Elevated abeta42 in skeletal muscle of Alzheimer disease patients suggests peripheral alterations of AbetaPP metabolism

Abstract

The levels of amyloid-beta40 (Abeta40) and Abeta42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Abeta42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Abeta40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Abeta40 and muscle Abeta42 levels were correlated with each other (P < 0.001), 2) muscle Abeta40 levels were positively correlated with age (P = 0. 036), and 3) muscle Abeta42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Abeta peptide were discovered by mass spectrometry, revealing the presence of Abeta starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Abeta and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Abeta in the temporalis muscles of AD patients suggest that alterations in AbetaPP and Abeta metabolism may be manifested in peripheral tissues.

Figure 1.

Chromatographic profile of the temporalis muscle proteins. The solid trace depicts the chromatographic contour of the muscle proteins produced by a size exclusion Superose 12 column (1 × 30 cm) developed with 80% glass-distilled formic acid. The separation was carried out using a FPLC apparatus (Pharmacia Biotechnology) at a flow rate of 15 ml per hour at room temperature and monitored at 280 nm. Fractions were automatically collected every 2 minutes. To define the retention time of the Aβ-containing fractions in the muscle preparation, an unused column was calibrated with synthetic Aβ reverse amino acid sequence 40–1 (molecular mass, 4331) which is indicated by the hyphened trace. After this calibration, extreme care was taken to decontaminate the injection system by multiple formic acid injections until, in a mock run, there were no traces of Aβ in the eluant as detected by EuIA. The Aβ eluted between 56 and 62 minutes was collected, pooled, and mixed with betaine (see Materials and Methods). The addition of this zwitterion prevents undesirable adsorption of Aβ to the glass during the drying process. The arrows indicate the elution times of calibration markers separated under the same denaturing conditions (in order from left to right): bovine serum albumin, ovalbumin, chymotrypsinogen, cytochrome C, Aβ dimer, Aβ monomer, and bacitracin.

Figure 2.

Western blot of the chromatographically separated 4.5-kd fraction from AD and control temporalis muscles. In each case, six chromatographic separations were pooled, totally dried by vacuum centrifugation, dissolved in tricine-SDS sample buffer (Novex), and separated in a 10% to 20% tricine gel. The peptides were transferred onto a PVDF membrane (BioRad) and reacted with a mixture of Aβ antibodies 4G8 and 6E10. The membranes were developed as previously published. The numbers on the right margin indicate the position of molecular weight markers.

Figure 3.

The relative levels of AβPP in the temporalis muscle and brain. A: A representative Western blot of AβPP stained by 22C11 from muscle obtained from control (C) and Alzheimer (A) tissues is shown. B and C: Histograms showing the relative levels of the 100- and 110-kd AβPP in the temporalis muscle (B) and the brain (C). The Western blots were densitometrically scanned, and the relative band intensities of the AD and control groups were averaged. There was no statistically significant difference between the two cohorts.