Combination therapy of lovastatin and AMP-activated protein kinase activator improves mitochondrial and peroxisomal functions and clinical disease in experimental autoimmune encephalomyelitis model.

Recent studies report that loss and dysfunction of mitochondria and peroxisomes contribute to the myelin and axonal damage in multiple sclerosis (MS). In this study, we investigated the efficacy of a combination of lovastatin and AMP-activated protein kinase (AMPK) activator (AICAR) on the loss and dysfunction of mitochondria and peroxisomes and myelin and axonal damage in spinal cords, relative to the clinical disease symptoms, using a mouse model of experimental autoimmune encephalomyelitis (EAE, a model for MS). We observed that lovastatin and AICAR treatments individually provided partial protection of mitochondria/peroxisomes and myelin/axons, and therefore partial attenuation of clinical disease in EAE mice. However, treatment of EAE mice with the lovastatin and AICAR combination provided greater protection of mitochondria/peroxisomes and myelin/axons, and greater improvement in clinical disease compared with individual drug treatments. In spinal cords of EAE mice, lovastatin-mediated inhibition of RhoA and AICAR-mediated activation of AMPK cooperatively enhanced the expression of the transcription factors and regulators (e.g. PPARα/ß, SIRT-1, NRF-1, and TFAM) required for biogenesis and the functions of mitochondria (e.g. OXPHOS, MnSOD) and peroxisomes (e.g. PMP70 and catalase). In summary, these studies document that oral medication with a combination of lovastatin and AICAR, which are individually known to have immunomodulatory effects, provides potent protection and repair of inflammation-induced loss and dysfunction of mitochondria and peroxisomes as well as myelin and axonal abnormalities in EAE. As statins are known to provide protection in progressive MS (Phase II study), these studies support that supplementation statin treatment with an AMPK activator may provide greater efficacy against MS.

Effect of vitamin D3 intake on the onset of disease in a murine model of human Krabbe disease.

Low vitamin D level is a risk factor for various late-onset CNS demyelinating disorders. We investigated whether vitamin D deficiency influences disease in twitcher mice (GALC(twi/twi) ; twi), a murine model of Krabbe disease (KD), an inherited disorder caused by galactocerebrosidase (GALC) deficiency that leads to psychosine accumulation, oligodendrocyte (OL) loss, and CNS demyelination. We found that the in situ 1,25-dihydroxyvitamin D3 level was reduced, with a parallel increase in the expression of inflammatory cytokines and vitamin D-catabolizing enzymes in the brains of KD and twi mice compared with age-matched controls. Pups maintained on milk from lactating heterozygous (GALC(twi/+) ) mothers that were fed a vitamin D3-supplemented diet until weaning and then fed a vitamin D3-supplemented diet demonstrated delayed body weight loss and development of disease in twi mice. This delayed the onset of tremors and locomotor disabilities that eventually impacted the life span of twi mice (50 ± 2 days). Accordingly, the expression of antioxidant enzymes was increased with delayed psychosine accumulation, lipid peroxidation, and inflammatory response that eventually protected CNS myelin and axonal integrity in twi mice. In vitro studies revealed that 1,25-dihydroxyvitamin D3 enhances antioxidant defenses in OLs deficient for GALC or incubated with psychosine. Together these data provide the first evidence that vitamin D deficiency affects disease development in twi mice and that vitamin D3 supplementation has the potential to improve the efficacy of KD therapeutics.

Post-trauma Lipitor treatment prevents endothelial dysfunction, facilitates neuroprotection, and promotes locomotor recovery following spinal cord injury.

We have previously reported neuroprotection in spinal cord injury (SCI) by Lipitor [atorvastatin (AT)]-pre-treatment. Though informative, pre-treatment studies find only limited clinical application as trauma occurrence is unpredictable. Therefore, this study investigates the efficacy of AT treatment post-SCI. In a rat model of contusion-SCI resulting in complete hindlimb paralysis, AT treatment (5 mg/kg; gavage) was begun 2, 4, or 6 h post-SCI followed by a once daily dose thereafter for 6 weeks. While the placebo vehicle (VHC)-SCI rats showed substantial functional deficit, AT-SCI animals exhibited significant functional recovery. AT diminished injury-induced blood-spinal cord barrier (BSCB) dysfunction with significantly reduced infiltration and tumor necrosis factor-alpha/interleukin-1beta/inducible nitric oxide synthase expression at site of injury. BSCB protection in AT-SCI was attributable to attenuated matrix metalloproteinase-9 (MMP9) expression - a central player in BSCB disruption. Furthermore, endothelial MMP9 expression was found to be RhoA/ROCK pathway-mediated and regulated by AT through an isoprenoid-dependent mechanism. Attenuation of these early inflammatory events reduced secondary damage. Significant reduction in axonal degeneration, myelin degradation, gliosis, and neuronal apoptosis with resultant enhancement in tissue sparing was observed in AT-SCI compared with VHC-SCI. In summary, this novel report presenting the efficacy of post-injury AT treatment might be of critical therapeutic value as effective treatments are currently unavailable for SCI.

Immunomodulatory effect of combination therapy with lovastatin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside alleviates neurodegeneration in experimental autoimmune encephalomyelitis.

Combination therapy with multiple sclerosis (MS) therapeutics is gaining momentum over monotherapy for improving MS. Lovastatin, an HMG-CoA reductase inhibitor (statin), was immunomodulatory in an experimental autoimmune encephalomyelitis (EAE) model of MS. Lovastatin biases the immune response from Th1 to a protective Th2 response in EAE by a different mechanism than 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, an immunomodulating agent that activates AMP-activated protein kinase. Here we tested these agents in combination in an EAE model of MS. Suboptimal doses of these drugs in combination were additive in efficacy against the induction of EAE; clinical symptoms were delayed and severity and duration of disease was reduced. In the central nervous system, the cellular infiltration and proinflammatory immune response was decreased while the anti-inflammatory immune response was increased. Combination treatment biased the class of elicited myelin basic protein antibodies from IgG2a to IgG1 and IgG2b, suggesting a shift from Th1 to Th2 response. In addition, combination therapy lessened inflammation-associated neurodegeneration in the central nervous system of EAE animals. These effects were absent in EAE animals treated with either drug alone at the same dose. Thus, our data suggest that agents with different mechanisms of action such as lovastatin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, when used in combination, could improve therapy for central nervous system demyelinating diseases and provide a rationale for testing them in MS patients.

Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment.

Peroxisomes are ubiquitous subcellular organelles and abnormality in their biogenesis and specific gene defects leads to fatal demyelinating disorders. We report that neuroinflammatory disease in brain of experimental autoimmune encephalomyelitis (EAE) rats decreased the peroxisomal functions. Degradation of very long chain fatty acids decreased by 47% and resulted in its accumulation (C26:0, 40%). Decreased activity (66% of control) of dihydroxyacetonephosphate acyltransferase (DHAP-AT), first enzyme in plasmalogens biosynthesis, resulted in decreased levels of plasmalogens (16-30%). Catalase activity, a peroxisomal enzyme, was also reduced (37%). Gene microarray analysis of EAE spinal cord showed significant decrease in transcripts encoding peroxisomal proteins including catalase (folds 3.2; p<0.001) and DHAP-AT (folds 2.6; p<0.001). These changes were confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, suggesting that decrease of peroxisomal functions in the central nervous system will have negative consequences for myelin integrity and repair because these lipids are major constituents of myelin. However, lovastatin (a cholesterol lowering and anti-inflammatory drug) administered during EAE induction provided protection against loss/down-regulation of peroxisomal functions. Attenuation of induction of neuroinflammatory mediators by statins in cultured brain cells [J. Clin. Invest. 100 (1997) 2671-2679], and in central nervous system of EAE animals and thus the EAE disease [J. Neurosci. Res. 66 (2001) 155-162] and the studies described here indicate that inflammatory mediators have a marked negative effect on peroxisomal functions and thus on myelin assembly and that these effects can be prevented by treatment with statins. These observations are of importance because statins are presently being tested as therapeutic agents against a number of neuroinflammatory demyelinating diseases.

A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases.

In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-gamma caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-gamma-induced iNOS, TNF-alpha, and IL-1beta gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IkappaB phosphorylation, NF-kappaB DNA binding activity, and NF-kappaB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI.