ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons in the central nervous system. The main cause of the disease remains elusive, but several mutations have been associated with the disease process. In particular, mutant superoxide dismutase 1 (SOD1) protein causes oxidative stress by activating glia cells and contributes to motor neuron degeneration. KCHO-1, a novel herbal combination compound, contains 30% ethanol and the extracts of nine herbs that have been commonly used in traditional medicine to prevent fatigue or inflammation. In this study, we investigated whether KCHO-1 administration could reduce oxidative stress in an ALS model. KCHO-1 administered to ALS model mice improved motor function and delayed disease onset. Furthermore, KCHO-1 administration reduced oxidative stress through gp91(phox) and the MAPK pathway in both classically activated microglia and the spinal cord of hSOD1(G93A) transgenic mice. The results suggest that KCHO-1 can function as an effective therapeutic agent for ALS by reducing oxidative stress.
Subject(s)
Animals , Mice , Amyotrophic Lateral Sclerosis , Central Nervous System , Ethanol , Fatigue , Inflammation , Medicine, Traditional , Mice, Transgenic , Microglia , Models, Animal , Motor Neurons , Neurodegenerative Diseases , Neuroglia , Oxidative Stress , Spinal Cord , Superoxide DismutaseABSTRACT
Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 microg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91(phox), which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways.
Subject(s)
Adenine , Cell Death , Cell Line , Coculture Techniques , Culture Media, Conditioned , Down-Regulation , Microglia , NADP , NADPH Oxidases , Neuroblastoma , Neurodegenerative Diseases , Neurons , Neuroprotective Agents , Niacinamide , Phosphorylation , Phosphotransferases , Reactive Oxygen SpeciesABSTRACT
Introducción: La Enfermedad Granulomatosa Crónica (EGC) se presenta como consecuencia de mutaciones en los genes que codifican 5 de las subunidades del sistema NADPH oxidasa humano. Su forma más común es causada por cambios en el gen CYBB que codifica gp91 phox. Objetivo: Identificar el defecto molecular que lleva a la presentación de la EGC. Caso clínico: Paciente de sexo masculino con antecedentes de enfermedad diarreica aguda y abscesos perianales recurrentes desde los 2 meses. A los 6 meses, presentó una inflamación crónica del colon y colitis bacteriana. A los 3 años tenía infecciones en las vías respiratorias inferiores y perianales. Estudio compatible con EGC. El análisis del ADNc identificó expresión anormal del ARNm, lo cual se confirmó al realizar la secuenciación. Específicamente se observó la ausencia del exón 2. Adicionalmente, los datos de la secuenciación del ADNg identificaron una alteración en el sitio aceptor de "splicing" del intrón 1, que incluye una deleción seguida de la inserción de 3 nucleótidos (c.46-14_-11delTTCT insGAA). Conclusiones: Se presenta el primer estudio molecular de un paciente con EGC por defecto de "splicing" reportado en Colombia. La definición de la mutación y su correlación con el fenotipo es importante para proveer una apropiada consejería genética al paciente y su familia.
Chronic granulomatous disease (CGD) is caused by mutations in the genes that encode five of the subunits of the human NADPH oxidase. The most common form is caused by mutations in CYBB, the human gene encoding gp 91 phox. Objective: To identify the molecular defects causing CGD. Case report: A male patient with a history of acute diarrhea and recurrent perianal abscess since two months old. At 6 months, the patient presented a chronic inflammatory disease of the colon and bacterial colitis. After three years, he developed infections in the lower and perianal respiratory tract. The cDNA analysis identified abnormal mRNA expression, which was confirmed by sequencing. Specifically the exclusion of exon 2 was observed. Additionally, gDNA sequencing identified an alteration in the acceptor splice site of intron 1, including a deletion followed by insertion of three nucleotides (c.46-14_-11delTTCT insGAA). Conclusions: The first molecular study of a patient with CGD due to splicing pattern change, reported in Colombia, is presented. The definition of the mutation and its correlation with the phenotype is essential to provide appropriate genetic counseling to patients and their families.
Subject(s)
Humans , Male , Infant , Chromosomes, Human, X , Granulomatous Disease, Chronic/genetics , Mutation , NADPH Oxidases/genetics , DNA, Complementary/genetics , RNA, Messenger/genetics , Base Sequence , Cell Separation , Exons , Granulomatous Disease, Chronic/diagnosis , Flow Cytometry , Phenotype , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , RNA SplicingABSTRACT
Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).