Leukocytes were separated from mice 2 months after transplantation with control bone marrow cells, NEP-lentivirus transduced bone marrow cells, or inactive NEPx-lentivirus transduced bone marrow cells, n = 5 for each group. Aβ 1–40 (2 ng/mL) was then incubated with 10⁷ leukocytes from each group at 37°C, and the Aβ remaining measured by Aβ ELISA. Rates were calculated as the difference between the starting amount of Aβ and the amount of Aβ remaining after one hour.
Error bars represent SEM (**p < 0.01 relative to NEPx and the control).

Bone marrow transplantation was conducted on 8-month old 3×Tg-AD mice and Aβ levels were determined 4 months post-transplantation when the mice were 12 months old.
(A). Aβ immunoreactivity in the brain of mice expressing active NEP compared with mice expressing inactive NEPx and control mice. Scale bar, 500µm.
(B). Quantitative analysis of Aβ immunoreactivity from each mouse brain (6 slices per mouse) reveals a ~50% reduction of Aβ deposited in the cortex and hippocampus of NEP expressing mice compared with NEPx expressing mice.
(C). Detergent soluble Aβ levels are decreased in the brain of mice expressing active NEP compared with the levels in mice expressing inactive NEPx and control untreated 3×Tg-AD mice.
(D). Detergent insoluble Aβ levels are unaffected in the brain of mice expressing active NEP compared with the levels in mice expressing inactive NEPx and control untreated 3×Tg-AD mice.
Error bars represent SEM (n = 10 for NEP and NEPx expressing mice, 12 for control mice) (*p < 0.05, **p < 0.01 relative to NEPx or the control).

HEK293T cells were transduced with lentivirus carrying NEP, GFP, or GFP-NEP and subjected to FACS analysis. The NEP signal was visualized with PE-conjugated antibody while the GFP signal was directly visualized. Cells in the lower left quadrant represent cells negative for NEP and GFP, cells in the upper left quadrant are NEP positive cells identified with anti-NEP antibody. Cells in the lower right quadrant are directly visualized GFP positive cells. Cells in the upper right quadrant express both NEP and GFP shown by antibody staining for NEP and direct visualization for GFP.
Note that the intensity of GFP signal is well correlated with the intensity of NEP signal and that virtually all cells show both NEP and GFP signals. The data shown are representative of three independent experiments.

Blood samples were collected 2 months after transplantation of NEP, or NEPx lentivirus transduced bone marrow cells, or untransduced control bone marrow cells and leukocyte counts (A) and their differential (B) determined as described in Materials and methods. No differences were observed among these three groups.
Error bars represent SEM (n = 5).

Bone marrow cells harvested from CD45.1+ donor mice were transduced with lentivirus expressing NEP and transplanted into CD45.1⁻ recipient mice. Peripheral blood samples were obtained from recipient mice 4 weeks after transplantation and tested for cell surface NEP expression by FACS analysis using anti-NEP antisera.
(A). FACS analysis of NEP expression on erythrocytes using fluorescein-conjugated anti-NEP. No NEP positive cells are observed.
The black line represents control mice while the grey line represents mice that received NEP lentivirus transduced bone marrow cells.
(B). FACS analysis of NEP expression on leukocytes using fluorescein-conjugated anti-NEP shows 25–40% NEP-positive cells. The lines are as described in A.
(C). Dual-color FACS analysis of CD45.1 and NEP expression on leukocytes after bone marrow transplantation. Cells were screened for CD45.1 antigen or NEP as described in methods. Cells in the upper two quadrants represent cells expressing CD45.1 (~50%), while cells in the right two quadrants represent cells expressing NEP. Those cells in the upper right quadrant (~27%) express both CD45.1 and NEP. Thus ~50% of the recipient mouse’s leukocytes express the CD45.1 antigen, and of these CD45.1⁺ cells ~50% express NEP. In repeated experiments we found that 30–60% of the recipient mice’s leukocytes express the CD45.1 antigen, and 50–80% of the CD45.1⁺ transplanted cells expressed NEP.  The data shown are representative of one mouse from 18 mice involved in three independent experiments.

Bone marrow transplantation was conducted on 2-month old 3×Tg-AD mice and Aβ levels were determined 6 months post-transplantation when the mice were 8 months old.
(A). Aβ immunoreactivity in brain sections of mice expressing active NEP compared with mice expressing inactive NEPx and control untreated 3×Tg-AD mice. Scale bar, 500 µm.
(B). Quantitative analysis of Aβ immunoreactivity from 6 slices of each mouse brain reveals a ~60% reduction of Aβ deposits in the cortex and hippocampus of NEP expressing mice compared with NEPx expressing mice.
(C). Detergent soluble Aβ levels are decreased in the brain of mice expressing active NEP compared with the levels in mice expressing inactive NEPx and control untreated 3×Tg-AD mice.
Error bars represent SEM (n = 8) (** p < 0.01 relative to NEPx or the control).

(A and B). Spatial learning phase of the Morris Water Maze assay that includes 4 days of training with the submerged platform. 3×Tg-AD mice expressing active NEP showed significantly decreased latency (A) as well as distance (B) traveled to the platform compared with 3×Tg-AD mice expressing inactive NEPx or control untreated 3×Tg-AD mice.
(C)(D). Memory portion of the Morris Water Maze assay with the platform removed to evaluate the time of animals spent in the target quadrant (C) and entries into target quadrant (D) where the platform was located. 3×Tg-AD mice expressing active NEP showed significantly increased time mice spent in target quadrant (C) and the number of target quadrant entries (D) compared with 3×Tg-AD mice expressing inactive NEPx or untreated 3×Tg-AD control mice. Horizontal line at 7.5 seconds represents random for a 30 second probe test.
Error bars represent SEM (n = 10 for NEP and NEPx expressing mice and nonTg mice, 12 for control mice) (** p < 0.01 relative to NEPx or the control).

Extracts were prepared from mouse brain following transplantation with NEP or NEPx lentivirus transduced bone marrow cells, or untransduced control bone marrow cells. NEP activity was determined as described in Materials and methods. No statistically significant differences are observed in the specific activities of NEP from brains of mice expressing active NEP or inactive NEPx and control 3×Tg-AD mice.
Error bars represent SEM (n = 5).

Systolic blood pressure in 3×Tg-AD mice was determined as described in Materials and methods one month following transplantation with NEP, or NEPx lentivirus transduced bone marrow cells, or untransduced control bone marrow cells. As shown peripheral NEP expression does not alter systolic blood pressure. Data are the mean ± SEM of weekly measurements (n = 8).