Herein we present a novel method utilizing immunoprecipitation and LC-tandem MS to independently measure the in vivo sAPPβ and sAPPα kinetics in the CSF of humans who had underwent a SILK study. To date, reported measures of sAPP proteins in the CSF of humans have been absolute concentrations of static values using traditional methods such as ELISA. Our approach differs from these methods as a kinetic curve allows measurement of the dynamic nature of proteins in a living system, such as the production and clearance (turnover) of a protein, in order to better understand both the physiological mechanisms of the protein, as well as pathophysiological changes in a disease setting.
Our method allows for the quantitation of sAPPβ and sAPPα using highly specific antibodies to initially isolate each protein of interest and then the highly sensitive LC/tandem MS approach is used to quantify a common mid-domain APP peptide which not only occurs in both proteins but is found in all three APP isoforms from which sAPPβ and sAPPα may arise after proteolytic processing. The LC/tandem MS method is incredibly robust and reproducible. The precision of the assay (< 3–5%) is much better than traditional methods (typical %CV < 20%) when samples are assayed a year apart and the instrument had undergone maintenance and consumables from varying lots were used. Additionally, we report that absolute concentrations of sAPPβ and sAPPα can be calculated using internal standards and IP/MS with our approach. Previous studies using various immunoassay techniques (in-home ELISAs, IBL ELISAs and MSD assays) have reported a wide range of concentrations of these proteins in human CSF (sAPPβ = 50–1600 ng/mL and sAPPα = 35–2200 ng/mL) 10,19−21. Our reported values are 1.4–1.8-fold higher than the upper end of the range for both analytes. This large range found in past studies, as well as our current reported values being higher, may be attributed to various sources of antibodies and calibrators/standards used between assays, as well as different stocks of antibodies and calibrators within the same assay. Concentrations may also vary based on amyloid status of a subject as well as the time of day the CSF was collected10.
Our experiments show that the newer-generation QqQ, the TSQ Altis, has significantly better signal-to-noise ratio and thus, kinetic curves generated on the Altis are smoother and not erratic when compared to samples measured on the Quantum Ultra. The Ultra, additionally, had much higher variability on repeat injections. Going forward with future studies, we utilized the Altis, due to its superiority.
We also show that CSF used to measure sAPP analytes may be processed by two different Aβ immunoprecipitation protocols and the results for sAPP kinetics do not vary significantly. We intend to measure the metabolism of sAPPβ and sAPPα in 100 human subjects whose CSF had previously been immunoprecipitated for Aβ by HJ5.1 (and a small subset by 21F12/2G3) using this reported method and combine these results with historically available Aβ kinetics for these subjects to better understand the APP processing in healthy humans as well as determine important changes in the whole system that occur in the setting of AD.
This approach using our method could potentially be used to determine how to dose therapies for individual clinical trial participants, as well as monitor therapeutic effects. In addition, in vivo CSF sAPPβ and sAPPα kinetics could reveal novel insights into pathophysiological mechanisms of AD, such as increased BACE1 processing and potential for decreased α-secretase processing of APP22.
This method could further be modified and applied to study the metabolism of non-APP BACE1 substrates, that have been implicated in chronic adverse events, such as cognitive decline, reported in Phase III BACE1 inhibitor clinical trials23–25. BACE1 cleavage of neural cell adhesion molecules, L1 and neural cell adhesion protein close homolog of L1 (CHL1), appears to play a role in synaptic plasticity and learning26–27. Conditional BACE1 knockout mouse studies reported defects in axonal organization correlated with a reduction in the BACE1-mediated cleavage of CHL128. Another mouse study reported that Seizure protein 6 (SEZ6) maintains normal dendritic spine dynamics and suggests that aberrations to BACE1-mediated cleavage of SEZ6 upon BACE1 inhibition results in alterations to synaptic function29. The adverse effects reported in the clinical trials may be a result of over-inhibition of BACE1 that negatively affects the processing of non-APP substrates. Thus, finding a BACE1 dose that doesn’t alter the cleavage and turnover of these other substrates to a degree that impairs cognition, but still inhibits the β-pathway of APP enough to prevent symptoms of AD, is paramount.
Lastly, our method could be applied to study the unique setting of AD in the Down syndrome (DS) population. Although the gross pathological hallmarks of AD in DS are similar to late-onset AD (LOAD) and DIAD, there are critically important differences in the genetic mechanisms that result in this disease. In DS, there is an extra copy of chromosome 21, and, thereby, an additional copy of the APP gene, located on this chromosome30. Therefore, an overproduction of APP, and consequently of its cleavage products, is a lifelong process31. The additional copy of APP complicates pharmacokinetic and pharmacodynamic analyses when standard static measures of proteins are utilized to study the mechanisms underlying the development of AD in this population. Thus, kinetic analyses of sAPPβ and sAPPα employing our described method may prove to be beneficial when applied to DS.