title: Recombinant adenovirus is an appropriate vector for endocytotic protein trafficking studies in cultured neurons.
abstract: Endocytosis of full-length beta-amyloid precursor protein (APP) from the plasma membrane contributes to beta-amyloid peptide (Abeta) secretion, and, hence, potentially contributes to the molecular pathogenesis of Alzheimer's disease. We recently have demonstrated that central neuronal APP is endocytosed in a common vesicular compartment with recycling synaptic vesicle integral membrane proteins, but is then sorted away from synaptic vesicles for retrograde transport to the neuronal soma. For this report, we explore whether recombinant adenovirus can be used to modulate APP expression in cultured central neurons to study APP processing by the endocytotic pathway in these cells. Using a replication-deficient recombinant adenovirus that expresses a lacZ reporter (Ad5/CMV-lacZ), we demonstrate high efficiency of transfection (30-35%) at low viral titer (10-20 MOI), with no significant neuronal toxicity or cytoarchitectural change. In addition, we demonstrate that infection with the control virus does not result in re-direction of endogenous neuronal APP from usual endocytotic pathways. We have prepared, using the same genomic background as the control virus, an adenoviral vector that expresses the neuronal isoform of human APP (Ad5/CMV-APP). Infection with Ad5/CMV-APP at 10-20 MOI results in significantly increased immunoreactivity for endocytosed APP with preservation of usual endocytotic trafficking. These results demonstrate that recombinant adenovirus at low titer is an appropriate and effective vector for protein trafficking/processing studies in cultured central neurons.
authors.affiliation: Department of Pathology, Northwestern University Medical School, Chicago, IL 60611, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yuan
authors.forename: P
authors.initials: P
authors.lastname: Zhai
authors.forename: L M
authors.initials: LM
authors.lastname: Anderson
authors.forename: J
authors.initials: J
authors.lastname: Pan
authors.forename: B
authors.initials: B
authors.lastname: Thimmapaya
authors.forename: E H
authors.initials: EH
authors.lastname: Koo
authors.forename: N R
authors.initials: NR
authors.lastname: Marquez-Sterling
mesh_terms: D000256:Adenoviridae; D000257:Adenoviridae Infections; D000544:Alzheimer Disease / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004705:Endocytosis / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D008854:Microscopy, Electron; D009474:Neurons / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins