Reducing Amyloid Plaque Burden via Ex Vivo Gene Delivery of an Aβ-Degrading Protease: A Novel Therapeutic Approach to Alzheimer Disease (original) (raw)
Figure 1
Tissue Distribution of NEP and Generation of Secreted NEP
(A) Identical amounts of protein from transfected CHO cells and the indicated mouse tissues were probed by NEP immunoblot. CHO cells were transiently transfected with empty vector (CHO-control) or human NEP (CHO-NEP). The indicated peripheral tissues and brain regions (Bg/Bs, basal ganglia/brainstem; Cb, cerebellum; Ctx, cortex; Hip, hippocampus) were homogenized and probed by Western blot for NEP. The lower blot is a longer exposure of the brain samples, demonstrating low levels of NEP in the brain. Each sample was either left untreated (−) or deglycosylated with PNGase F (+) to remove N-linked sugars.
(B) Schematic representation of wild-type NEP and sNEP proteins. The sNEP construct was generated by replacing the NEP transmembrane (TM) domain and cytosolic N terminus with a signal peptide (SP) ending at the luminal residue 52 of NEP. Cleavage of the signal peptide produces a soluble, secreted form of NEP.
(C) NEP immunoblot of cellular lysates from CHO cells transfected with empty vector, NEP, or sNEP constructs.
(D) NEP activity assay in which the fluorogenic NEP substrate DAGNPG was incubated with lysates from CHO cells stably transfected with the indicated constructs. NEP enzymatic activity was inhibited by phosphoramidon.
(E) NEP immunoblot of conditioned medium from CHO cells transfected with the indicated constructs.
(F) NEP activity assay on conditioned media from cells transfected with the indicated constructs. NEP activity assays of conditioned media (F) were performed using the same relative amounts of material as for the lysates (D).
Immunoblots are representative of at least three experiments; NEP activity assays report the mean ± SEM of six experiments.