Quantification of Butyrylcholinesterase Activity as a Sensitive and Specific Biomarker of Alzheimer's Disease

Abstract

Amyloid-β (Aβ) plaques are a neuropathological hallmark of Alzheimer's disease (AD); however, a significant number of cognitively normal older adults can also have Aβ plaques. Thus, distinguishing AD from cognitively normal individuals with Aβ plaques (NwAβ) based on Aβ plaque detection is challenging. It has been observed that butyrylcholinesterase (BChE) accumulates in plaques preferentially in AD. Thus, detecting BChE-associated plaques has the potential as an improved AD biomarker. We present Aβ, thioflavin-S, and BChE quantification of 26 postmortem brain tissues; AD (n = 8), NwAβ (n = 6), cognitively normal without plaques (n = 8), and other common dementias including corticobasal degeneration, frontotemporal dementia with tau, dementia with Lewy bodies, and vascular dementia. Pathology burden in the orbitofrontal cortex, entorhinal cortex, amygdala, and hippocampal formation was determined and compared. The predictive value of Aβ and BChE quantification was determined, via receiver-operating characteristic plots, to evaluate their AD diagnostic performance using sensitivity, specificity, and area under curve (AUC) metrics. In general, Aβ and BChE-associated pathology were greater in AD, particularly in the orbitofrontal cortex. In this region, the largest increase (9.3-fold) was in BChE-associated pathology, observed between NwAβ and AD, due to the virtual absence of BChE-associated plaques in NwAβ brains. Furthermore, BChE did not associate with pathology of the other dementias. In this sample, BChE-associated pathology provided better diagnostic performance (AUC = 1.0, sensitivity/specificity = 100% /100%) when compared to Aβ (AUC = 0.98, 100% /85.7%). These findings highlight the predictive value of BChE as a biomarker for AD that could facilitate timely disease diagnosis and management.

Keywords: Acetylcholinesterase; Alzheimer’s disease; amyloid-β; butyrylcholinesterase; tauopathies; thioflavin-S; α-synucleinopathy.

Fig.1

Photomicrographs of postmortem human orbitofrontal cortex from normal (A,D,G), cognitively normal with Aβ plaques (NwAβ, B,E,H), and AD (C,F,I) brains stained for amyloid-β (A,B,C), thioflavin-S (D,E,F), and butyrylcholinesterase (BChE, G,H,I). Note, no BChE staining in normal orbitofrontal cortex (G), paucity of BChE activity associated with NwAβ brain pathology (H), and significant BChE activity in AD (I). Scale bar for all frames (A-I) = 250 μm.

Fig.2

Comparisons of percentage area covered by amyloid-β (Aβ; A), thioflavin-S (Th-S; B), or butyrylcholinesterase (BChE; C) pathology in the orbitofrontal cortex, entorhinal cortex, amygdala, and hippocampal formation of normal, cognitively normal with Aβ plaques (NwAβ), and Alzheimer’s disease (AD) brains. A) In AD, significantly greater Aβ was present in the orbitofrontal cortex and amygdala compared to NwAβ brains, whereas the entorhinal cortex and hippocampal formation were not significantly different between AD and NwAβ. B) Th-S pathology occupied considerably less percentage of area in each of the brain regions compared to those stained for Aβ or BChE. However, Th-S pathology burden in AD was significantly greater than NwAβ brains. C) In normal and NwAβ brains, the presence of BChE was negligible. In AD, BChE-associated pathology burden was significantly greater in all regions analyzed when compared to NwAβ, especially in the orbitofrontal cortex with a nearly 9.3-fold increase observed. Significant differences (Bonferroni correction after multiple comparisons) are denoted as follows: ^*p < 0.0167; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Fig.3

Ratio of percentage of area covered by butyrylcholinesterase (BChE) to amyloid-β (Aβ) stained pathology in the orbitofrontal cortex (A), entorhinal cortex (B), amygdala (C), and hippocampal formation (D) of cognitively normal with Aβ (NwAβ) and Alzheimer’s disease (AD) brains. Note, AD brains had a much greater ratio compared to NwAβ demonstrating that BChE activity is greatly increased in the AD brain compared to NwAβ. **Denotes statistically significant difference (p < 0.01).

Fig.4

Receiver-operating characteristic (ROC) plot (sensitivity versus 1-specificity) of amyloid-β (Aβ), thioflavin-S (Th-S), and butyrylcholinesterase (BChE) quantification metrics of the orbitofrontal cortex. Empirical data shown as solid lines and fitted curves as dashed lines of the same color. Chance association shown as diagonal line indicates no discriminative capability of a diagnostic test. The area under the curve (AUC) serves as a summary measure of the diagnostic performance of each metric. BChE showed high diagnostic accuracy. See Table 2 for a complete list of ROC summary measures.

Fig.5

Photomicrographs of postmortem human entorhinal cortex from corticobasal degeneration (A,B), frontotemporal dementia with tau (C,D), dementia with Lewy bodies (E,F), and vascular dementia (G,H) stained for tau 3R (A), tau 4R (C), α-synuclein (E), Aβ (G), and butyrylcholinesterase (BChE) activity (B,D,F,H). Note, insets are higher magnification photomicrographs demonstrating examples of the pathology observed in each of the neurodegenerative diseases including neurofibrillary tangles (A), neuropil threads and degenerating neurites (C), Lewy bodies (E), and intraneuronal inclusions (G). Note, BChE staining was limited to a few scattered cortical neurons (insets B,D,F,H) and did not label pathological structures in these neurodegenerative diseases. Scale bars = 250 μm, insets 50 μm.