Amyloid-β42 alters apolipoprotein E solubility in brains of mice with five familial AD mutations

Abstract

Amyloid plaques composed of the 42 amino acid form of amyloid-beta peptide (Aβ42) are a pathological hallmark of Alzheimer's disease (AD), but soluble and intraneuronal Aβ42 are the more proximal causes of synaptic dysfunction and neurotoxicity. Apolipoprotein E (apoE) modulates this disease process, as inheritance of the ε4 allele of the apoE gene is the primary genetic risk factor for AD. To address the solubility of Aβ42 and apoE, a protein extraction protocol in the presence of minimal to extensive Aβ42 pathology was optimized. 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 Aβ42 and m-apoE protein was increased with FA compared to GuHCl. The 5 FAD mutations significantly increase production of Aβ42, 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 Aβ42 were observed in all extraction fractions, particularly in the cortex and hippocampus. In both young and old 5xFAD mice, Aβ42 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 extraction profile of Aβ42 paralleled that of m-apoE in 5xFAD mice. As now characterized, this method identifies the extraction profile for disease-relevant brain proteins, both normal or modified due to neuropathological processes.

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples*

“…IHC co-localization of apoE and A␤ at the synapse is a measure of primarily insoluble apoE/A␤ (35,36), and data demonstrate insoluble apoE/A␤ appears to form preferentially with apoE4 compared with apoE3 (36). Previous results have shown that the detergent and guanidine extraction pattern of mouse apoE parallels that of A␤42 in 5ϫFAD mice (5), and the proportion of apoE/A␤ in insoluble fractions was increased in AD synaptosomes compared with controls. 6 Importantly, insoluble apoE4/A␤ complex may accumulate in autophagic structures within synaptic terminals (66,67).…”

“…Tissue Preparation-Brain tissue isolation and serial protein extraction were conducted as described previously (5,15). Briefly, 6-month-old male EFAD mice were anesthetized with sodium pentobarbital (50 mg/kg) and transcardially perfused (PBS plus protease inhibitors (Calbiochem, set 3)), and brains were removed and dissected at the midline.…”

“…Thus, apoE was thought to facilitate the process of A␤ deposition as amyloid. Biochemical analyses validate IHC measures, as the levels of apoE and A␤ are equivalent in the insoluble extraction fraction from brains of transgenic mice expressing familial AD (FAD) mutations (FAD-Tg), specifically the 5ϫFAD-Tg mice (5). The association of APOE4 with AD risk was first described in 1993 (6,7), leading to research efforts focused on the effects of the APOE genotype on plaque burden and the structural relationship between apoE and amyloid.…”

Amyloid-β Pathology and APOE Genotype Modulate Retinoid X Receptor Agonist Activity in Vivo

“…TBSX employs a nonionic detergent to release apoE from lipoproteins without inducing the formation of new micelles, as can occur with ionic detergents such as SDS (31,58), and thus it indicates the levels of lipoprotein-associated apoE. In EFAD mouse brain homogenates, total brain apoE levels are lower with APOE4, but this difference is seen only in the TBSX fraction, indicating that apoE4-containing lipoproteins are less lipoprotein-associated and/or less lipidated (31).…”

APOE4-specific Changes in Aβ Accumulation in a New Transgenic Mouse Model of Alzheimer Disease

“…TBS containing 1% Triton X-100 (TBSX)) without inducing the formation of new micelles, as can occur with ionic detergents such as SDS (35)(36)(37)(38)41). To address this issue, a three-step sequential protein extraction protocol was optimized to account for the extraction conditions for lipoprotein-associated apoE, as well as insoluble protein from dense-core amyloid plaques (42).…”