Using stable isotope labeling to advance our understanding of Alzheimer's disease etiology and pathology

Abstract

Stable isotope labeling with mass spectrometry (MS)-based proteomic analysis has become a powerful strategy to assess protein steady-state levels, protein turnover, and protein localization. Applying these analyses platforms to neurodegenerative disorders may uncover new aspects of the etiology of these devastating diseases. Recently, stable isotopes-MS has been used to investigate early pathological mechanisms of Alzheimer's disease (AD) with mouse models of AD-like pathology. In this review, we summarize these stable isotope-MS experimental designs and the recent application in the context of AD pathology. We also describe our current efforts aimed at using nuclear magnetic resonance (NMR) analysis of stable isotope-labeled amyloid fibrils from AD mouse model brains. Collectively, these methodologies offer new opportunities to study proteome changes in AD and other neurodegenerative diseases by elucidating mechanisms to target for treatment and prevention.

Keywords: APP Knock-In Mice; Alzheimer's disease; Amyloid-β; mass spectrometry; proteomics; stable isotopes.

Figure 1:. Using stable isotope labeling to measure relative protein abundance in mouse models of AD-like pathology.

(A) Direct comparison of relative protein levels between two mouse models. A mouse model of AD-like pathology remains unlabeled (¹⁴N) and a relevant control mouse model is labeled with stable isotopes (¹⁵N). Brains are dissected and extracts are mixed 1:1 before proceeding for analysis by LC-MS/MS. (B) Comparison of relative protein levels using an internal standard. Two different mouse models of AD-like pathology remain unlabeled (¹⁴N). One control mouse model is labeled with stable isotopes (¹⁵N). Brains are dissected from all three mouse models. Control brain extracts are mixed 1:1 with AD model 1 as well as AD model 2. The two mixtures are then processed separately for LC-MS/MS. Analysis enables comparison of the two unlabeled AD models. (C) Multiscale analysis using stable isotopes. A mouse model of AD-like pathology remains unlabeled (¹⁴N) and a relevant control mouse model is labeled with stable isotopes (¹⁵N). Brains are dissected and extracts are mixed 1:1. Before proceeding for MS analysis, mixed extracts are processed further, including but not limited to, isolation of specific cell types, purification of organelles, or fractionation for synaptosomes. After the processing steps, samples are analyzed with LC-MS/MS.

Figure 2:. Pulse-chase stable isotope labeling to measure protein turnover in mouse models of AD-like pathology.

(A) Single generation labeling with stable isotopes. Mice from a model of AD-like pathology are put on a diet of ¹⁵N enriched Spirulina chow for months. After several months, brains are dissected and extracted for LC-MS/MS analysis in order to identify and quantify the proteins that remain labeled with ¹⁴N. (B) Two generation labeling with stable isotopes. Female mice from a mouse model of AD-like pathology are put on a diet of ¹⁵N enriched Spirulina chow for months. Mice of the same genotype are introduced for breeding. Homozygous pups are born labeled with ¹⁵N. These second-generation mice are subsequently chased with a chow containing the natural ¹⁴N for several months. Brains are dissected and extracted for LC-MS/MS analysis in order to identify and quantify the proteins that remain labeled with ¹⁵N. (C) Pulse chase experimental designs can be affected by altering the timing of the labeling or chase. Based on the two-generational labeling, different labeling or chase periods allows for investigation into different stages of disease progression, including but not limited to Aβ accumulation, plaque deposition and Aβ turnover, or advanced aging.

Figure 3:. Using stable isotopes and NMR to determine structures of amyloid in vivo.

(A) Assessing how amyloid structures differ amongst various mouse models of AD-like pathology. Several mouse models of AD-like pathology, including but not limited to, App KI mice, App transgenic mice, App/PS1 transgenic mice, and 5XFAD mice are labeled with ¹⁵N. Amyloid fibrils are then purified from each of these mouse models and amyloid structure is assessed with solid state nuclear magnetic resonance imaging (NMR). (B) Assessing how Aβ seeds from different sources influence Aβ structure. Amyloid fibrils are purified from human AD brains with different degrees of pathology, and seeds are injected into an isotopically labeled mouse model of AD-like pathology. After seeding, ¹⁵N labeled amyloid is purified from the mouse and the amyloid structure changes as a result of the injected seeds is assessed with solid state NMR.