Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis

Abstract

Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as 'lipid rafts' contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p<0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain.

Fig. 1

Characterization of synaptic lipid rafts isolated from rat brain. The levels of GM1, raft marker proteins Thy-1 and FLT-1, and the non-raft marker Na⁺/K⁺-ATPase were determined in the density gradient fractions using dot blots or immunoblots. Dilutions of the antibodies were: anti-Thy-1, 1:250; anti-FLT-1, 1:250; and anti-Na⁺/K⁺-ATPase, 1:500. The amounts of protein loaded on the gels were: Thy-1 and FLT-1 (15 µg), and Na⁺/K⁺-ATPase (10 µg). Representative blots from five independent experiments with similar results are shown.

Fig. 2

Enlarged images of representative protein spots illustrating age-dependent differences in protein levels. Progenesis software was used to generate magnified images of the protein spots and images are shown of two spots exhibiting differential expression with age. Spot no. 931 identified as IMMT, was decreased with age. Spot no. 1648 corresponding to GFAP was increased in aged samples. Only one representative example of a protein that decreased and one that increased with age is shown for clarity. However, Fig. 1 in Supplementary Material shows a full array of differentially expressed proteins that were identified and are listed in Table 1.

Fig. 3

Representative mass spectra of MALDI-TOF and LTQ-FT used for protein identification by peptide mass fingerprint and MS/MS ion search. (A) The MALDI-TOF spectrum of NADH dehydrogenase (ubiquinone) Fe-S protein 1 identified in the peptide mass fingerprint search. The MS spectrum of spot #1104 was internally calibrated using the two ions of trypsin autolytic peptides at 842.51 and 2211.10 m/z. The monoisotopic mass list was submitted for a peptide mass fingerprint search in Mascot, resulting in one significant hit with a top score of 147 for NADH dehydrogenase (ubiquinone) Fe-S protein 1 as reported (Table 1). Matched peptide masses are assigned to the peaks in the range of 800–2500 m/z. (B and C) The LTQ-FT spectra of the ERC protein in the MS/MS ion search. The protein identification was based on a single peptide with a resultant Mascot score of 60 (Table 1). The double-charged precursor ion at 650.369 m/z was detected in the MS1 with a deviation of 3 ppm (B) and fragmented in the MS2 (C). The MS/MS data with a series of b and y ions were searched and scored in Mascot. As a result, the fragmentation spectrum was matched to the peptide AAILQTEVDALR of either ERC1 or isoform 1 of ERC2. In (C) the matched b and y ions as well as their detected masses are marked. The unique sequence of this peptide was matched only to isoform 1 of ERC2 protein.

Fig. 4

Evaluation of mitochondrial contamination by immunoblotting. Representative immunoblots show the levels of two mitochondrial markers in synaptic rafts vs. in mitochondria, both isolated from young (Y) and old (O) rat brains. Fifty micrograms of protein per lane were separated on an SDS-PAGE gel, transferred to PVDF membranes, and probed with anti-cytochrome c (1:200) and anti-GDH (1:800) antibodies. Flotillin (1:250) and MnSOD (1:2500) were used as loading controls for SPM raft and mitochondrial preparations, respectively.

Fig. 5

Immunoblot confirmation of the age-associated differences in levels of selected proteins. (A) Representative immunoblots showing age-related differences in protein levels in young (Y) vs. old (O) rafts. (B) Densitometric analysis of the protein alterations shown in (A). Data represent mean ± S.E.M. immunoreactivities in three separate preparations, with the indicated fold difference (Y/O) and P value above each set of samples. The dilutions used for the various antibodies were: anti-NDUFS3 (1:1,600), anti-ATPB (1:400), anti-VDAC1 (1:1,600), anti-IMMT (1:100), anti-GFAP (1:500), and anti-FLT-1 (1:250). The protein loading amounts were: NDUFS3 (25 µg), ATPB, VDAC1 and GFAP (30 µg), IMMT and FLT-1 (50 µg).