High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation

Abstract

Amyloid plaques are a neuropathological hallmark of Alzheimer's disease (AD), but their relationship to neurodegeneration and dementia remains controversial. In contrast, there is a good correlation in AD between cognitive decline and loss of synaptophysin-immunoreactive (SYN-IR) presynaptic terminals in specific brain regions. We used expression-matched transgenic mouse lines to compare the effects of different human amyloid protein precursors (hAPP) and their products on plaque formation and SYN-IR presynaptic terminals. Four distinct minigenes were generated encoding wild-type hAPP or hAPP carrying mutations that alter the production of amyloidogenic Abeta peptides. The platelet-derived growth factor beta chain promoter was used to express these constructs in neurons. hAPP mutations associated with familial AD (FAD) increased cerebral Abeta(1-42) levels, whereas an experimental mutation of the beta-secretase cleavage site (671(M-->I)) eliminated production of human Abeta. High levels of Abeta(1-42) resulted in age-dependent formation of amyloid plaques in FAD-mutant hAPP mice but not in expression-matched wild-type hAPP mice. Yet, significant decreases in the density of SYN-IR presynaptic terminals were found in both groups of mice. Across mice from different transgenic lines, the density of SYN-IR presynaptic terminals correlated inversely with Abeta levels but not with hAPP levels or plaque load. We conclude that Abeta is synaptotoxic even in the absence of plaques and that high levels of Abeta(1-42) are insufficient to induce plaque formation in mice expressing wild-type hAPP. Our results support the emerging view that plaque-independent Abeta toxicity plays an important role in the development of synaptic deficits in AD and related conditions.

Fig. 1.

Summary of transgenic lines. A, Diagram of hAPP indicating the mutations expressed in transgenic mice. FAD-linked mutations are commonly referred to by place of discovery or residence of affected kindred. The 670/671_KM→NL double mutation affects a large pedigree in Sweden (Mullan et al., 1992), and the 717_V→F mutation was identified in Indiana (Murrell et al., 1991) (numbers refer to amino acids in APP770). Mutations at position 717 are often collectively referred to as “London mutations” based on the first report of the FAD-linked 717_V→I mutation (Goate et al., 1991); however, the latter mutation was not studied here. The sequence of Aβ is indicated inbold in single-letter amino acid code.KPI, Kunitz-type protease inhibitor domain. Elements are not drawn to scale. B, Relative levels of cerebral transgene expression (values in parentheses) were determined in different lines of PDGF-hAPP mice as illustrated in Figure 2. The expression level in line I63 was arbitrarily defined as 1.0.

Fig. 2.

Identification of wild-type and FAD-mutant hAPP mice with matching levels of cerebral transgene expression.A, Representative autoradiograph showing results of an RNase protection assay. Total RNA was extracted from entire hemibrains. The left lane shows signals of undigested radiolabeled riboprobes; the other lanes contained the same riboprobes plus brain RNA samples, digested with RNases. Each samplelane contains RNA from a different mouse. The hAPP probe detects human but not mouse APP; it also recognizes an SV40 segment (S) of transgene-derived mRNAs.Non-tg, Nontransgenic. B, Phosphorimager quantitation of signals shown in A. Values represent group means ± SD.

Fig. 3.

Comparison of human Aβ levels in hippocampi of mice expressing wild-type or FAD-mutant hAPP. Aβ_1-x and Aβ_1–42 were quantitated by ELISA in mice from different transgenic lines (n = 6–9 mice per line) at 2–4 months of age. Values represent group means ± SD. No plaques were detected in the opposite hemibrains of these mice by immunostaining with the 3D6 antibody (data not shown).

Fig. 4.

Hippocampal plaque formation in different lines of FAD-mutant hAPP mice. Aβ deposits were detected by immunostaining of brain sections (n = 3 per mouse) with the 3D6 antibody as described in Materials and Methods. Six to 18 mice per line were analyzed at 2–4, 8–10, and 21–25 months of age, and 1–6 (mean = 4.3) mice per line were analyzed at 5–7 and 11–16 months of age.

Fig. 5.

Age-related cerebral Aβ deposition occurs in mice expressing FAD-mutant hAPP but not in mice expressing wild-type hAPP. Brain sections were immunoperoxidase-stained for Aβ with the 3D6 antibody and imaged by light microscopy (A–D) or double-labeled with antibodies against Aβ (R1280; red) and monoclonal antibodies against phosphorylated neurofilaments (SMI312; green) and imaged by laser scanning confocal microscopy (E,F). Hippocampal sections of transgenic mice are shown: A, APP_Ind line H6 (18 months);B, APP_Wt line I63 (15 months);C, APP_{Sw, Ind} line J9 (10 months);D, APP_Sw,Ind line J20 (10 months);E, APP_Ind line H6 (10 months).F, Midfrontal gyrus from a human AD brain. Magnifications: A–D, 4×; E,F, 930×.

Fig. 6.

Density of SYN-IR presynaptic terminals does not correlate with plaque load in mice from different FAD-mutant hAPP lines. Plaque load and density of SYN-IR presynaptic terminals in the hippocampus were determined in 31 transgenic mice from APP_Ind lines H6, H9, and H40 and APP_{Sw, Ind}line J9 at 21–27 months of age. No correlation was found between the two variables.

Fig. 7.

Comparison of hippocampal Aβ levels, plaque formation, and density of SYN-IR presynaptic terminals in the high expresser lines APP_Wt I63 and APP_Ind H6. Note that the cerebral hAPP mRNA levels in these lines are very well matched (Figs. 1, 2). A, Levels of human Aβ were determined at 2–4 months of age in 8–9 mice per line by ELISA.Circles represent values in individual mice;horizontal lines indicate group means. B, Proportion of mice in which 3D6-immunoreactive plaques were identified (black) at the ages indicated (n = 4–18 mice per line and age range). C, The density of SYN-IR presynaptic terminals was determined in 4–41 mice per genotype and age range. Data represent group means ± SD. *p < 0.05, **p < 0.01 versus nontransgenic controls (Tukey–Kramer post hoctest).

Fig. 8.

Density of SYN-IR presynaptic terminals does not correlate with hAPP levels across hAPP mice from different lines. Levels of full-length plus α-secreted hAPP and density of SYN-IR presynaptic terminals in the hippocampus were determined in 36 transgenic mice from APP_Wt lines I5, I7, and I63, APP_Ind lines H6 and H40, and APP_{Sw, Ind} line J9 at 2–4 months of age. No correlation was identified between the two variables. No plaques were detected in the opposite hemibrains of these mice by immunostaining with the 3D6 antibody (data not shown).

Fig. 9.

Inverse correlation between density of SYN-IR presynaptic terminals and levels of Aβ. At 2–4 months of age, hippocampal levels of Aβ_1-x and Aβ_1–42 in one hemibrain were correlated with the hippocampal density of SYN-IR presynaptic terminals in the opposite hemibrain in mice from lines expressing APP_Wt, APP_Ind, or APP_{Sw, Ind} at different levels (4–9 mice per line). None of these mice had plaques by immunostaining with the 3D6 antibody (data not shown). Arrows indicate the normal density of SYN-IR presynaptic terminals in age-matched nontransgenic controls (mean of 29 mice).