App gene dosage modulates endosomal abnormalities of Alzheimer's disease in a segmental trisomy 16 mouse model of down syndrome

Abstract

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimer's disease (AD) and Down syndrome (DS) brain and has been linked to increased Abeta production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

Figure 1.

Early endosomal enlargement in neurons from Ts65Dn mice. Representative neurons from the brain of a 2-month-old Ts65Dn mouse (B) labeled with rab5, showing the presence of enlarged early endosomes (arrow) as seen in DS (C, arrow) and AD brain. In contrast to trisomic mice, neurons from age-matched 2N control mice exhibit the typical small uniformly sized rab5-positive endosomes (A, arrow).

Figure 2.

Endocytic uptake and recycling are increased in neurons of Ts65Dn mice. Neurons of the cingulate cortex of a 6-month-old Ts65Dn mouse labeled with rab5 (B) and rabaptin5 (D) show increased immunoreactivity (arrows) and enlarged endosomes (D, inset, arrow) compared with an age-matched 2N control mouse (A, C, C, inset, arrows). Serial adjacent sections immunolabeled with rab4 show increased immunoreactivity (F, arrow) in neurons of trisomic mice compared with controls (E, arrow), which is consistent with increased endosomal reflux to the plasma membrane. Western blot analysis (G) of whole-brain homogenates (50 μg per lane) prepared from 2N control mice (n = 4; lanes 1-4) and Ts65Dn mice (n = 4; lanes 5-8) confirm the immunocytochemical findings and revealed an increase in rab4-immunoreactive protein (M_r ∼25-28) in the Ts65Dn mouse brains.

Figure 3.

Endocytic abnormalities target neurons of the septohippocampal system. A, In tissue sections from 2-month-old Ts65Dn mice, endocytic abnormalities are most prominent in neurons of the medial septal nucleus (MSN). Increased levels of rab5 immunoreactivity and atypically large rab5-positive endosomes (right inset, arrows) are detected in most neurons in this region, which is composed primarily of cholinergic neurons that undergo age-related neurodegeneration in Ts65Dn mice. In neurons of the basal ganglia (BG), in contrast, rab5 immunolabeling in neurons is associated with the typical small early endosomal profiles located in close proximity to the cell surface (left inset, arrows). IC, Internal capsule. B, Age-related endosomal alterations represented by enlarged rab5-positive endosomes and elevated levels of rab5 immunolabeling are apparent in neurons of the MSN as well as other regions of the septohippocampal system, including related populations in the neocortex and hippocampus and the NBM.

Figure 4.

App triplication promotes endosomal abnormalities in Ts65Dn mice. Rab5-positive early endosomes in representative basal forebrain neurons from a Ts65Dn mouse with three copies of the App gene (A) are abnormally large (arrow) compared with those in control 2N mice with two copies of App (Fig. 1). Neurons from the same neuronal populations in presenilin (PS)-APP transgenic mice (B) and from two strains of trisomic mice with two copies of App, Ts1Cje (C), and Ts65Dn-App^+/+/- (D) do not exhibit endosomal abnormalities but, like 2N control mice, contained early endosomes of normal size (arrows). Mice transgenic for mutant forms of human APP (E, F) that express two- to sevenfold higher protein levels also show endosomes of normal size implying that App dosage alone does not promote the endocytic response.