Vulnerability of Synaptosomes from ApoE Knock-Out Mice to Structural and Oxidative Modifications Induced by Aβ(1−40):  Implications for Alzheimer's Disease

Abstract

Apolipoprotein E (apoE) plays an important role in the response to central nervous system injury. The e4 allele of apoE and amyloid beta-peptide (Abeta) are associated with Alzheimer's disease (AD) and may be central to the pathogenesis of this disorder. Recent studies demonstrate evidence for neurodegeneration and increased lipid peroxidation in transgenic mice lacking apoE (KO). In the current study, synaptosomes were prepared from apoE KO mice to determine the role of apoE in synaptic membrane structure and to determine susceptibility to oxidative damage by Abeta(1-40). ApoE KO mice exhibited structural modifications to lipid and protein components of synaptosomal membranes as determined by electron paramagnetic resonance in conjunction with lipid- and protein- specific spin labels. Incubation with 5 microM Abeta(1-40) resulted in more severe oxidative modifications to proteins and lipids in apoE KO synaptosomes as measured by protein carbonyls, an index of protein oxidation, and TBARs and protein-bound 4-hydroxynonenal (HNE), markers of lipid oxidation. Together, these data support a role for apoE in the modulation of oxidative injury and in the maintenance of synaptic integrity and are discussed with reference to alterations in AD brain.

Effects of apolipoprotein E on the human immunodeficiency virus protein tat in neuronal cultures and synaptosomes

“…Synaptosomes isolated from WT mice contain apoE as shown by Lauderback et al (2001) and Keller et al (2000), and WT and apoE-KO mice have similar levels of the LRP receptor (data not shown), which is consistent with previous reports (Games et al, 1995). To measure ROS formation by Tat and the influence of apoE in Tat-induced formation of ROS, synaptosomes were incubated with nonfluorescent DCFH-DA, which, upon oxidation, produces the highly fluorescent DCF.…”

“…An Oxidized Protein Detection Kit (Oxyblot; Oncor, Gaithersburg, MD; catalog No. S7150-Kit) was used, as previously described (Butterfield et al, 1999;Lauderback et al, 2001Lauderback et al, , 2002. Samples were incubated for 20 min with 12% sodium dodecyl sulfate (SDS) and 2,4-dinitrophenylhydrazine (DNPH) in 10% trifluoroacetic acid, vortexed every 5 min, and then neutralized with Oxyblot Neutralization solution.…”

Widespread distribution of PLTP in human CNS: evidence for PLTP synthesis by glia and neurons, and increased levels in Alzheimer's disease

“…Such findings are not atypical. KO mouse models for some important molecules for neurophysiological processes, such as apoE (40), beta site amyloid precursor protein cleaving enzyme (41), and postnatal prion protein (42), also appear to have normal brain macrostructure and function under basal conditions when compensatory mechanisms may be adequate to maintain apparently normal structure and function. Abnormalities could become evident, however, in states of increased metabolic demand, such as recovery from neuronal injury or disease, or when accumulation of subtle defects due to aging overcomes compensatory mechanisms.…”

Hypoxia Induces Intracellular Ca2+ Release by Causing Reactive Oxygen Species-Mediated Dissociation of FK506-Binding Protein 12.6 from Ryanodine Receptor 2 in Pulmonary Artery Myocytes

“…FKBP12.6 and RyR2 oxidation detection FKBP12.6 oxidation was determined by assessing ROSmediated formation of carbonyls, which was detected by a reaction with 2, 4-dinitrophenyl hydrazine (DNPH) using an OxyBlotÔ Protein Oxidation Detection Kit (Chemicon International, Inc.), as previously described (5). According to the manufacturer's instructions, 5 ml of soluble SR membrane or cytosol fraction samples were denatured by 5 ml 12% of SDS and then derivatized with 10 ml of 1ÂDNPH (20 mM DNPH in 10% trifluoracetic acid) for 15 min at room temperature.…”