Amyloid precursor protein (APP) regulates synaptic structure and function

Abstract

The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by β- and γ-secretases to generate the amyloid β-protein (Aβ), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aβ peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15 months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aβ-mediated synaptic toxicity.

Figure 1

APP−/− hippocampal neurons show a decrease in spine number. (A) Representative images of primary hippocampal neurons expressing eGFP. Top: APP+/− (left) and APP−/− (Holcomb et al., 1998) neurons were from the same parental interbreeding of APP mice. The images were presented in grey and inverted color. Panels at the bottom of each image show high-magnification renderings of dendritic segments. The dendritic images were taken with 0.3-μm step z-section and then stacked with maximum projection. Scale bar: 150 μm (top) and 5 μm (bottom). (B) Quantification of spine density. There was no difference in spine number between APP+/+ and APP+/− (p > 0.05). APP−/− neurons showed highly significant decrease in spine density compared to APP+/+ or APP+/− neurons (**, p < 0.01 and ***, p < 0.001). Data are expressed as mean ± SEM and tested using a one-way ANOVA with Tukey’s post-hoc test.

Figure 2

3-Dimensional reconstructions of CA1 neurons. (A-D) Representative examples of 3-dimensional reconstructions of neurons and the corresponding apical dendrograms (arrow indicates the apical dendrite). Dendrograms of CA1 pyramidal layer neurons in 13 months old APP+/− and APP−/− mice (A and B); and in young APP+/− and APP−/− mice (C and D; 2-4 months old). (E) and (F) Dendrogram and quantification of dendritic length of CA1 neurons in APP−/− mice. Thirteen month-old APP−/− exhibited a significant decrease in apical length compared to APP+/−, whereas young APP−/− mice showed no significant difference in apical dendritic length compared to APP+/− mice (E). There is no difference in basal dendritic length among APP+/− and APP−/− mice of both age groups (F). Data are expressed as mean ± SEM; asterisks indicate significant differences between groups (*, p < 0.05; unpaired t-test).

Figure 3

Quantification of spine density in APP animal. (A) Representative dendrite images after 3-dimensional deconvolution and processing with NeuronStudio software (see methods). Scale bars: 5 μm. (B) The spine density was decreased (left) ~15% in old APP−/− mice compared to their littermate controls (APP+/−). Young APP−/− mice showed no significant difference in spine density compared to APP+/− mice. The young mice were 2-4 months old. Data are expressed as means ± SEM; asterisks indicate significant differences between groups (**, p < 0.01; unpaired t-test).

Figure 4

APP−/− mice showed impaired electrical properties in acute hippocampal slices of old APP−/− mice. (A) 12-15 months old APP−/− mice (closed triangles) showed impairment in LTP but not in APP+/− mice (open circle). (B) Young APP−/− mice showed normal LTP as did their littermate controls. fEPSPs were recorded in stratum radiatum (dendritic area) of the CA1 region following stimulation of the Schaffer collateral pathway. LTP were induced with 4 tetanus stimulation delivered at 20-s intervals, each at 100 Hz for 1 s. (C) and (D) Basal synaptic transmission was assayed by measuring fEPSPs within the stratum radiatum of the CA1 region of the hippocampus evoked by a bipolar electrode placed at the CA3–CA1 border. For each level of stimulation, the maximum amplitude of the presynaptic fiber volley (FV; arrow) and the initial slope of the fEPSP were measured (see also supplemental Fig. 1). Scatter plot of the fEPSP slope versus fiber volley of every recording in old APP−/− mice (closed triangles, black line; n = 24 slices, 9 animals) and APP+/− mice (16 slices, 7 animals; open circle, dash line), fit by linear regression. There is no significant difference in basal transmission among APP+/− and APP−/− mice in both age groups. (E) and (F) Paired-pulse facilitation at the Schaffer collateral CA1 synapse was measured by dividing the amplitude of the second fEPSP by the amplitude of the first elicited by a pair of two 50-ms spaced stimuli. No significant change in PPF ratio was observed in both age groups between APP+/− and APP−/− mice.

Figure 5

APP deficient effects on spines are prevented by adding conditioned medium (CM) from APP wild type neurons (APP+/+ or APP+/−). (A) Representative culture APP−/− hippocampal neurons expressing eGFP: APP−/− neurons are shown at right and wild type CM-treated APP−/− neurons at left. Scale bars: 20 (top) and 5 (bottom) μm. Wild type CM was replaced with medium on the second day after plating the neurons. The medium was changed every other day. The bottom of each image shows high-magnification micrographs of dendrites. (B) The quantification of spine density. Wild types CM rescue the decrease in spine density of APP−/− neurons (*, p < 0.05; **, p < 0.01, unpaired t-test).

Figure 6

Soluble APPα but not soluble APPβ rescued partially deficits in dendritic spine number and morphology. (A) The scheme of soluble mouse APP (sAPPα and sAPPβ) and full length of mouse APP (APP). (B) left, 63G antibody (the polyclonal antibody against the middle region of APP) Western blot from medium of B103 cells transfected with either sAPPα ( lanes 1 and 4 from two of single clone), sAPPβ (lane 5) or mouse APP695 (APP) constructs (lane 3) and the non-transfected control (lane 2); Right: CT15 immunoblot from cell lysates of untransfected (lane 6), mouse APP-transfected (lane 7), sAPPα-transfected (lane 8) and sAPPβ-transfected B103 cells (lane 9). (c) Quantification of spine density. APP−/− neurons were treated with medium from either sAPPα-, sAPPβ- and vehicle-transfected B103 cells or from the conditioned medium of APP WT (APP+/+ or APP+/−) neurons (each treatment is indicated in the bar legend). The spine density was restored partially in sAPPα-treated APP−/− neurons compared to APP−/− neurons (*, p < 0.05, unpaired t-test). The sAPPβ-CM did not restore spine density in APP−/− neurons (p > 0.5, unpaired t-test).