Increased in Vivo Amyloid-β42 Production, Exchange, and Loss in Presenilin Mutation Carriers

Abstract

Alzheimer’s disease is hypothesized to be caused by an over-production or reduced clearance of amyloid-beta (Aβ) peptide. Autosomal Dominant Alzheimer’s Disease (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aβ42 compared to Aβ40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers We used compartmental modeling of stable isotope labeling kinetic (SILK) studies in human carriers of PSEN mutations and related non-carriers to evaluate the pathophysiological effects of PSEN1 and PSEN2 mutations on the production and turnover of Aβ isoforms. We compared these findings by mutation status and amount of fibrillar amyloid deposition as measured by positron emission tomography (PET) using the amyloid tracer, Pittsburgh compound B (PiB). CNS Aβ42 to Aβ40 production rates were 24% higher in mutation carriers compared to non-carriers and this was independent of fibrillar amyloid deposits quantified by PET PiB imaging. The fractional turnover rate of soluble Aβ42 relative to Aβ40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aβ42 into plaques leading to reduced recovery of Aβ42 in cerebrospinal fluid (CSF). Reversible exchange of Aβ42 peptides with pre-existing unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with presenilin mutations that cause AD, and demonstrate that soluble Aβ42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aβ42 concentrations in the CSF.

Characterization of AD-like phenotype in aged APPSwe/PS1dE9 mice

“…This result indicates that there is decreased Aß clearance, without increased Aß production, in the late stage of this AD model. This phenotype is consistent with previous human studies that Aβ clearance is impaired in both early-onset and late-onset forms of AD (Mawuenyega et al 2010;Potter et al 2013). Based on this, it seems that aged APP/PS1 mice may be available for screening potential anti-AD agents targeting at Aβ clearance.…”

The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function

“…Given the substantial increase in Aβ observed in Rag5xfAD mice, we next sought to determine whether these findings arose from increased Aβ production or decreased clearance. Although autosomal-dominant AD is characterized primarily by mutations that increase production of Aβ or Aβ42/40 ratio (21,22), recent studies demonstrate that sporadic AD patients primarily accumulate Aβ as a result of impaired clearance (23-25).…”

In vivo kinetic approach reveals slow SOD1 turnover in the CNS

“…However, measuring turnover rates of proteins in an immortalized, rapidly dividing cell culture model has limitations and shows discrepancies when turnover rates of identical proteins are compared between tissues and cell culture models (4). The development of stable isotope-labeling kinetics (SILK) has enabled the study of protein turnover rates in vivo using a safe, stable isotope amino acid tracer that is incorporated into newly synthesized proteins and can be quantitatively measured by mass spectrometry -a technique that has been highly successful in studies of amyloid-β in human cerebral spinal fluid (CSF) and in brains from animal models (5)(6)(7)(8)(9)(10)(11)(12). In these studies, a relatively short (9-hour) intravenous infusion of the stable isotope 13 C 6 -leucine resulted in adequate labeling of the rapidly turned over amyloid-β (half-life of 8 hours).…”

“…The calculation of protein turnover by administering stable isotope-labeled amino acids or D 2 O over time has been demonstrated in numerous cell culture models and in human CSF and plasma proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)23). Stable isotope-labeled amino acids are biologically identical to their naturally occurring counterparts and, unlike radiolabeled amino acids, are innocuous to both the system being studied and the experimental environment.…”

“…Animal protocols were approved by the Institutional Animal Care and Use Committee at Washington University and conducted according to the NIH Guide for the Care and Use of Laboraprecipitated with 10% trichloroacetic acid overnight at 4°C, the protein pellet was retained for quantification of bound 13 C 6 -leucine, and the supernatant was removed after centrifugation at 21,000 g for 10 minutes. The supernatant was chemically derivatized to form the N-heptafluorobutyryl n-propyl esters of plasma-free amino acids, and 13 C 6 -leucine enrichment was quantified using capillary gas chromatography-negative chemical ionization-quadrupole mass spectrometry (GC-MS; Agilent 6890N Gas Chromatograph and Agilent 5973N Mass Selective Detector) with the m/z of 355 as compared with 349 as described previously (11,39). Protein-bound 13 C 6 -leucine abundance was quantified in the TCA-precipitated proteins after sonicating the pellet in a cold 10% TCA solution twice.…”