Analysis of microdissected neurons by ¹⁸O mass spectrometry reveals altered protein expression in Alzheimer's disease

Abstract

It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post-mortem brains by laser capture microdissection. By using 18O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat-shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron-specific analysis. Hence, we conclude that cell-specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis.

The role of protein glycosylation in Alzheimer disease

“…Alterations in lectin affinity for proteins involved in, for instance, Glc metabolism (a-enolase, c-enolase, and glutamate dehydrogenase), chaperone functions (Glc-regulated protein 78, heat shock protein 90, protein disulfide isomerase, and Glc-regulated protein 96), cytoskeletal maintenance [GFAP, tropomyosin (TPM)1, TPM2, TPM3, 14-3-3-c,e,f, gelsolin, and calreticulin], synaptic function (dihydropyrimidase 2, Rab GDP dissociation inhibitor XAP4, and b-synuclein) and cell signaling (protein phosphatase-related protein SDS22), and calmodulin) were identified. Similarly, a recent proteomics study on dissected hippocampal neurons revealed alterations in proteins involved in, for instance, tran-scription and nucleotide binding, glycolysis, the heat shock response, microtubule stabilization, axonal transport, and inflammation [98], suggesting overlapping pathways. Proteins that were identified in both the lectin-binding study and the proteomics study include GFAP and 14-3-3e.…”

Loss of neprilysin alters protein expression in the brain of Alzheimer's disease model mice

“…Briefly, the frozen dissected cortices were homogenized in 8 M Urea and 400 mM ammonium bicarbonate followed by trypsinization of 50 μg homogenate. For accurate quantification by MS/MS analysis an 18 O‐labeled internal standard made from a pool of the cortices derived from the 8 individuals was used, as 18 O‐isotope labeling is a robust technique for quantitative proteome analysis . The reproducibility and a dynamic range of at least threefold was confirmed by injecting a 1:1 and a 1:3 mixture of sample and 18 O‐labeled internal standard (Fig.…”

Evidence for the accumulation of Abeta immunoreactive material in the human brain and in transgenic animal models

“…With these approaches, intraneuronal Aβ was found to be significantly increased in neurons from AD brains compared to controls (Hashimoto et al, 2010;Hashimoto et al, 2011).…”