Proteomics-determined differences in the concanavalin-A-fractionated proteome of hippocampus and inferior parietal lobule in subjects with Alzheimer's disease and mild cognitive impairment: implications for progression of AD

Abstract

Alzheimer's disease (AD) is the most common type of dementia, comprising 60-80% of all reported cases, and currently affects 5.2 million Americans. AD is characterized pathologically by the accumulation of senile plaques (SPs), neurofibrillary tangles (NFTs), and synapse loss. The early stages of memory loss associated with AD have been studied in a condition known as amnestic mild cognitive impairment (MCI), arguably the earliest form of AD. In spite of extensive research across a variety of disciplines, the cause of AD remains elusive. Proteomics techniques have helped to advance knowledge about AD by identifying irregularities in protein expression and post-translational modifications (PTMs) in AD brain. Glycosylation is a less studied PTM with regards to AD and MCI. This PTM is important to study because glycosylation is involved in proper protein folding, protein anchoring to cell membranes, and the delivery of proteins to organelles, and these processes are impaired in AD. Concanavalin-A (Con-A) binds to N-linked glycoproteins, but hydrophobic sites on nonglycoproteins are also known to bind Con-A. To our knowledge, the present study is the first to examine Con-A-associated brain proteins in MCI and AD with focus on the hippocampus and inferior parietal lobule (IPL) brain regions. Proteins found in AD hippocampus with altered levels are glutamate dehydrogenase (GDH), glial fibrillary acidic protein (GFAP), tropomyosin 3 (TPM3), Rab GDP-dissociation inhibitor XAP-4 (XAP4), and heat shock protein 90 (HSP90). Proteins found with altered levels in AD IPL are alpha-enolase, gamma-enolase, and XAP-4. MCI hippocampal proteins with altered levels are dihydropyrimidase-2 (DRP2), glucose-regulated protein 78 (GRP-78), protein phosphatase related protein Sds-22 (Sds22), and GFAP and the only protein found with altered levels in MCI IPL was beta-synuclein. These results are discussed with reference to biochemical and pathological alterations in and progression of AD.

Figure 1

Method Overview. The use of Con-A lectin-affinity chromatography coupled to 2D electrophoresis allows the separation and the resolution of Con-A-associated proteins. Alzheimer’s disease (AD) is the most common type of dementia, comprising 60–80% of all reported cases, and currently affects 5.2 million Americans. Mild cognitive impairment (MCI) arguably is the earliest for of AD. The present study identifies Con-A-fractionated brain proteins AD and MCI in two brain regions. The identity of proteins with altered levels in AD and MCI brain are consistent with biochemical and/or pathological alterations in both disorders.

Figure 2

Sypro-stained 4–16% polyacrylamide representative 2D gels of Con-A-eluted proteins from AD hippocampus and age-matched control with statistically significant protein spot identities. For protein spot p-values please refer to Table 3.

Figure 3

Sypro-stained 4–16% polyacrylamide representative 2D gels of Con-A-eluted proteins from MCI hippocampus and age-matched control with statistically significant protein spot identities. For protein spot p-values please refer to Table 3.

Figure 4

Sypro-stained 4–16% polyacrylamide representative 2D gels of Con-A-eluted proteins from AD IPL and age-matched control with statistically significant protein spot identities. For protein spot p-values please refer to Table 4.

Figure 5

Sypro-stained 4–16% polyacrylamide representative 2D gels of Con-A-eluted proteins from MCI IPL and age-matched control with statistically significant protein spot identities. For protein spot p-values please refer to Table 4.

Figure 6

Enolase 1-D Western blot probed with Con-A/biotin conjugate. Equal amonts of un-oxidized enolase, oxidized enolase, PNGase F-treated un-oxidized enolase, and PNGase F-treated oxidized enolase were probed with Con-A/biotin.

Figure 7

Con-A/biotin binding of Con-A column fractions in the presence or absence of competitive haptens and the non-ionic detergent NP-40. C = Control H = Hapten (0.1 M α-methyl glucopyranoside, 0.5 M α-methyl mannopyranoside) NP40 = Non-ionic detergent.