title: Knockdown of amyloid precursor protein normalizes cholinergic function in a cell line derived from the cerebral cortex of a trisomy 16 mouse: An animal model of down syndrome.
abstract: We have generated immortal neuronal cell lines from normal and trisomy 16 (Ts16) mice, a model for Down syndrome (DS). Ts16 lines overexpress DS-related genes (App, amyloid precursor protein; Sod1, Cu/Zn superoxide dismutase) and show altered cholinergic function (reduced choline uptake, ChAT expression and fractional choline release after stimulation). As previous evidence has related amyloid to cholinergic dysfunction, we reduced APP expression using specific mRNA antisense sequences in our neuronal cell line named CTb, derived from Ts16 cerebral cortex, compared to a cell line derived from a normal animal, named CNh. After transfection, Western blot studies showed APP expression knockdown in CTb cells of 36% (24 hr), 40.4% (48 hr), and 50.2% (72 hr) compared to CNh. Under these reduced APP levels, we studied 3H-choline uptake in CTb and CNh cells. CTb, as reported previously, expressed reduced choline uptake compared to CNh cells (75%, 90%, and 69% reduction at 1, 2, and 5 min incubation, respectively). At 72 hr of APP knockdown, choline uptake levels were essentially similar in both cell types. Further, fractional release of 3H-choline in response to glutamate, nicotine, and depolarization with KCl showed a progressive increase after APP knockdown, reaching values similar to those of CNh after 72 hr of transfection. The results suggest that APP overexpression in CTb cells contributes to impaired cholinergic function, and that gene knockdown in CTb cells is a relevant tool to study DS-related dysfunction.
authors.affiliation: Program of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile.
authors.forename: Patricia
authors.initials: P
authors.lastname: Opazo
authors.forename: Katherine
authors.initials: K
authors.lastname: Saud
authors.forename: Michelle
authors.initials: M
authors.lastname: de Saint Pierre
authors.forename: Ana María
authors.initials: AM
authors.lastname: Cárdenas
authors.forename: David D
authors.initials: DD
authors.lastname: Allen
authors.forename: Juan
authors.initials: J
authors.lastname: Segura-Aguilar
authors.forename: Raúl
authors.initials: R
authors.lastname: Caviedes
authors.forename: Pablo
authors.initials: P
authors.lastname: Caviedes
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000465:Algorithms; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002540:Cerebral Cortex / Q000502:physiology*; D002772:Cholera Toxin / Q000494:pharmacology; D002794:Choline / Q000378:metabolism; D004314:Down Syndrome / Q000235:genetics* / Q000503:physiopathology*; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D009538:Nicotine / Q000378:metabolism; D010275:Parasympathetic Nervous System / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D016372:RNA, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014314:Trisomy / Q000235:genetics* / Q000503:physiopathology*
chemical_list: D016564:Amyloid beta-Protein Precursor; D016372:RNA, Antisense; D012333:RNA, Messenger; D018698:Glutamic Acid; D011189:Potassium Chloride; D009538:Nicotine; D002772:Cholera Toxin; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D002794:Choline; D000109:Acetylcholine