Altered levels of variant cholinesterase transcripts contribute to the imbalanced cholinergic signaling in Alzheimer's and Parkinson's disease

Abstract

Acetylcholinesterase and butyrylcholinesterase (AChE and BChE) are involved in modulating cholinergic signaling, but their roles in Alzheimer's and Parkinson's diseases (AD and PD) remain unclear. We identified a higher frequency of the functionally impaired BCHE-K variant (rs1803274) in AD and PD compared to controls and lower than in the GTEx dataset of healthy individuals (n = 651); in comparison, the prevalence of the 5'-UTR (rs1126680) and intron 2 (rs55781031) single-nucleotide polymorphisms (SNPs) of BCHE and ACHE's 3'-UTR (rs17228616) which disrupt AChE mRNA targeting by miR-608 remained unchanged. qPCR validations confirmed lower levels of the dominant splice variant encoding the "synaptic" membrane-bound ACHE-S in human post-mortem superior temporal gyrus samples from AD and in substantia nigra (but not amygdala) samples from PD patients (n = 79, n = 67) compared to controls, potentially reflecting region-specific loss of cholinergic neurons. In contradistinction, the non-dominant "readthrough" AChE-R mRNA variant encoding for soluble AChE was elevated (p < 0.05) in the AD superior temporal gyrus and the PD amygdala, but not in the neuron-deprived substantia nigra. Elevated levels of BChE (p < 0.001) were seen in AD superior temporal gyrus. Finally, all three ACHE splice variants, AChE-S, AChE-R, and N-extended AChE, were elevated in cholinergic-differentiated human neuroblastoma cells, with exposure to the oxidative stress agent paraquat strongly downregulating AChE-S and BChE, inverse to their upregulation under exposure to the antioxidant simvastatin. The multi-leveled changes in cholinesterase balance highlight the role of post-transcriptional regulation in neurodegeneration. (235).

Keywords: Alzheimer’s disease; Parkinson’s disease; SNPs (single-nucleotide polymorphisms); acetylcholinesterase; butyrylcholinesterase; splice variants.

FIGURE 1

Our study’s outline. Cholinergic transcripts were analyzed by qPCR, Sanger sequencing, and enzyme activity assays from different regions of human post-mortem brains from AD and PD patients, SH-SY5Y and LAN-5 cells, and GTEx cohort datasets.

FIGURE 2

Altered prevalence of ACHE and BCHE SNPs in AD and PD patients compared to non-demented controls. (A) Loci of SNPs in the BCHE (K allele, rs1803274), 5′-UTR (rs1126680), and intron 2 (rs55781031), and of 3′-UTR ACHE SNP (rs17228616). SNPs were studied in AD (n = 79) and PD (n = 67) patients and in the genomic dataset of the GTEx healthy donors (n = 651). (B) The SNPs in the 5′-UTR (rs1126680) and intron 2 (rs55781031) of the BCHE gene, as well as the 3′-UTR ACHE SNP (rs17228616), showed similar occurrence in the diseased and healthy cohorts of AD and PD. (C) In the GTEx cohort, carriers of the 5′-UTR BCHE SNP (rs1126680) tended to carry the BCHE SNP in intron 2 (rs55781031) but did not show distinct prevalence of the coding sequence BCHE-K allele (rs1803274). (D) Both AD and PD donors of our small cohorts showed higher prevalence of the BCHE-K minor allele (rs1803274) compared to controls.

FIGURE 3

ACHE and BCHE mRNA transcripts and AChE enzyme activity show changed levels in the superior temporal gyrus of AD patients vs. controls. (A) Lower AChE-S mRNA (p < 4e-6), higher AChE-R mRNA (p < 3e-6). Unchanged AChE-Next mRNA, and higher BChE mRNA (p < 8e-9) in AD compared with controls, (B) qPCR-obtained Cq values for each cholinesterase show the predominance of the AChE-S splice variant over the AChE-R and AChE-Next transcripts in the superior temporal gyrus. (C) Decreased AChE enzyme activity (p < 0.05) in AD compared to control brains. Normalized expression represents ΔΔCq, and enzyme activity is shown in nmol ATCh/min/mg protein, ^∗p < 0.05 and ^∗∗∗p < 0.001.

FIGURE 4

PD-modified levels of brain cholinesterase transcripts and AChE enzyme activity in the amygdala and substantia nigra. (A) Elevated mRNA levels of AChE-R (p < 0.023) and AChE-Next (p < 0.05) variants. (B) Unchanged AChE enzyme activity in PD compared with controls in the amygdala. (C) Decreased AChE-S mRNA levels (p < 0.05). (D) Decreased AChE enzyme activity (p = 0.078) in PD compared to controls in the substantia nigra. Normalized expression represents ΔΔCq, and enzyme activity is in nmol ATCh/min/mg protein, ^∗p < 0.05, ^∗∗p < 0.01.

FIGURE 5

Cholinergic differentiation of neuroblastoma cell lines elevates the levels of AChE-S, AChE-R, and AChE-Next, but not BChE transcripts. Cholinergic differentiation induced by 4 days of treatment with CNTF and ATRA led to elevation of cholinergic markers CHAT, SLC18A3 (VAChT), and SLC5A7 (CHT) (Supplementary Figure 1). (A) Specifically, LAN-5 cells showed upregulation of all AChE variants (AChE-S, p-value < 0.0004; AChE-R, p-value < 0.004, AChE-Next, p-value < 0.02). (B) SH-SY5Y cells showed upregulated AChE-S (p-value < 0.0001), ACHE-R (p < 0.01), and ACHE-Next (p < 0.05). BChE mRNA levels remained unchanged in both cell lines. (C) RT-qPCR Cq values show the predicted predominance of AChE-S over BChE or AChE-R and AChE-Next transcripts in cholinergic-differentiated LAN-5 and SH-SY5Y cells. Normalized expression represents ΔΔCq. T-test p-values, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

FIGURE 6

Oxidative stress and antioxidant treatment shift cholinesterase mRNA levels inversely in neuroblastoma cells. (A) SH-SY5Y cells were exposed to the oxidative stress-inducing agent paraquat (PQ; 50 μM) or to (B) antioxidant simvastatin (STT; 10 μM) for 24 h. Note PQ-induced decreases in AChE-S (p < 0.015), BChE (p < 0.015), AChE-R (p = 0.09), and AChE-Next (p = 0.234), and STT-induced increase in AChE-S (p < 0.0003), BChE (p = 0.23), AChE-R (p < 0.06), and AChE-Next (p < 0.002). Normalized expression represents ΔΔCq, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.