Reducing amyloid plaque burden via ex vivo gene delivery of an Abeta-degrading protease: a novel therapeutic approach to Alzheimer disease

Abstract

Background: Understanding the mechanisms of amyloid-beta protein (Abeta) production and clearance in the brain has been essential to elucidating the etiology of Alzheimer disease (AD). Chronically decreasing brain Abeta levels is an emerging therapeutic approach for AD, but no such disease-modifying agents have achieved clinical validation. Certain proteases are responsible for the catabolism of brain Abeta in vivo, and some experimental evidence suggests they could be used as therapeutic tools to reduce Abeta levels in AD. The objective of this study was to determine if enhancing the clearance of Abeta in the brain by ex vivo gene delivery of an Abeta-degrading protease can reduce amyloid plaque burden.

Methods and findings: We generated a secreted form of the Abeta-degrading protease neprilysin, which significantly lowers the levels of naturally secreted Abeta in cell culture. We then used an ex vivo gene delivery approach utilizing primary fibroblasts to introduce this soluble protease into the brains of beta-amyloid precursor protein (APP) transgenic mice with advanced plaque deposition. Brain examination after cell implantation revealed robust clearance of plaques at the site of engraftment (72% reduction, p = 0.0269), as well as significant reductions in plaque burden in both the medial and lateral hippocampus distal to the implantation site (34% reduction, p = 0.0020; and 55% reduction, p = 0.0081, respectively).

Conclusions: Ex vivo gene delivery of an Abeta-degrading protease reduces amyloid plaque burden in transgenic mice expressing human APP. These results support the use of Abeta-degrading proteases as a means to therapeutically lower Abeta levels and encourage further exploration of ex vivo gene delivery for the treatment of Alzheimer disease.

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.

Figure 2. Clearance of the Aβ Peptide by NEP and sNEP

(A) CHO cells overexpressing APP were cocultured with CHO cells stably expressing either empty vector, NEP, or sNEP constructs. Equal numbers of cells were seeded, grown to confluence, and media were conditioned for 18 h. Conditioned media were analyzed for total Aβ levels by ELISA. (B) Cellular lysates from the coculture in (A) were probed by Western blotting for APP (which presents in both mature and immature glycosylated forms), demonstrating equal amounts of APP expression. Each condition is shown in duplicate. (C) Conditioned media from CHO cells stably transfected with APP were incubated in vitro with conditioned media from CHO cells expressing empty vector, NEP, or sNEP. The conditioned media were combined and incubated at 37 °C for 18 h. Aβ levels were determined by ELISA. The immunoblot for APP (B) is representative of four experiments; ELISA values for Aβ represent the mean ± SEM of five experiments. For comparisons to the empty vector condition, ** p < 0.01 and *** p < 0.001.

Figure 3. Characterization of a sNEP Lentiviral Construct

(A) Schematic representation of the lentiviral constructs expressing sNEP and GFP. Blocks indicate lentiviral genetic components. Internal promoters and transgenes are indicated by arrows. (B) HEK cells stably transfected with APP₆₉₅ K595N/M596L (HEKAPP₆₉₅) and CHO cells stably transfected with APP₇₅₁ V717F (CHOAPP₇₅₁) were transduced with the lentiviral constructs in (A). Lysates and conditioned media (CM) were collected and probed for sNEP, GFP, APP, and Aβ. (C) Media from the lentivirally transduced cells were conditioned, and Aβ levels were determined by ELISA. Immunoblots are representative of four experiments; Aβ ELISAs represent the mean ± SEM of four experiments, compared to GFP transduced cells: *** p < 0.001

Figure 4. Generation of Primary Mouse Fibroblasts Expressing GFP and sNEP

Fibroblast cultures were generated from wild-type littermates of J20 APP transgenic mice. (A) Live-cell fluorescent imaging of primary fibroblasts transduced with lentiviral vectors expressing GFP (top) or sNEP (bottom). (B) Conditioned media from the primary fibroblasts were probed for the presence of sNEP. The immunoblot is representative of three experiments.

Figure 5. Reduction in Hippocampal Plaque Burden following sNEP Cell Engraftment

Aged J20 APP transgenic mice were implanted with fibroblasts expressing either sNEP (n = 5) (A, C, E, and G) or GFP (n = 3) (B, D, F, and H). Cells were stereotaxically placed into the right (ipsilateral, right) hippocampus, with the uninjected left (contralateral, left) hippocampus serving as control. Brains were harvested for analysis 28 d after surgery. Grafted cells were immunoreactive for either NEP (A) or GFP (B), with superior and inferior aspects of the graft, respectively, indicated by arrows. Images (C–H) show staining for plaque burden at sites medial to the graft (C and D), at the site of the graft (E and F), and at sites lateral to the graft (G and H). These fields were quantified by calculating the ratio of the area covered by plaques on the ipsilateral versus the contralateral side; quantification was done in a blinded fashion. This ratio was determined separately for the sNEP and GFP groups and compared at the hippocampus medial to the graft (I), the graft site (J), and the hippocampus lateral to the graft (K). Data represent the ratio's mean ± SEM, compared to GFP control: ** p = 0.0020 for the medial hippocampus (I); * p = 0.0269 for the graft site (J); and ** p = 0.0081 for the lateral hippocampus (K).

Figure 6. Reduction in Hippocampal Thioflavin Staining Following sNEP Cell Engraftment

Brain sections from mice engrafted with cells expressing sNEP or GFP were stained with thioflavin-S to quantify fibrillar plaques. Photomicrographs (A–D) show thioflavin staining at sites medial to the graft (A and B) and at the site of the graft (C and D). Thioflavin-positive plaques were counted, and the ratios of plaques on the ipsilateral versus contralateral hemispheres were calculated for sNEP and GFP groups for the medial hippocampus (E), the graft site (F), and the lateral hippocampus (G). Data represent the mean ± SEM, compared to GFP control: *p = 0.0432 for the medial hippocampus (E), and *p = 0.0337 for the graft site (F). The difference at the lateral hippocampus was not significant.

Figure 7. No Change in Overall Astrocytosis following Cell Implantation

Brain sections from engrafted mice were stained for GFAP to probe for changes in astrocytosis. (A and B) Compared to the contralateral hemisphere (A), the graft site (B) demonstrated an absence of GFAP staining within the graft and modestly increased astrocyte staining along the border of the graft. The needle track is indicated by arrows and the graft by an asterisk. This staining pattern was observed for both sNEP and GFP conditions (only sNEP is shown). (C–E) The area staining for GFAP on the ipsilateral versus contralateral hippocampus was determined for the medial hippocampus (C), the graft site (D), and the lateral hippocampus (E). Data represent the mean ± SEM.