Neprilysin gene transfer reduces human amyloid pathology in transgenic mice

Abstract

The degenerative process of Alzheimer's disease is linked to a shift in the balance between amyloid-beta (Abeta) production, clearance, and degradation. Neprilysin has recently been implicated as a major extracellular Abeta degrading enzyme in the brain. However, there has been no direct demonstration that neprilysin antagonizes the deposition of amyloid-beta in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in transgenic mouse models of amyloidosis. We show that unilateral intracerebral injection of Lenti-Nep reduced amyloid-beta deposits by half relative to the untreated side. Furthermore, Lenti-Nep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice. These data further support a role for neprilysin in regulating cerebral amyloid deposition and suggest that gene transfer approaches might have potential for the development of alternative therapies for Alzheimer's disease.

Fig. 1.

a, Vector design showing the neprilysin, inactive neprilysin (NepX), and green fluorescent protein (GFP) expressing lentiviral constructs. The internal promoters driving the transgenes are indicated by arrows (human CMV). LTR sequences are shown on the ends (packaging signal, Ψ). b, 293T cells transduced with Lenti-Nep (10 nl per well, or 0.015 pg p24 gag-antigen per cell) were immunostained for neprilysin expression and analyzed by flow cytometry.c, Immunoblot on 293T cells and differentiated rat neural progenitors (HCN) transduced with the Lenti-Nep or Lenti-GFP vector. d, 293T cells transduced with serial dilutions of Lenti-Nep could degrade Aβ_1–42 in a dose-dependent manner. Lenti-GFP transduced cells were used as a negative control (GFP), whereas medium alone was used to determine the baseline (Medium). e, Differentiated neural progenitors (HCN) infected with Lenti-Nep (Nep; n = 2), Lenti-NepX (NepX; n = 2) or Lenti-GFP (GFP; n = 2) were used to degrade Aβ_1–42 (as above). Values are presented as the percentage of the average value from the GFP samples. Thiorphan was also used as a negative control (Nep + Thior.;n = 1).

Fig. 2.

Expression of neprilysin in mouse brains.a, Detection of human neprilysin expression by immunoblot. Lenti-GFP and saline injections were used as negative controls. The quantity of total protein extracted from mouse brains was similar among all samples (data not shown). b, Detection of human neprilysin by immunohistochemistry (Lenti-Nep, top panels, arrows indicate examples of Nep-positive cells; Lenti-GFP, bottom panels, APPtg, J9M; Nontg, nontransgenic mouse; nep, Lenti-Nep injected; vector, Lenti-GFP injected).

Fig. 3.

Reduced amyloid plaque formation in mice injected with Lenti-Nep. Shown are hippocampal sections from a representative transgenic-APP mouse (TASD41) injected with Lenti-Nep into the hippocampus and frontal cortex (right side injected: A,C, D, G, H; contralateral side: B, E,F, I, J) (amyloid = green). Double immunolabeling for neprilysin and amyloid (G–J) (nep = red).

Fig. 4.

Comparison of plaque burdens in Lenti-Nep and control-treated transgenic mice. a, Mean absolute values for amyloid staining in Lenti-Nep (Neprilysin) and control-treated animals on the contralateral and ipsilateral hemispheres (*p = 0.0007;^#p = 0.06; t test). Error bars represent SE. b, Relative ratios of ipsilateral (injected) versus contralateral (noninjected) plaque burdens. Nep, Lenti-Nep injected; GFP, Lenti-GFP injected; NepX, Lenti-NepX injected;INep, heat inactivated Lenti-Nep injected;Saline, saline injected. c, Scatter plot showing individual and the average relative values of the neprilysin (n = 12) and control (n = 14) groups; p = 0.000001 (t test) (TASD41: red squares, p = 0.01; J9M:blue circles, p = 0.004;t test). Error bars represent SD.