Immunoassay for pyruvate kinase M1/2 as an Alzheimer's biomarker in CSF

Abstract

Alzheimer's disease (AD) is characterized by amyloid-beta plaques and tau tangles in the brain, but these markers alone do not predict disease progression. The intersection of these pathologies with other processes including metabolic changes may contribute to disease progression. Brain glucose metabolism changes are among the earliest detectable events in AD. Pyruvate kinase (PKM) has been implicated as a potential biomarker to track these metabolic changes. We have developed an enzyme-linked immunosorbent assay (ELISA) to assess PKM levels in cerebrospinal fluid (CSF). First, we verified the relationship of CSF PKM levels with cognitive decline, revealing a correlation between elevated CSF PKM levels and accelerated cognitive decline in preclinical AD patients in a tau-dependent manner. We developed the ELISA using two PKM-specific antibodies and validated it through quality control steps, indicating robust quantification of PKM. We showed that ELISA measurements of PKM correlate with mass spectrometry values in matching samples. When tested on an independent cohort, the assay confirmed elevation of PKM in AD. These findings support the use of PKM as a potential biomarker for tracking early metabolic changes in AD, offering a novel tool for investigating metabolic alterations and their intersection with other underlying pathologies in AD progression.

Keywords: Alzheimer’s disease; biomarker; glucose metabolism; immunoassay; pyruvate kinase.

Figure 1

Preclinical AD patients with elevated PKM levels at baseline show accelerated cognitive decline. (a) Data from Tijms et al. [2] shows PKM levels in CSF are elevated in patients that are amyloid (A) and tau (T) positive compared to both controls and amyloid positive but tau negative patients (p-value = 2 × 10⁻¹⁶). This elevation in PKM was also independent of cognitive AD disease stage (NC A+T− vs A+T+ p-value = 4.22 × 10⁻¹⁶; MCI A+T− vs A+T+ p-value = 1.40 × 10⁻⁸; Dementia A+T− vs A+T+ p-value = 8.77 × 10⁻¹⁶). (b) Subjects with preclinical AD were split into three groups based on tertiles of CSF PKM abundance levels at baseline. When tracked over time the subjects with high baseline PKM levels showed a faster decline on the memory delayed recall score (linear mixed effects p = 0.046).

Figure 2

Development of MSD ELISA targeting PKM in CSF. (a) Example standard curves taken from mass spectrometry correlation experiment. Standard curve of the assay ranges from 1,000–4.1 ng/mL in dilution steps of 2.5×. Dotted line indicates the LLOQ, which was determined at 10.24 ng/mL. Grey dots indicate individual samples measured. All samples fell within the detectable range of the assay. (b) Average signal of PKM shows good dilution linearity across 2×, 3×, and 4× dilutions. The calculated concentration of PKM adjusted for the dilution factor is unchanged between the different dilutions confirming dilution linearity. Dots indicate the mean signal and concentration. Error bars indicate the standard deviation. (c) Spike recovery of PKM. Difference in average PKM signal between spiked and unspiked samples was calculated and compared to spiked diluent. Dotted lines indicate recovery ranges between 80 and 120%. (d) FT stability of PKM. Two AT positive and three AT negative samples were tested at three different FT points. No FT effect was observed for PKM.

Figure 3

Correlation between mass spectrometry PKM abundance and ELISA concentration. A significant positive correlation was observed between the normalized PKM abundance measured by mass spectrometry and logged PKM concentration measured by MSD ELISA (spearman rho = 0.51, p = 9.4 × 10⁻⁶). Red dots indicate AT negative participants and blue dots indicate AT positive participants.

Figure 4

Validation of PKM assay on an independent cohort. 31 AT negative and 59 AT positive samples were measured. PKM was found to be elevated in the AT positive group compared to the AT negative group (p-value = 0.003). Boxplots indicate median and interquartile range. Black line indicates the mean and grey dots show the individual sample measurements.