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In vitro AAV5‐mediated expression of metalloendopeptidase neurolysin in mouse brain primary cultures
Author(s) -
Wangler Naomi J.,
Sumners Colin,
Speth Robert C.,
Karamyan Vardan T.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.690.7
Subject(s) - in vitro , blot , microbiology and biotechnology , cytoplasm , biology , chemistry , biochemistry , gene
The aim of this study was to characterize a newly developed adeno‐associated virus serotype 5 (AAV5) vector encoding mouse neurolysin in primary cultures of neurons and astroglia from mouse brains. Cortical neurons and astroglia were prepared from E16 embryos and 1‐day‐old CD‐1 mice, respectively. Neurons were transduced on in vitro day (ivd) 7, and astroglial cells on ivd 21 using AAV5‐neurolysin or control AAV5‐eGFP vectors with chicken β‐actin promoter at a concentration of 1.8 × 10 11 genome copies per 100 mm dish. Activity of neurolysin (sensitive to inhibition by 10 mM Pro‐Ile) in cytosolic and membrane fractions was determined two weeks after transduction by cleavage of a specific quenched fluorogenic substrate. Relative quantity of neurolysin in the same samples was determined by Western Blotting using a specific antibody. In neuronal membranes, neurolysin activity and protein were significantly upregulated: 50% increase in activity, and 40% increase in density. Cytoplasmic increases in neurolysin activity and protein were 198% and 248%, respectively. In contrast, neurolysin activity and protein amount in astroglial membranes and cytoplasm showed a marginal, non‐significant increase. These results demonstrate the ability of newly developed vector to transduce functional neurolysin in neurons making it an excellent tool to study (patho)physiological function of this peptidase.