Natural Docosahexaenoic Acid in the Triglyceride Form Attenuates In Vitro Microglial Activation and Ameliorates Autoimmune Encephalomyelitis in Mice.

Many neurodegenerative diseases are associated, at least in part, to an inflammatory process in which microglia plays a major role. The effect of the triglyceride form of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (TG-DHA) was assayed in vitro and in vivo to assess the protective and anti-inflammatory activity of this compound. In the in vitro study, BV-2 microglia cells were previously treated with TG-DHA and then activated with Lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ). TG-DHA treatment protected BV-2 microglia cells from oxidative stress toxicity attenuating NO production and suppressing the induction of inflammatory cytokines. When compared with DHA in the ethyl-ester form, a significant difference in the ability to inhibit NO production in favor of TG-DHA was observed. TG-DHA inhibited significantly splenocyte proliferation but isolated CD4+ lymphocyte proliferation was unaffected. In a mice model of autoimmune encephalomyelitis (EAE), 250 mg/kg/day oral TG-DHA treatment was associated with a significant amelioration of the course and severity of the disease as compared to untreated animals. TG-DHA-treated EAE mice showed a better weight profile, which is a symptom related to a better course of encephalomyelitis. TG-DHA may be a promising therapeutic agent in neuroinflammatory processes and merit to be more extensively studied in human neurodegenerative disorders.

K(ATP) channel opener diazoxide prevents neurodegeneration: a new mechanism of action via antioxidative pathway activation.

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis.

CCAAT/enhancer binding protein δ regulates glial proinflammatory gene expression.

The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ) is expressed in activated astrocytes and microglia and can regulate the expression of potentially detrimental proinflammatory genes. The objective of this study was to determine the role of C/EBPδ in glial activation. To this end, glial activation was analyzed in primary glial cultures and in the central nervous system from wild type and C/EBPδ(-/-) mice. In vitro studies showed that the expression of proinflammatory genes nitric oxide (NO)synthase-2, cyclooxygenase-2, and interleukin (IL)-6 in glial cultures, and the neurotoxicity elicited by microglia in neuron-microglia cocultures, were decreased in the absence of C/EBPδ when cultures were treated with lipopolysaccharide (LPS) and interferon γ, but not with LPS alone. In C/EBPδ(-/-) mice, systemic LPS-induced brain expression of NO synthase-2, tumor necrosis factor-α, IL-1ß, and IL-6 was attenuated. Finally, increased C/EBPδ nuclear expression was observed in microglial cells from amyotrophic lateral sclerosis patients and G93A-SOD1 mice spinal cord. These results demonstrate that C/EBPδ plays a key role in the regulation of proinflammatory gene expression in glial activation and suggest that C/EBPδ inhibition has potential for the treatment of neurodegenerative disorders, in particular, amyotrophic lateral sclerosis.

Correction: KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation.

[This corrects the article DOI: 10.1371/journal.pone.0075189.].

Effect of genetic background on onset and disease progression in the SOD1-G93A model of amyotrophic lateral sclerosis.

Knowledge of the potential effect of genetic background in disease models is important. The SOD1-G93A transgenic mouse is the most widely used model in amyotrophic lateral sclerosis (ALS). Since these animals show considerable variability both in the onset and the progression of the disease, this study aimed to characterize the potential differences between the two most widely used strains, C56BL/6 (B6) and B6SJL. A rotarod test was carried out to assess strength and motor coordination, while electrophysiology tests were performed to evaluate the function of upper and lower motor neurons. Survival of the animals and motor neuron loss were also studied. The results did not show any background effect regarding the rotarod test, despite the differences in the pattern of decline in central and peripheral motor conduction. The onset of motor neuron abnormalities was later in B6SJL mice, but progressed more rapidly. Lifespan was longer for B6 than for B6SJL animals. In conclusion, background differences in disease onset and progression are important. The characteristics of the strain should be taken into account in experimental design of therapeutic studies.

C/EBPß expression in activated microglia in amyotrophic lateral sclerosis.

Neuroinflammation is thought to play a pathogenic role in many neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). In this study we demonstrate that the expression of nitric oxide (NO) synthase-2 (NOS2), and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) with interferon-γ is higher in microglial-enriched cultures from G93A-SOD1 mice, an ALS animal model, than from wild type mice. The levels of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor that regulates proinflammatory gene expression, are also upregulated in activated G93A-SOD1 microglial cells. In vivo, systemic lipopolysaccharide also induces an exacerbated neuroinflammatory response in G93A-SOD1 mice versus wild type mice, with increased expression of glial fibrillary acidic protein (GFAP), CD11b, nitric oxide synthase-2, cyclooxygenase-2, proinflammatory cytokines, and C/EBPß. Finally, we report that C/EBPß is expressed by microglia in the spinal cord of ALS patients. This is the first demonstration to our knowledge of microglial C/EBPß expression in human disease. Altogether these findings indicate that G93A-SOD1 expression results in an exacerbated pattern of neuroinflammation and suggest that C/EBPß is a candidate to regulate the expression of potentially neurotoxic genes in microglial cells in ALS.

Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. METHODS: Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNγ)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35₋55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. RESULTS: Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. CONCLUSION: Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

CCAAT/enhancer binding protein delta in microglial activation.

The transcription factor CCAAT/enhancer binding protein delta (C/EBP delta) regulates transcription of genes that play important roles in glial activation. Previous studies have shown the astroglial expression of C/EBP delta but the microglial expression of C/EBP delta remains virtually unexplored, with the exception of two microarray studies. In this report, using murine primary cultures and BV2 cells we clearly demonstrate that C/EBP delta is expressed by microglia and it is upregulated in microglial activation. Lipopolysaccharide upregulates C/EBP delta both in microglia and in astrocytes. This effect is time-dependent, with a maximum effect at 3 hr at mRNA level and at 4-8 hr at protein level, and concentration-dependent, with a maximum effect at 100 ng/mL. The lipopolysaccharide-induced C/EBP delta upregulation in BV2 microglia is mimicked by agonists of the toll-like receptors 2, 3 and 9 and can be prevented by an inhibitor of extracellular signal-regulated kinase activation. C/EBP delta from activated BV2 microglia binds to the cyclooxygenase-2 promoter and forms complexes with C/EBP beta isoforms. These results point to C/EBP delta as a putative key regulator of proinflammatory gene expression in microglial activation.

Upregulation of p21Cip1 in activated glial cells.

The cdk inhibitor p21(Cip1), also named p21(Cip1/Waf1), is intimately involved in coupling growth arrest to cellular differentiation in several cell types. p21(Cip1) is a multifunctional protein that might regulate cell-cycle progression at different levels. In a recent study, we found no differences in the rate of proliferation between glial cells from wild-type and p21(Cip1-/-) mice. In the present study, we examined differences in glial activation between glial cells from wild-type and p21(Cip1-/-) mice, using mixed glial cultures, microglia-enriched cultures, and astrocyte-enriched cultures. We compared the effect of lipopolysaccharide and two forms (oligomeric and fibrillar) of the 1-42 beta-amyloid peptide on glial activation. We observed an attenuation of nuclear translocation of the nuclear factor kappa-B in p21(Cip1-/-) glial cells, when compared with glial cells from wild-type mice. In contrast, tumor necrosis factor-alpha release was enhanced in p21(Cip1-/-)microglial cells. In addition glial activation induced by lipopolysaccharide and the fibrillar form of the 1-42 beta-amyloid peptide upregulated p21(Cip1). Our results support a role for p21(Cip1) in the activation of glial cells, particularly in microglia.