Partial BACE1 reduction in a Down syndrome mouse model blocks Alzheimer-related endosomal anomalies and cholinergic neurodegeneration: role of APP-CTF

Abstract

β-amyloid precursor protein (APP) and amyloid beta peptide (Aβ) are strongly implicated in Alzheimer's disease (AD) pathogenesis, although recent evidence has linked APP-βCTF generated by BACE1 (β-APP cleaving enzyme 1) to the development of endocytic abnormalities and cholinergic neurodegeneration in early AD. We show that partial BACE1 genetic reduction prevents these AD-related pathological features in the Ts2 mouse model of Down syndrome. Partially reducing BACE1 by deleting one BACE1 allele blocked development of age-related endosome enlargement in the medial septal nucleus, cerebral cortex, and hippocampus and loss of choline acetyltransferase (ChAT)-positive medial septal nucleus neurons. BACE1 reduction normalized APP-βCTF elevation but did not alter Aβ40 and Aβ42 peptide levels in brain, supporting a critical role in vivo for APP-βCTF in the development of these abnormalities. Although ameliorative effects of BACE1 inhibition on β-amyloidosis and synaptic proteins levels have been previously noted in AD mouse models, our results highlight the additional potential value of BACE1 modulation in therapeutic targeting of endocytic dysfunction and cholinergic neurodegeneration in Down syndrome and AD.

Keywords: APP-βCTF; Alzheimer's disease; BACE1; Basal forebrain cholinergic neurons; Endosomes; Trisomic mice.

Figure 1. Ts2 mice display endosomal abnormalities similar to Ts65Dn mice

Representative neurons labeled with anti-rab5 antibody in the MSN (A-F) and cingulate cortex (G-I) of 9 month old 2N (A, D, G), Ts2 (B, E, H) and Ts65 (C, F, I) mice. rab5-positive early endosomes are enlarged in Ts2 mice (E, H inset) compared to 2N (D, G inset), and resemble those seen in Ts65Dn mice (F, I inset).

Figure 2. Ts2 mice have a degenerative BFCN phenotype similar to Ts65Dn mice

Morphometric analyses of the MSN reveals decreased numbers of ChAT-immunoreactive BFCNs at 9 months of age in representative images of Ts2 mice (B, E) and Ts65Dn mice (C, F) compared to 2N mice (A, D).

Figure 3. Upregulation of APP holoprotein, APP-αCTF, and APP-βCTF in Ts2 and Ts65Dn mice

Representative Western blots (A) and quantitation (B) of full-length APP (APP-FL), APP-αCTF, and APP-βCTF of mouse brain cortex homogenates at 8-9 months of age (n=4 for each genotype). GAPDH is shown as a loading control. Statistical significance was determined by Student's t-test (*, p<0.05; **, p<0.01; ***, p<0.001). Graphs show mean ± SEM.

Figure 4. Ts2 mice develop neuronal endosomal pathology preceding cholinergic neurodegenerative changes

Quantitative analysis revealed that ChAT-positive MSN numbers remain unchanged in 4 month old Ts2 mice compared to 2N mice (Fig. A, B). Representative neurons labeled with anti-rab5 antibody are shown in the MSN (C-F) and cingulate cortex (G, H) 4 month old 2N and Ts2 mice. Rab5-positive early endosomes are enlarged in Ts2 mice (F, H inset) compared to 2N (E, G inset). Increased rab5-positive endosome numbers and sizes are seen in mice from 4 months old to 16 months of age (I, J, n=9-10, for each genotype and age group) (*, p<0.05, **, p<0.01). Graphs show mean ± SEM.

Figure 5. Genetic reduction of BACE1 decreases APP-CTFs, but not Aβ levels in Ts2 mice

Representative Western blots (A) and quantitation (B) reveals decreased expression of BACE1, APP-αCTF, APP-βCTF, and sAPPβ in Ts2.BACE^+/- mouse brain homogenates (n=12 for each genotype) at 8-9 months of age. Elevated levels of Aβ40 and Aβ42 are not reduced by BACE1 reduction in Ts2 mice compared to 2N mice and 2N mice with BACE1 reduction (C; n=6 for each genotype) (*, p<0.05; **, p<0.01; ***, p<0.001).

Figure 6. Genetic reduction of BACE1 prevents neuronal endosomal pathology in Ts2 mice

Representative neurons labeled with anti-rab5 antibody in the MSN (A) and quantitative analysis within the MSN (B, C) and hippocampal CA1 region (HC; D, E) of 9 month old mice (n=8-10 for each genotype). BACE1 genetic reduction in Ts2 mice shows endosome profiles similar to 2N mice, which are significantly reduced compared to Ts2 mice without BACE1 knockdown (*, p<0.05; **, p<0.01; ***, p<0.001).

Figure 7. BACE1 reduction rescues ChAT-immunoreactive BFCNs in the MSN of Ts2 mice

Representative images show double immunofluorescence labeling of rab5-positive endosomes (A) and ChAT (B) in 2N, Ts2 mice and Ts2.BACE1^+/- mice, with restoration of ChAT levels and normalization of rab5 expression by BACE1 partial deletion. DAB staining of ChAT-positive BFCNs in the MSN of 9 month old 2N (C), Ts2 (D) and Ts2.BACE^+/- (E) mice was quantified by stereological analysis (F; n=6-7 for each genotype), which revealed rescue of phenotypic expression by BACE1 reduction in Ts2 mice (**, p<0.01; ***, p<0.001). Graphs show mean ± SEM.