Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway

Abstract

Alzheimer's disease (AD) represents the most common age-related neurodegenerative disorder. It is characterized by the invariant accumulation of the beta-amyloid peptide (Abeta), which mediates synapse loss and cognitive impairment in AD. Current therapeutic approaches concentrate on reducing Abeta levels and amyloid plaque load via modifying or inhibiting the generation of Abeta. Based on in vivo two-photon imaging, we present evidence that side effects on the level of dendritic spines may counteract the beneficial potential of these approaches. Two potent gamma-secretase inhibitors (GSIs), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) and LY450139 (hydroxylvaleryl monobenzocaprolactam), were found to reduce the density of dendritic spines in wild-type mice. In mice deficient for the amyloid precursor protein (APP), both GSIs had no effect on dendritic spine density, demonstrating that gamma-secretase inhibition decreases dendritic spine density via APP. Independent of the effects of gamma-secretase inhibition, we observed a twofold higher density of dendritic spines in the cerebral cortex of adult APP-deficient mice. This observation further supports the notion that APP is involved in the modulation of dendritic spine density--shown here for the first time in vivo.

Figure 1.

The fate of individual dendritic spines before and after GSI treatment in WT and APP KO mice (APP ^−/−). A–D, Over a time period of 25 d, representative two-photon in vivo images were taken of the same dendritic element in 4- to 6-month-old mice. Numbers indicate time points in days relative to day 0 when the treatment (LY450139; 30 mg/kg) started for 4 d. Blue arrows exemplarily mark spines that were stable at that exact time point; green arrows indicate gained spines, whereas red arrows represent lost spines at that time point. Scale bars, 3 μm. A, GSI-treated WT mice; B, vehicle-treated WT mice; C, GSI-treated APP KO mice; and D, vehicle-treated APP KO mice.

Figure 2.

Dendritic spine density decreased after GSI treatment by either DAPT or LY450139, exclusively in WT, but not in APP KO, mice, indicating an APP-dependent mechanism. A–D, Time point values represent mean values of individual mice (n = 5 per group). The gray column indicates the 4 d treatment period (days 0–3). Error bars represent mean ± SEM. A, The average dendritic spine density decreased significantly in WT mice after GSI treatment compared with vehicle-treated WT mice (repeated-measures ANOVA; p < 0.001), whereas in APP KO mice, GSI treatment had no effect on the dendritic spine density. In addition, the baseline dendritic spine density in APP KO mice was elevated by twofold. The severity of the induced dendritic spine loss in WT was stronger by DAPT compared with LY450139. B, The average survival fraction of spines significantly decreased (repeated-measures ANOVA; p < 0.001) exclusively in WT mice after GSI treatment. The decrease in dendritic spine density manifested only in a loss of persistent spines (repeated-measures ANOVA; p < 0.001) (C), whereas in transient spines, no significant difference was detected (D).

Figure 3.

Dendritic spine density and spine shape are dependent on APP expression in a dose-dependent manner. A, Representative images of dendritic branches of 4- to 6-month-old WT, heterozygous APP (APP ^+/−), and APP KO (APP ^−/−) mice. Dendritic elements were three-dimensionally reconstructed to classify dendritic spines regarding their shape using a computer-based algorithm. Dendritic spine shapes are exemplarily indicated by t (thin), s (stubby), and m (mushroom). Scale bars, 3 μm. B, The dendritic spine density was significantly higher in APP ^−/− mice compared with APP ^+/− littermates and WT mice (one-way ANOVA, p < 0.001). C, The increase in dendritic spine density in APP ^−/− mice compared with APP ^+/− and WT mice was mainly caused by an increase in stubby- and mushroom-shaped spines (one-way ANOVA). Error bars (B, C) indicate SEM.