Proteomic study on neurite responses to oxidative stress: search for differentially expressed proteins in isolated neurites of N1E-115 cells

Abstract

Reactive oxygen species attack several living organs and induce cell death. Previously, we found axonal/dendrite degeneration before the induction of cell death in hydrogen peroxide treated neuro blastoma: N1E 115 cells and primary neurons. This phenomenon may be connected with membrane oxidation, microtubule desta bilization and disruption of intracellular calcium homeostasis. However, its detailed mechanisms are not fully understood. Here, we identified proteins after treatment with hydrogen peroxide using isolated neurites by liquid chromatography matrix assisted laser desorption/ionization time of flight/time of flight analysis. Twenty one proteins were increased after treatment with hydro gen peroxide. Specifically, 5 proteins which were secretogranin 1, heat shock protein family D member 1, Brain acid soluble protein 1, heat shock 70 kDa protein 5 and superoxide dismutase 1, were identified of all experiments and increased in isolated neurites of hydrogen peroxide treated cells compared to the controls. Further more, secretogranin 1 and heat shock protein family D member 1 protein expressions were significantly increased in normal aged and Alzheimer's transgenic mice brains. These results indicate that secretogranin 1 and heat shock protein family D member 1 might contribute to reactive oxygen species induced neurite degeneration. Both proteins have been related to neurodegenerative disorders, so their study may shed light on neurite dysfunction.

Relationship between Cognitive Dysfunction and Age-Related Variability in Oxidative Markers in Isolated Mitochondria of Alzheimer’s Disease Transgenic Mouse Brains

“…All samples except isolated mitochondria samples were homogenized in lysis buffer and used in Western blotting as described previously [ 27 ], with some modifications. Sample lysates were centrifuged, and protein contents were determined using the Bradford assay (Bio-Rad protein assay, #500-0006JA, Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s protocol.…”

Twendee X, a mixed antioxidant supplement, improves cognitive function, coordination, and neurotrophic factor expression in long-term vitamin E-deficient mice

“…Cerebral cortex and hippocampus were homogenized in radioimmunoprecipitation assay (RIPA) buffer and used in Western blotting as described previously. (16) Protein contents were determined using the Bradford assay (Bio-Rad protein assay, #500-0006JA; Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer's protocol. Twenty micrograms of protein extracts were separated on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to cellulose nitrate membranes (CelarTrans, 0.2 μm, #030-25643; FUJIFILM Wako Pure Chemical Corp.).…”

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“…Western blotting was performed as described previously with some modifications [ 47 ]. Twenty micrograms of sample protein were loaded onto 12% sodium lauryl sulfate-polyacrylamide gels.…”