Increased Expression of Readthrough Acetylcholinesterase Variants in the Brains of Alzheimer's Disease Patients

Abstract

Alzheimer's disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). AChE is expressed as multiple splice variants, which may serve both cholinergic degradative functions and non-cholinergic functions unrelated with their capacity to hydrolyze acetylcholine. We have recently demonstrated that a prominent pool of enzymatically inactive AChE protein exists in the AD brain. In this study, we analyzed protein and transcript levels of individual AChE variants in human frontal cortex from AD patients by western blot analysis using specific anti-AChE antibodies and by quantitative real-time PCR (qRT-PCR). We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchoring subunit, proline-rich membrane anchor (PRiMA-1) in frontal cortex obtained from AD patients and non-demented controls. Interestingly, we found an increase in the protein and transcript levels of the non-cholinergic "readthrough" AChE (AChE-R) variants in AD patients compared to controls. Similar increases were detected by western blot using an antibody raised against the specific N-terminal domain, exclusive of alternative N-extended variants of AChE (N-AChE). In accordance with a subset of AChE-R monomers that display amphiphilic properties that are upregulated in the AD brain, we demonstrate that the increase of N-AChE species is due, at least in part, to N-AChE-R variants. In conclusion, we demonstrate selective alterations in specific AChE variants in AD cortex, with no correlation in enzymatic activity. Therefore, differential expression of AChE variants in AD may reflect changes in the pathophysiological role of AChE, independent of cholinergic impairment or its role in degrading acetylcholine.

Keywords: AChE splice variants; Acetylcholinesterase; Alzheimer’s disease; human brain; readthrough AChE variants.

Figure 1. Decrease of acetylcholinesterase (AChE) activity levels in the frontal cortex of Alzheimer's disease (AD) patients

(A) AChE specific activity (mU/mg of total protein) was measured in protein extracts from brain cortices of non-demented controls (NDC; n=22) and AD patients (AD; n=19). One milliunit (mU) of AChE activity was defined as the number of nmoles of acetylthiocholine hydrolyzed per min at 22 °C. (B) Equal amount of protein from NDC and AD brain extracts were separated by ultracentrifugation on sucrose gradient and molecular forms of AChE (tetramers: G₄; and light monomers: G₁) were identified in each fraction by comparison with the position of molecular weight markers catalase (C; 11.4S) and alkaline phosphatase (P; 6.1S). Representative profiles for NDC (●) and AD (○) are shown (left panel) in which the G₄/(G₁) ratio were calculated (right panel, n=6 for each group). Represented values are means ± SEM. *Significantly decreased (p< 0.05) from NDC as assessed by a Student's t-test.

Figure 2. Increased protein levels of AChE splice variants in the frontal cortex of AD patients

Immunodetection of AChE variants and the membrane anchor subunit PRiMA-1 in frontal cortex of non-demented control (NDC, n=12) and AD patients (n=12). Fifty μg of protein from brain extracts (equal amount of protein in each lane) were resolved by electrophoresis and probed with specific primary antibodies raised to: (A) the C-terminus of the “tailed” AChE-T variant; (B) the C-terminus of “readthrough” AChE-R variant; (C) the extended N-terminus of N-AChE variants; and (D) the anchor subunit PRiMA-1. Representative blots and densitometric quantification of the immunoreactive bands are shown and expressed as percentage (%) relative to immunoreactive of the 55 kDa AChE band or to the 22 kDa PRiMA band, from the NDC group. For semiquantitative analysis, levels were normalized to the housekeeping protein GAPDH. The results were confirmed in two independent determinations. Values are means ± SEM. * p< 0.05 significantly upregulated compared to NDC by a Student's t-test.

Figure 3. Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects

Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by qRT-PCR in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power SYBR® Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2^−ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased (p< 0.05) compared to NDC as assessed by a Student's t test.

Figure 4. An amphiphilic subset of the “readthrough” acetylcholinesterase (AChE-R) variant is increased in the frontal cortex of Alzheimer's patients

(A) Light AChE monomers (G₁) were fractioned and identified by sucrose gradient centrifugation from frontal extracts of NDC (●; n= 6) and Alzheimer's subjects (○; n= 6). Fractions containing AChE monomers were pooled, dialyzed and concentrated by ultrafiltration. Representative sedimentation profiles illustrating the fractions selected for (G₁) peak isolation are shown. Equal amount of protein from enriched AChE light forms fractions were then assayed by immunoblotting using specific antibodies to AChE-R (B) and the N-extended (N-AChE) (C) variants. Representative blot and densitometric quantification of the AChE-immunoreactive bands for each antibody, expressed in arbitrary units (a.u.), are shown. The results were confirmed in two independent determinations with equivalent amounts of protein loaded in each lane. (D) The enriched AChE monomeric fractions were also characterized by hydrophobic interaction on a phenyl-agarose matrix. The original enriched peak (input), the bound (amphiphilic isoforms eluted with Triton X-100), and the unbound (hydrophilic isoforms) were then assayed by Western blotting using the anti-AChE-R antibody, to compare differences in AChE-R amphiphilic behaviour between groups. A representative blot is included (left panel). Immunoreactive bands were quantified and the percentage (%) of the bound fraction respect the input was calculated (right panel). Values are means ± SEM. * p< 0.05 significantly different from NDC group, (Student's t-test).