1-L, GTX, and PPAR-{delta} in LOV-treated EAE animals indicates that LOV treatment enhances the increase in number of postmitotic oligodendrocytes in the spinal cord of recovering EAE animals. The expression of MyT1 and GTX mRNA is shown to be associated with expression of myelin proteins in differentiating oligodendrocytes (32) . Likewise, PPAR-{delta} agonists are documented to induce the differentiation of OPs, whereas the attenuation of PPAR-{delta} mRNA expression in OPs when treated with TNF-{alpha} results in impaired remyelination (33 , 34) . In agreement with these results, the observed increase in survival, proliferation, and differentiation of OPs by LOV treatment both in the spinal cord of treated EAE animals and in cytokine cocktail treated mixed glial cell cultures indicate that LOV attenuates both the inflammatory responses and cytotoxic effects of inflammatory cytokines on OP survival in CNS. Together, these observations indicate that LOV treatment attenuates inflammatory demyelination and restores remyelination by promoting the normalized biosynthesis and expression of myelin constituents in postmitotic oligodendrocytes and their progenitors, i.e., proteins and lipids.

The prerequisite for enhancing the remyelination process in the CNS of EAE animals is the recruitment of OPs to the demyelinated lesions followed by their differentiation into postmitotic oligodendrocytes. A shortage of OPs in the demyelinated areas of the white matter is considered an important factor for the impaired remyelination. The observed decrease in number of OPs in the spinal cord of untreated EAE animals in parallel to cytokine cocktail-treated mixed glial cells indicates that inflammatory mediators are responsible for the loss of OPs under CNS inflammatory disease conditions. These observations are consistent with our recent study, in which we showed that endotoxin-induced inflammatory mediators are responsible for the major loss of OPs and hypomyelination in the developing fetal rat brain (35) . The recruitment of OPs after a demyelinating episode leaves a zone with diminished numbers of OPs in the vicinity of the demyelinated area, and the impaired capacity for repair after recurring demyelinating attack results in impaired remyelination. The significant increase in number of both NG2+/BrdU– and NG2+/BrdU+ cells corresponding to the expression of PDGF-{alpha}R and A2B5 proteins in the spinal cord of LOV-treated EAE animals during remission indicates that LOV may promote both the proliferation and recruitment of OPs in the CNS. In support of this, the increase in expression for SOX10 in the spinal cord of LOV-treated EAE animals further confirms our hypothesis that LOV augments the proliferation of OPs in the spinal cord of treated EAE animals. SOX10 is a member of the high mobility group domain family of DNA binding proteins and is expressed only in OPs and not in neurons or astrocytes (36) . This increase in OP numbers during remission may be due to their proliferation and recruitment from adjacent areas or from transformation of neuronal stem cells in the CNS. There are reports that indicate that neural stem cells present in the ependymal cell layer (around the central canal in the spinal cord) and subventricular zone (brain cortex) or transplanted embryonic stem cells have the potential to transform into OPs and premyelinating oligodendrocytes (10 , 37) . Thus, both in vivo and in vitro data confirm the potential of LOV to protect and enhance the proliferation of OPs in neuroinflammatory disease conditions.

The pro-regenerative properties of cytokine-activated astrocytes include the supply of neurotrophic factors for survival of neurons and oligodendrocytes, which help to restore the blood-brain barrier, CNS homeostasis and remyelination (38) . Neurotrophic factors such as CNTF, LIF, IGF-1, PDGF, and FGF-2 secreted by glial cells are reported to have promyelinating effects to induce the increased proliferation of OPs and their maturation into postmitotic oligodendrocytes (6 , 7 , 13) . For instance, FGF-2 transgenic mice, when induced for development of chronic EAE had a less severe clinical course and only mild pathological changes, but had enhanced numbers of OPs in brain (39) . Consistent with this, the observed increase in expression of neurotrophic factors that occurs parallel to the decrease in GFAP expression in the spinal cord of LOV-treated EAE animals and treated activated mixed glial cells is suggestive of a pro-remyelinating environment induced by LOV. In agreement with these findings, reactive gliosis has been shown to be associated with hypomyelination due to the absence of MBP expressing oligodendrocytes and an increase in GFAP expressing glial cells in the white matter of the shiverer, quaking, and phenylketonuric mice brain (40) . The observed increase in secretion of neurotrophic factors by LOV-treated activated glial cells indicates that LOV attenuates reactive gliosis and thereby increases the secretion of neurotrophic factors to generate a pro-remyelinating environment in the CNS.

These pleiotropic effects of LOV are in addition to its cholesterol-lowering effects, mediated through inhibition of the synthesis of isoprenoids, intermediate products of the cholesterol biosynthesis pathway (Fig. 8 ). These isoprenoids are involved in the post-translational modification of a large number of proteins involved in intracellular signal transduction pathways including small GTPases, which are reported to play a crucial role in the regulation of cell growth and differentiation, gene expression, protein and lipid trafficking, nuclear transport, and host defense (41 , 42) . First, the trans-localization and inactivation of small GTPases including RhoA, have been shown to regulate the NF-{kappa}B dependent transcription of inflammatory genes including iNOS in both immune and glial cells thereby inhibiting the loss of oligodendrocytes and OPs in the CNS (43 , 44) . The increase in expression of iNOS in MS lesions and the CNS of EAE animals through infiltration of immune cells resulting in the activation of astrocytes and microglia in MS lesions and EAE, and the subsequent attenuation of iNOS expression in the CNS of LOV-treated EAE animals coinciding with disease severity suggest a possible role of RhoA in LOV-treated EAE animals (16 , 45) . In agreement with our previous observations with purified astrocytes and microglia (46) , the observed attenuation of iNOS expression and NO release in LOV-treated activated mixed glial cells represent the NF-{kappa}B dependent decrease in iNOS expression by LOV in glial cells (data not shown). Also, the inactivation of RhoA has also been shown to enhance stress fiber formation in differentiating oligodendrocytes (47) . In agreement with this, the observed increase in differentiation of OPs in LOV-treated mixed glial cell cultures or purified OPs treated with increasing concentration of LOV (data not shown) indicates a direct relationship between oligodendrocyte differentiation and inactivation of these small GTPases (RhoA). Detailed studied are presently under way in our laboratory to address this issue. Collectively, these observations imply that effects of LOV such as attenuation of inflammatory disease and induction of remyelination through induction of pro-remyelinating environment may be mediated through these small GTPases.



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  		Figure 8. Possible mechanism of action of lovastatin-mediated OP survival and their differentiation into mature oligodendrocytes in the CNS. Lovastatin inhibits the synthesis of isoprenoids required for isoprenylation of small G-proteins (Rho family GTPase viz. RhoA, CDC42, and Rac1). These isoprenylated small G-proteins regulate the expression of inflammatory mediators (CM and iNOS) by trans-activation of NF-{kappa}B and AP-1 in immune cells (43 , 44) and CNS resident immune cells, i.e., microglia (MC) and astrocytes (AS). The inactivation of small G-proteins (e.g., RhoA) has been associated with differentiation of maturing oligodendrocytes (47) . Lovastatin treatment attenuates the inflammatory response-mediated reactive gliosis and induces a pro-remyelinating environment, i.e., release of neurotrophic factors in the CNS.

In summary, we provide an evidence for the first time that LOV treatment attenuates the development of inflammatory demyelination similar to that observed in chronic lesions of MS and restores the remyelination process in EAE. In support of this, in a recent clinical trial with simvastatin conducted on 30 patients with remitting-relapsing MS for 6 months, the investigators reported a 45% reduction in the number and volume of Gd-positive lesions in the MS brain (17) . LOV provides protection to OPs against the inflammatory response and promotes their proliferation and differentiation through attenuation of reactive gliosis and induction of a pro-remyelinating environment in CNS. Small GTPase-dependent effects of LOV may regulate both the release of inflammatory mediators in CNS and immune cells, and the differentiation of OPs by terminating their proliferation. There was no significant change in the levels of these myelin-lipids and proteins in the spinal cord of LOV-treated controls, which is indicative of no LOV-dose related adverse effects (therapeutic human dose, 20–80 mg/day) and positively predictive of the use of statins during remyelination. In accordance with these results, no adverse effect of different statins was observed regarding the expression of proteins involved directly in cholesterol synthesis, although the cholesterol levels were slightly lowered in the cerebral cortex of the mouse brain when treated chronically (48) . Together, these pro-remyelinating properties of statins make them a viable option for use in CNS demyelinating diseases such as MS, Alzheimer’s disease, stroke, X-ALD and HIV dementia.


   ACKNOWLEDGMENTS
 
We thank our laboratory colleagues for their valuable comments and help during the course of this study. We also thank the UCI DNA Micro array facility for microarray analysis, the animal care facility of the Institute, and Ms. Joyce Brian, Ms. Hope Terry and Ms. Carrie Barnes for their technical assistance. We especially thank Drs. Shalendra Giri and Jennifer G. Schnellmann for critical reading of this manuscript. This study was supported in part by grants from the National Institutes of Health Sciences: NS-22576, NS-34741, NS-37766, and NS-40144.


   FOOTNOTES
 
1 These authors contributed equally to this study.

Received for publication February 9, 2005. Accepted for publication May 5, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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