TY - JOUR
T1 - A cellular model of amyloid precursor protein processing and amyloid-β peptide production
AU - Macias, Mi Mi P.
AU - Gonzales, Amanda M.
AU - Siniard, Ashley L.
AU - Walker, Aaron W.
AU - Corneveaux, Jason J.
AU - Huentelman, Matthew J.
AU - Sabbagh, Marwan N.
AU - Decourt, Boris
N1 - Funding Information:
Financial support for the conduct of the research was provided by the National Institute on Aging grants R01AG034155 , P30AG019610-09 , and 5P30AG019610-12 , in part by NIH-NINDS grant R01NS059873 , and by grants from the Arizona Alzheimer's Consortium and Department of Health Services (AARC MaciasBBB DHSFYE14/MatchFYE14), and from the Alzheimer's Association ( NIRG-12-237512 ). The funding sources had no involvement in study design, collection, analysis, and interpretation of data, or writing or submission of the report.
Funding Information:
This study was supported by the National Institute on Aging grants R01AG034155 , P30AG019610-09 , and 5P30AG019610-12 , in part by NIH-NINDS grant R01NS059873 , and by a grant from the Alzheimer's Association ( NIRG-12-237512 ). We thank Chera Maarouf for SIG39152 MAb and helpful discussions, Jennifer Nolz for gift of RNA ladder, Diego Mastroeni and Andy Grover for discussion of SK-N-BE(2) and gift of 100 mM RA.
PY - 2014/2/15
Y1 - 2014/2/15
N2 - Background: A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration. New method: We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aβ production, and (2) high-throughput screening assays of new pharmacotherapeutics. Results: In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. Comparison with existing method(s): In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. Conclusions: Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.
AB - Background: A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration. New method: We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aβ production, and (2) high-throughput screening assays of new pharmacotherapeutics. Results: In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. Comparison with existing method(s): In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. Conclusions: Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.
KW - Alzheimer's
KW - Amyloid-beta
KW - BE(2)-M17
KW - In vitro
KW - Secretase
KW - TNF
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U2 - 10.1016/j.jneumeth.2013.11.024
DO - 10.1016/j.jneumeth.2013.11.024
M3 - Article
C2 - 24333289
AN - SCOPUS:84891362775
SN - 0165-0270
VL - 223
SP - 114
EP - 122
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
ER -