Differentiation of Alzheimer's Disease Subgroups using sAPPβ and sAPPα as Cerebrospinal Fluid Biomarkers of BACE1 activity

Project: Research project

Description

Using highly sensitive stable isotope labeling kinetics (SILK)/immunoprecipitation (IP)/liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to measure sAPPβ and sAPPα kinetic rates, we propose to determine if and by how much BACE1 activity is increased in late-onset AD (LOAD) subjects and in what subgroups of LOAD the shift toward increased BACE1 processing of APP occurs. We hypothesize that a subgroup of the LOAD and non-demented Amyloid+ populations overproduce Aβ because of increased BACE1 activity. We anticipate that kinetic rates of sAPPβ and sAPPα as biomarkers of BACE1 activity, together with kinetic rates of Aβ isoforms, will allow differentiation of AD subgroups based on different pathogenic mechanisms.

Our Specific Aims are:
Aim 1) Determine absolute quantitation of newly generated sAPPβ and sAPPα as measures of BACE1 and α-secretase activities in CSF of 27 control (amyloid-negative) and 27 LOAD (amyloid-positive) subjects that have undergone stable isotope labeling kinetics (SILK). We propose to determine if differences in BACE1 processing of Amyloid Precursor Protein are apparent in Amyloid- vs Amyloid+ individuals.
Aim 2) Determine if control and LOAD subjects stratify into subgroups indicative of elevated BACE1 activity and amyloid positivity. Extensive modeling and statistical analyses will be completed to take into account Apolipoprotein E (ApoE) status as well as historical Aβ kinetics and absolute concentrations, to determine if subgroups exist within amyloid-positive individuals that would differentiate diverse pathogenic mechanisms of AD.

Proposed Research Methods:
sAPPβ and sAPPα will be measured in banked CSF from 54 individuals: 27 Amyloid- (MCBP<0.18; CSF Aβ42/Aβ40>0.12; CSF Aβ42>500pg/mL) and 27 Amyloid+ (MCBP>0.18; CSF Aβ42/Aβ40<0.12; CSF Aβ42<500pg/mL). sAPPβ and sAPPα 13C-Leu labeled standards from H4APPwt cell culture media and serially-sampled CSF (hour 7 as baseline, and h8-36, bihourly) with 15N-sAPPβ or 15N-sAPPα spiked-in internal standards will undergo sequential IP to isolate sAPPβ (using a neo-epitope sAPPβ-specific antibody-bead complex) and then sAPPα (using a W02-antibody bead complex). Peptides resulting from tryptic digest of the purified sAPPβ or sAPPα will be quantified by LC-MS/MS using the Dionex UltiMate 3000/TSQ Altis system. Absolute concentrations of proteins will be measured using the MesoScale Discovery platform. Kinetic rates and newly generated metabolites will be calculated and the Population Kinetics companion application to the SAAM II modeling program will be used in modeling efforts.
StatusActive
Effective start/end date7/1/196/30/22

Funding

  • BrightFocus Foundation (Agmt 7/9/19)

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Amyloid
Cerebrospinal Fluid
Alzheimer Disease
Biomarkers
Isotope Labeling
Immunoprecipitation
Amyloid Precursor Protein Secretases
Antibodies
Amyloid beta-Protein Precursor
Apolipoproteins E
Tandem Mass Spectrometry
Liquid Chromatography
Population
Culture Media
Epitopes
Protein Isoforms
Cell Culture Techniques
Peptides
Research