Journal
JOURNAL OF NEUROSCIENCE METHODS
Volume 196, Issue 1, Pages 51-59Publisher
ELSEVIER
DOI: 10.1016/j.jneumeth.2010.12.025
Keywords
Alzheimer's disease; apoE; Detergent; 5xFAD; Solubility
Categories
Funding
- NIH (NIA) [P01AG03012801]
- Alzheimer's Association [ZEN-08-99900]
- UIC CCTS [UL1RR029879]
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Amyloid plaques composed of the 42 amino acid form of amyloid-beta peptide (A beta 42) are a pathological hallmark of Alzheimer's disease (AD). but soluble and intraneuronal A beta 42 are the more proximal causes of synaptic dysfunction and neurotoxicity. Apolipoprotein E (apoE) modulates this disease process, as inheritance of the epsilon 4 allele of the apoE gene is the primary genetic risk factor for AD. To address the solubility of A beta 42 and apoE, the 5xFAD-specific extraction profile for A beta 42 was optimized, a protein extraction protocol was optimized in the presence of minimal to extensive A beta 42 pathology. Sequential extractions with TBS, TBS + Triton X-100 (TBSX), and guanidine-HCl (GuHCl) or formic acid (FA) were used with tissue from young and old wild type or mice expressing 5 familial AD mutations (5xFAD), in disease-susceptible or -resistant brain regions. In older 5xFAD mice, the extraction of insoluble A beta 42 and m-apoE protein was increased with FA compared to GuHCl. The 5xFAD mutations significantly increase production of A beta 42, recapitulating AD-like pathology at a greatly accelerated rate. Consistent protein extraction and the specificity of extractions for soluble or membrane-associated proteins were demonstrated. Age-dependent increases in A beta 42 were observed in all extraction fractions, particularly in the cortex and hippocampus. In both young and old 5xFAD mice, A beta 42 is TBS- or GuHCl-soluble. While in WT mice m-apoE is TBSX-soluble, in 5xFAD mice m-apoE is TBS- or GuHCl-soluble. Thus, the 5xFAD-specific extraction profile of A beta 42 paralleled that of m-apoE. As now characterized, this method identifies the extraction profile for disease relevant apoE and A beta in the brain, both normal or modified due to neuropathological processes. (C) 2011 Elsevier B.V. All rights reserved.
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