Author Of 1 Presentation
P0933 - Inhibition of protein disulfide isomerase has neuroprotective effects in a mouse model of experimental autoimmune encephalomyelitis (ID 1402)
Abstract
Background
background: Endoplasmic reticulum (ER) stress is strictly linked to neuroinflammation and involves in the development of neurodegenerative disorders. Protein disulfide isomerase (PDI) is an enzyme that catalyzes formation and isomerization of disulfide bonds and also acts as a chaperone that survives the cells against cell death by removal of misfolded proteins.
Objectives
Our previous work revealed that PDI is explicitly upregulated in response to myelin oligodendrocyte glycoprotein (MOG)-induced ER stress in the brain of experimental autoimmune encephalomyelitis (EAE) mice. The significance of overexpression of PDI in the apoptosis of neural cells prompted us to study the effect of CCF642, an efficient inhibitor of PDI, in the recovery of EAE clinical symptoms.
Methods
The animal model of EAE was produced by the “Salari Institute of Cognitive and Behavioral Disorders”. The hippocampal tissues were homogenized in RIPA buffer, and the total protein concentration was determined by Bradford assay. PDI activity was measured by the reduction of insulin in the presence of dithiothreitol (DTT; Merck, Germany) as a reducing agent.To prepare protein samples, the hippocampus tissues were homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl (pH = 8), 150 mM NaCl, 1% SDS, 1 mM EDTA and 0.5% sodium deoxycholate supplemented with 1 μg/ml pepstatin, 10 μg/ml leupeptin, 60 μg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride (PMSF; all chemicals obtained from Sigma). The activity of Caspase-12 in the hippocampus of mice was determined using a fluorometric assay according to the specified manufacturer’s protocol for Caspase 12 Assay Kit (ab65664, Abcam, Cambridge, MA, USA). Double staining of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) and neuronal marker (NeuN) was carried out to explore colocalization of apoptotic cells and neurons. Hematoxylin (Sigma, USA) and eosin (Sigma, USA) staining were used to evaluate inflammation in the hippocampus of mice. Frozen hippocampus tissues (n = 10 each group) were homogenized in ice-cold lysis buffer used in western blotting. The obtained lysates were used to measure IL-1β, IL-6, IL-17, IL-23, IFN-γ, and Bcl2 by commercially available ELISA kits. Total protein concentration in the supernatants was detected using the Bradford assay. Data are presented as the mean ± SD. The significant differences between the two groups were calculated by the Student′s t-test or Mann-Whitney U test where appropriate
Results
Our observations suggested that CCF642 administration attenuates EAE clinical symptoms and the expression of ER stress-related proteins. Further, it suppressed the inflammatory infiltration of CD4 + T cells and the activation of hippocampus-resident microglia and Th17 cells.
Conclusions
We reported here that the inhibition of PDI protected EAE mice against neuronal apoptosis induced by prolonged ER stress and resulted in neuroprotection.
Presenter Of 1 Presentation
P0933 - Inhibition of protein disulfide isomerase has neuroprotective effects in a mouse model of experimental autoimmune encephalomyelitis (ID 1402)
Abstract
Background
background: Endoplasmic reticulum (ER) stress is strictly linked to neuroinflammation and involves in the development of neurodegenerative disorders. Protein disulfide isomerase (PDI) is an enzyme that catalyzes formation and isomerization of disulfide bonds and also acts as a chaperone that survives the cells against cell death by removal of misfolded proteins.
Objectives
Our previous work revealed that PDI is explicitly upregulated in response to myelin oligodendrocyte glycoprotein (MOG)-induced ER stress in the brain of experimental autoimmune encephalomyelitis (EAE) mice. The significance of overexpression of PDI in the apoptosis of neural cells prompted us to study the effect of CCF642, an efficient inhibitor of PDI, in the recovery of EAE clinical symptoms.
Methods
The animal model of EAE was produced by the “Salari Institute of Cognitive and Behavioral Disorders”. The hippocampal tissues were homogenized in RIPA buffer, and the total protein concentration was determined by Bradford assay. PDI activity was measured by the reduction of insulin in the presence of dithiothreitol (DTT; Merck, Germany) as a reducing agent.To prepare protein samples, the hippocampus tissues were homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl (pH = 8), 150 mM NaCl, 1% SDS, 1 mM EDTA and 0.5% sodium deoxycholate supplemented with 1 μg/ml pepstatin, 10 μg/ml leupeptin, 60 μg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride (PMSF; all chemicals obtained from Sigma). The activity of Caspase-12 in the hippocampus of mice was determined using a fluorometric assay according to the specified manufacturer’s protocol for Caspase 12 Assay Kit (ab65664, Abcam, Cambridge, MA, USA). Double staining of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) and neuronal marker (NeuN) was carried out to explore colocalization of apoptotic cells and neurons. Hematoxylin (Sigma, USA) and eosin (Sigma, USA) staining were used to evaluate inflammation in the hippocampus of mice. Frozen hippocampus tissues (n = 10 each group) were homogenized in ice-cold lysis buffer used in western blotting. The obtained lysates were used to measure IL-1β, IL-6, IL-17, IL-23, IFN-γ, and Bcl2 by commercially available ELISA kits. Total protein concentration in the supernatants was detected using the Bradford assay. Data are presented as the mean ± SD. The significant differences between the two groups were calculated by the Student′s t-test or Mann-Whitney U test where appropriate
Results
Our observations suggested that CCF642 administration attenuates EAE clinical symptoms and the expression of ER stress-related proteins. Further, it suppressed the inflammatory infiltration of CD4 + T cells and the activation of hippocampus-resident microglia and Th17 cells.
Conclusions
We reported here that the inhibition of PDI protected EAE mice against neuronal apoptosis induced by prolonged ER stress and resulted in neuroprotection.