Knock-down of protein l-isoaspartyl o-methyltransferase increases β-amyloid production by decreasing adam10 and adam17 levels

ABSTRACT AIM: To examine the role of protein _L_-isoaspartyl _O_-methyltransferase (PIMT; EC 2.1.1.77) on the secretion of Aβ peptides. METHODS: HEK293 APPsw cells were treated with PIMT

siRNA or adenosine dialdehyde (AdOX), a broad-spectrum methyltransferase inhibitor. Under the conditions, the level of Aβ secretion and regulatory mechanism by PIMT were examined. RESULTS:

Knock-down of PIMT and treatment with AdOX significantly increased Aβ40 secretion. Reductions in levels of PIMT decreased the secretion of soluble amyloid precursor protein alpha (sAPPα)

without altering the total expression of APP or its membrane-bound C83 fragment. However, the levels of the C99 fragment generated by β-secretase were enhanced. Moreover, the decreased

secretion of sAPPα resulting from PIMT knock-down seemed to be linked with the suppression of the expression of α-secretase gene products, α-disintegrin and metalloprotease 10 (ADAM10) and

ADAM17, as indicated by Western blot analysis. In contrast, ADAM10 was not down-regulated in response to treatment with the protein arginine methyltransferase (PRMT) inhibitor, AMI-1.

CONCLUSION: This study demonstrates a novel role for PIMT, but not PRMT, as a negative regulator of Aβ peptide formation and a potential protective factor in the pathogenesis of AD. SIMILAR

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May 2024 INTRODUCTION Alzheimer's disease (AD), the most common neurodegenerative disease, is characterized by progressive memory loss and other cognitive impairments1, 2.

Neuropathological hallmarks of AD include the deposition of amyloid beta (Aβ) peptides, which are organized in senile plaques. In addition, AD is characterized by the accumulation of

phosphorylated tau proteins, which are arranged in neurofibrillary tangles (NFTs)2. Aβ peptides are generated through the proteolysis of the amyloid precursor protein (APP). In the

amyloidogenic pathway, β-secretase cleaves APP to produce soluble amyloid precursor protein beta sAPPβ and a C99 fragment. Membrane-bound C99 can be further processed by γ-secretase to

produce Aβ peptides3, 4. As an alternative, non-amyloidogenic pathway, α-secretase can cleave within the Aβ region to produce a sAPPα fragment and a C83 fragment4. High levels of

homocysteine (HCY) can lead to increased concentrations of _S_-adenosylhomocysteine (SAH), a strong methyltransferase inhibitor5. This up-regulation of SAH results in an overall decrease in

the activity of _S_-adenosylmethionine (SAM)-dependent methyltransferases. Increased SAH levels in the brain tissue of patients with AD has been associated with the inhibition of

catechol-_O_-methyltransferase (COMT) and phenylethanolamine-_N_-methyltransferase (PNMT), two enzymes that are widely distributed throughout the human brain6. Treatment of Neuro-2a

neuroblastoma cells with SAH has been shown to inhibit protein phosphatase 2A methyltransferase (PPMT), resulting in decreased methylation of protein phosphatase 2A7. SAH treatment has also

been associated with the increased accumulation of APP and phosphorylated tau and with increased Aβ secretion8. Protein _L_-isoaspartyl methylation is also essential for the maintenance of

neural activity in the central nervous system (CNS). Deficiency of protein _L_-isoaspartyl _O_-methyltransferase (PIMT, EC2.1.1.77), an enzyme that catalyzes the transfer of an active methyl

group from SAM to _L_-isoaspartate and _D_-isoaspartate, leads to fatal progressive epileptic disease9. Alterations in the SAM/SAH ratio, which is relevant to the overall excitatory state

of neurons, have been reported in PIMT-deficient mice10. Previous studies have identified deposits of Aβ peptides with isoaspartates in brain tissue isolated from AD patients and PIMT

knock-out mice, suggesting a potential pathophysiological role in progressive neurodegeneration10, 11. In patients with AD, PIMT is up-regulated in degenerating neurons and is localized in

NFTs10. Despite the increasing evidence supporting a role for PIMT in neurodegeneration, the mechanism by which PIMT modulates Aβ peptide generation in AD pathogenesis remains unclear. To

uncover the mechanism whereby PIMT exerts its effects, we examined the ability of PIMT to regulate Aβ secretion _in vitro_. MATERIALS AND METHODS ANTIBODIES AND REAGENTS Adenosine dialdehyde

(AdOX), SAM, and mouse anti-β-actin antibodies were purchased from Sigma-Aldrich Chemicals (St Louis, MO, USA). AMI-1 was obtained from Calbiochem (La Jolla, CA, USA). Dulbecco's

modified Eagle's medium (DMEM), Opti-MEM, Dulbecco's phosphate buffered saline (DPBS), penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad,

CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Calbiochem (La Jolla, CA, USA). Moloney Murine Leukemia virus (M-MLV) reverse transcriptase and

polymerase chain reaction (PCR) premix was purchased from Rexgene Biotech Co, Ltd (Ochang, Korea). All of the primers used for PCR were purchased from Bioneer (Daejeon, Korea). A mixture of

Stealth™ /siRNA duplex oligoribonucleotides against PIMT and Lipofectamine™ RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). Monoclonal mouse anti-APP (6E10) antibody was

obtained from Signet Laboratories (Dedham, MA, USA). Polyclonal rabbit antibodies to ADAM9, ADAM10, and ADAM17 were obtained from Chemicon International (Temecula, CA, USA). Monoclonal mouse

BACE1, monoclonal anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies, and anti-rabbit HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology

(Santa Cruz, CA, USA). Rabbit anti-PIMT antisera was produced against recombinant porcine PIMT proteins as described12. The Genbank nucleotide sequence database accession number of the

nucleotide sequence of the clone is AF239700. CELL CULTURE, DRUG TREATMENT, AND PROTEIN PREPARATION HEK293 APPsw and SH-SY5Y cells were plated on 100-mm culture dishes (Corning Incorporated,

Corning, NY, USA). The dishes were filled with DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. The cultures were maintained at 37 °C with 5% CO2 under

humidified conditions. Cells were treated with drugs (SAM, AdOX, and AMI-1) for indicated times. Vivaspin20 centrifugal filter devices (Satorius, Goettingen, Germany) were used to

concentrate conditioned media (CM) from HEK293 APPsw and SH-SY5Y cells collected after drug or siRNA treatments. Cells were washed with DPBS and lysed in Pro-Prep™ protein extraction buffer

for 20 min on ice. The protein concentration of each sample was quantified using a Bradford assay (Bio-Rad, Hercules, CA, USA). CELL VIABILITY ASSAY To determine cell viability, cells were

plated on 96-well plates at a density of 2×104 cells per well. The original media was then replaced with media containing MTT at a final concentration of 0.5 mg/mL13. Four hours later, the

medium was discarded, and DMSO was added for the colorimetric assay. Absorption values were determined using an _E_max microplate reader from Molecular Devices (Union City, CA, USA) with a

540-nm filter. REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION RNA was isolated from HEK293 APPsw cells treated with or without PIMT siRNA using TRIzol reagent (Gibco BRL) according to the

manufacturer's instructions. For each RT-PCR reaction, 1 μg of RNA was used. Each sample was pre-heated to 60 °C with oligo (dT)18 primers for 10 min. One unit per milliliter of M-MLV

reverse transcriptase was added. The reaction was then performed at 37 °C for 60 min with the following primers: PIMT, forward 5′-TCAGGAAGGACGATCCAACA-3′, reverse 5′-TCCTCCGGGCTTTAACTGAT-3′;

and GAPDH, forward 5′-AAGGGTCATCATCTCTGCCC-3′, reverse 5′-GTGATGGCATGGACTGTGGT-3′. Amplification was carried out for 20 to 30 cycles with the following parameters: 94 °C for 30 s, 55–57 °C

for 40 s, and 72 °C for 30 s. These steps were followed by a final 5 min extension step at 72 °C. SIRNA TRANSFECTION To conduct the PIMT siRNA transfection, 500 000 cells were seeded onto

100-mm culture plates. Cells were cultured for 48 h at 37 °C in culture medium containing serum, which allowed the cells to be approximately 80% confluent. Immediately prior to transfection,

lipofectamine RNAiMAX was incubated with the siRNA of interest in OPTI-MEM (Gibco) at room temperature for 10 min. The cells were then incubated in this mixture for 48 h at 37 °C in fresh

medium containing serum. IMMUNOBLOTTING Twenty micrograms of protein mixed with 5×loading buffer [0.313 mol/L Tris-HCl (pH 6.8), 10% SDS, 0.05% bromophenol blue, 50% glycerol], and

20×reducing agent (2 mol/L DTT: Fermentas, Hanover, MD, USA) were boiled for 5 min and loaded onto a 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to a

polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in 20 mmol/L Tris-HCl (pH 7.4) containing 150 mmol/L NaCl and 0.1%

Tween 20 (TBS-T). They were then incubated overnight at 4 °C with primary antibodies (1:2000 for 6E10, 1:1000 for 22C11, 1:3000 for β-actin, 1:2000 for ADAM10, 1:1000 for BACE1, 1:3000 for

PIMT) in non-fat milk. The membranes were washed for 10 min in TBS-T and then incubated for 2 h in non-fat milk at room temperature with horseradish peroxidase-conjugated anti-mouse/rabbit

secondary antibodies. Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Bioscience, Pittsburgh, PA, USA). CELL SURFACE BIOTINYLATION HEK 293 APPsw

cells were surface biotinylated by incubation with 2 mg/mL Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) in ice-cold PBS. After 30 min, the cells were washed and quenched with PBS

containing 100 mmol/L glycine. Cells were lysed in 1% NP-40 buffer and incubated with Neutravidin™ immobilized onto 6% cross-linked beaded agarose (Pierce). The beads were washed in NP-40

buffer, boiled in sample buffer, separated using SDS-PAGE, and immunoblotted with the indicated antibodies. AΒ40 ELISA ASSAY A variety of Aβ peptides, ranging from 38 to 43 amino acids in

length, have been shown to be secreted in response to γ-secretase activation14, 15. Aβ42 is the peptide most widely implicated in AD pathogenesis16; however, the antibody for Aβ40 was

selected based on its reproducibility and accuracy in the Aβ40 ELISA kit. The CM was cleared of debris, and the secreted Aβ40 was measured using a sandwich ELISA kit (Signet Laboratory,

Dedham, MA, USA) according to the manufacturer's instructions. STATISTICAL ANALYSIS Quantitative analysis of Western blotting was performed by calculating the relative density of

immunoreactive bands. The data are expressed as a percentage of the control values. Data are presented as the mean±SD. Each procedure was performed in three to five independent experiments.

A Student's _t_-test analysis was used to evaluate statistical significance. RESULTS PIMT SIRNA AND ADOX INDUCE AΒ SECRETION IN HEK293 APPSW CELLS As shown in Figure 1A and 1B, PIMT

siRNA transfection and AdOX treatment increased the secretion of Aβ40 in HEK293 APPsw cells approximately two fold. Compared to controls, PIMT siRNA induced a 35.6%±8.0% reduction in mRNA

and protein levels in HEK293 APPsw cells 48 h after transfection (Figure 1C). Importantly, the concentration of AdOX used was not cytotoxic (Figure 1D). PIMT SIRNA TRANSFECTION AND ADOX

DECREASES SAPPΑ SECRETION AND INCREASES C99 IN HEK293 APPSW CELLS To understand the molecular mechanism of PIMT siRNA-mediated Aβ40 secretion, we evaluated whether PIMT played a role in the

processing of APP. To do this, we measured the levels of the APP cleavage products: sAPPα, C99, and C83. Western blotting for sAPPα in the CM revealed that PIMT siRNA decreased the secretion

of sAPPα by 67.4%±3.4%. However, the overall expression of total APP and biotin-labeled membrane APP remained unchanged (Figure 2A), suggesting that the amount of sAPPβ, a critical

component for secretion of Aβ40, might be increased. Indeed, the C99 fragment, the cleavage product of β-secretase17, was increased in PIMT siRNA-treated cells. At the same time, C83 was

decreased in PIMT transfected cells (Figure 2B). Similar patterns of sAPPα and total APP were also observed in the AdOX treatment group (Figure 2C, upper panel). In contrast, the induction

of transmethylation with 100 μmol/L SAM, a methyl donor5, increased sAPPα in both cell types (Figure 2C lower panel), demonstrating a critical role of transmethylation in APP processing.

PIMT SIRNA TRANSFECTION DECREASES ADAM10 AND ADAM17 EXPRESSION A decrease in sAPPα without a corresponding reduction in total APP expression suggests a potential alteration in the expression

or activity of APP processing enzymes. To investigate whether PIMT siRNA altered the expression of APP processing enzymes, we measured the expression levels of several α-secretase

candidates: ADAM9, ADAM10, and ADAM17; and the β-secretase candidate, BACE1. As shown in Figure 3A, PIMT siRNA reduced the expression of both the mature and the immature forms of ADAM10 by

45% to 55% compared to controls. In contrast, PIMT siRNA did not significantly alter the levels of BACE1 (90.4%±14% compared to control). PIMT siRNA treatment down-regulated the total

protein levels of ADAM17, but not ADAM9 (Figure 3B). In agreement with these data, AdOX, but not AMI-1, a PRMT inhibitor, also strongly reduced ADAM10 levels (Figure 3C). These results

suggest that PIMT, but not PRMT, selectively modulates the protein levels of ADAM10 and ADAM17. DISCUSSION In this study, we used HEK293 APPsw cells and SH-SY5Y cells to investigate the

effects of protein _L_-isoaspartyl methylation on APP processing. HEK293 APPsw cells express high levels of Aβ18, and SH-SY5Y human neuroblastoma cells express considerable levels of APP and

secrete non-toxic, non-amyloidogenic sAPP17. Because of this, these cell lines have been widely used to study the regulation of APP processing related to the pathogenesis of AD19.

Therefore, we used these cells in our study to examine the regulatory role of transmethylation on APP processing. Interestingly, treatment of either cell type with PIMT siRNA and AdOX, a

well-known inhibitor of transmethylation20, remarkably induced the release of Aβ40 peptides (Figure 1), indicating the involvement of PIMT-mediated methylation in APP cleavage. Because

numerous papers have shown that the secretion of Aβ40 is accompanied by the release of additional γ-secretase-generated Aβ peptides, such as Aβ38, Aβ42, and Aβ43, it is likely that the

production of these peptides would also be regulated by PIMT siRNA treatment. To investigate the molecular mechanism underlying this phenomenon, the levels of the enzyme that generate Aβ40

peptides were first determined. Figures 2 and 3 reveal that PIMT knock-down modulates both the secretion and cleavage of sAPPα. In response to PIMT siRNA treatment, sAPPα secretion was

dramatically diminished, but the levels of total APP and membrane-bound biotinylated APP remained unchanged (Figure 2A). These results suggest that sAPPβ might be relatively enhanced by PIMT

siRNA. The C99 fragment, a β-secretase cleavage product of APP, was increased by PIMT knock-down, whereas C83 was dramatically diminished (Figure 2B). Overall, our results suggest that the

PIMT-mediated Aβ40 production pathway might be primarily associated with the sAPPα cleavage pathway. It has been reported that green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG)

exerts a beneficial role in reducing brain Aβ levels by promoting the cleavage of the C99 fragment of APP. The corresponding elevation of sAPPα21 and G-protein coupled signaling, a major

excitatory signal transduction pathway in neuronal cells, is known to activate a sAPPα generation pathway22. Therefore, PIMT knock-down could contribute to the down-regulation of sAPPα

during Aβ40 production. The improper production of APP isoforms or aberrant APP trafficking during AD pathogenesis is believed to favor the amyloidogenic pathway23. In addition, recent

reports have shown that Aβ production is influenced more by the location of APP cleavage than the total amount of secretase present within the cell24. However, in our study, neither the

total amount of APP nor the amount of membrane-associated APP (Figure 2A) was altered in response to PIMT siRNA treatment. These results imply that PIMT does not regulate the trafficking of

APP to the cell membrane or its synthesis. Instead, the protein levels of Aβ40 generating enzymes (Figure 3) clearly reveal an involvement of proteolytic processing in the observed decrease

in sAPPα and increase in Aβ40 peptides in response to PIMT siRNA. Indeed, the expression of the α-secretase gene products, α-disintegrin and metalloprotease 10 (ADAM10) and ADAM1721, was

reduced after PIMT siRNA transfection in HEK293 APPsw cells according to Western blot analysis (Figure 3). The facts that BACE1 expression was not altered (Figure 3A) and that the PRMT

inhibitor, AMI-1, did not affect ADAM10 levels (Figure 3C) seem to highlight the specificity of this pathway leading to Aβ40 generation. However, we cannot exclude the possibility that PIMT

knock-down leads to the direct activation of BACE1 despite not affecting its expression level. Indeed, previous work has shown that BACE1-inducible cells exhibit increased production of Aβ40

peptides25. To date, there is no experimental evidence suggesting that PIMT can regulate the enzyme activity of BACE1. However, several studies have reported that BACE1 can be modified by

S-palmitoylation26 and ubiquitination27, indicating the importance of post-translational modifications of BACE1. Future studies will determine whether PIMT-induced methylation of BACE1 at

aspartyl residues increases its enzyme activity. In conclusion, we have demonstrated that knock-down of PIMT increased Aβ production via the inhibition of the non-amyloidogenic α-secretase

pathway, an effect that is linked to a decrease in sAPPα and ADAM10/17 levels as summarized in Figure 4. Therefore, our study suggests a novel protective role for PIMT in the pathogenesis of

AD as a negative regulator of Aβ40 peptide formation. AUTHOR CONTRIBUTION Narkhyun BAE, Jae Youl CHO, and Sungyoul HONG designed research; Narkhyun BAE, Se Eun BYEON, Jihyuk SONG, and

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Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture,

Forestry and Fisheries, Republic of Korea. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Genetic Engineering, Sungkyunkwan University, 440–746, Suwon, Republic of Korea

Narkhyun Bae, Se Eun Byeon, Jihyuk Song, Sang-Jin Lee, Moosik Kwon, Jae Youl Cho & Sungyoul Hong * Department of Biochemistry and Cancer Research Institute, Seoul National University

College of Medicine, Seoul, 110–799, Republic of Korea Inhee Mook-Jung Authors * Narkhyun Bae View author publications You can also search for this author inPubMed Google Scholar * Se Eun

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PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bae, N., Byeon, S., Song, J. _et al._ Knock-down of protein _L_-isoaspartyl _O_-methyltransferase increases

β-amyloid production by decreasing ADAM10 and ADAM17 levels. _Acta Pharmacol Sin_ 32, 288–294 (2011). https://doi.org/10.1038/aps.2010.228 Download citation * Received: 26 September 2010 *

Accepted: 17 December 2010 * Published: 04 March 2011 * Issue Date: March 2011 * DOI: https://doi.org/10.1038/aps.2010.228 SHARE THIS ARTICLE Anyone you share the following link with will be

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initiative KEYWORDS * Alzheimer's disease * β-amyloid protein * _L_-isoaspartyl _O_-methyltransferase * soluble amyloid precursor protein alpha * ADAM10 * ADAM17