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Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus

Specific inhibitory effect of aptamer A1 on BACE1 activity in an AD cell model.(A) Western blot analysis of sAPPα, sAPPβ and BACE1 expressions in the A1, Gp30, U31 and blank control groups. (B-D) Quantitative analysis of the sAPPβ (B), sAPPα (C) and BACE1 (D) bands among groups. The data are represented as the mean±SD (n = 3). **P<0.01 compared with the Gp30 and blank groups. #P<0.05 compared with the U31 group.
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pone.0140733.g005: Specific inhibitory effect of aptamer A1 on BACE1 activity in an AD cell model.(A) Western blot analysis of sAPPα, sAPPβ and BACE1 expressions in the A1, Gp30, U31 and blank control groups. (B-D) Quantitative analysis of the sAPPβ (B), sAPPα (C) and BACE1 (D) bands among groups. The data are represented as the mean±SD (n = 3). **P<0.01 compared with the Gp30 and blank groups. #P<0.05 compared with the U31 group.

Mentions: MTT was used to evaluate the effect of different A1 concentrations on cell survival. M17-APPsw cell viability was not affected at a final concentration of 3 μM (Fig 4A). Thus, a 3 μM A1 concentration was applied for cell incubation. The resulting cell lysates and media were resolved on a double-antibody sandwich ELISA. A 3 μM U31 treated group and a blank control (without treatment) group served as the controls. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media compared with the control group (Fig 4B–4E). Western blot analysis indicated that sAPPβ expression was significantly decreased in the A1 treated group compared with the control group, whereas no substantial differences in sAPPα and BACE1 expression levels were identified among the groups (Fig 5). These results suggest that A1 can inhibit BACE1 activity in an AD cell model.


Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Specific inhibitory effect of aptamer A1 on BACE1 activity in an AD cell model.(A) Western blot analysis of sAPPα, sAPPβ and BACE1 expressions in the A1, Gp30, U31 and blank control groups. (B-D) Quantitative analysis of the sAPPβ (B), sAPPα (C) and BACE1 (D) bands among groups. The data are represented as the mean±SD (n = 3). **P<0.01 compared with the Gp30 and blank groups. #P<0.05 compared with the U31 group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608735&req=5

pone.0140733.g005: Specific inhibitory effect of aptamer A1 on BACE1 activity in an AD cell model.(A) Western blot analysis of sAPPα, sAPPβ and BACE1 expressions in the A1, Gp30, U31 and blank control groups. (B-D) Quantitative analysis of the sAPPβ (B), sAPPα (C) and BACE1 (D) bands among groups. The data are represented as the mean±SD (n = 3). **P<0.01 compared with the Gp30 and blank groups. #P<0.05 compared with the U31 group.
Mentions: MTT was used to evaluate the effect of different A1 concentrations on cell survival. M17-APPsw cell viability was not affected at a final concentration of 3 μM (Fig 4A). Thus, a 3 μM A1 concentration was applied for cell incubation. The resulting cell lysates and media were resolved on a double-antibody sandwich ELISA. A 3 μM U31 treated group and a blank control (without treatment) group served as the controls. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media compared with the control group (Fig 4B–4E). Western blot analysis indicated that sAPPβ expression was significantly decreased in the A1 treated group compared with the control group, whereas no substantial differences in sAPPα and BACE1 expression levels were identified among the groups (Fig 5). These results suggest that A1 can inhibit BACE1 activity in an AD cell model.

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus