Beta amyloid-independent role of amyloid precursor protein in generation and maintenance of dendritic spines

Abstract

Synapse loss induced by amyloid beta (Abeta) is thought to be a primary contributor to cognitive decline in Alzheimer's disease. Abeta is generated by proteolysis of amyloid precursor protein (APP), a synaptic receptor whose physiological function remains unclear. In the present study, we investigated the role of APP in dendritic spine formation, which is known to be important for learning and memory. We found that overexpression of APP increased spine number, whereas knockdown of APP reduced spine density in cultured hippocampal neurons. This spine-promoting effect of APP required both the extracellular and intracellular domains of APP, and was accompanied by specific upregulation of the GluR2, but not the GluR1, subunit of AMPA receptors. In an in vivo experiment, we found that cortical layers II/III and hippocampal CA1 pyramidal neurons in 1 year-old APP-deficient mice had fewer and shorter dendritic spines than wild-type littermates. In contrast, transgenic mice overexpressing mutant APP exhibited increased spine density compared to control animals, though only at a young age prior to overaccumulation of soluble amyloid. Additionally, increased glutamate synthesis was observed in young APP transgenic brains, whereas glutamate levels were decreased and GABA levels were increased in APP-deficient mice. These results demonstrate that APP is important for promoting spine formation and is required for proper spine development.

Figure 1

APP promotes dendritic spine formation. A–F. Cultured hippocampal neurons (DIV21) were transfected with expression vectors encoding EGFP and empty vector, APP, APP (M671V), or shRNA against APP as indicated. To rescue the RNAi effect, a shRNA-resistant form of APP (human APP) was coexpressed with APP shRNA. G. Quantification of spine density (10 neurons/group, **p<0.01; *** p<0.001). H. Quantification of spine density (10 neurons/group, *p<0.05). I. Serial APP deletion constructs with a C-terminal myc-tag were generated containing the following amino acid residues: 265–770 (ΔE1), 591–770 (ΔE1E2), and 671–770 (APP β-CTF). A rectangle in each construct represents a signaling peptide. J. Immunoblot analysis showing comparable expression levels of deletion mutants. Arrowhead, β-CTF; Arrow, α-CTF. K–N. Cultured hippocampal neurons (DIV21) were co-transfected with EGFP and each deletion construct as indicated. O. Quantification of spine density (10 neurons/group, *p<0.05; **p<0.01).

Figure 2

Effect of APP on spine formation in developing neurons. A. Cultured hippocampal neurons (DIV14) were transfected with expression vectors encoding EGFP and each deletion construct of APP (ΔE1, ΔE1E2, and β-CTF). B. Quantitative analysis of spine density (10 neurons/group, *p<0.05; **p<0.01).

Figure 3

APP affects clustering of synaptic proteins. A–F. Cultured hippocampal neurons (DIV21) were transfected with expression vectors encoding EGFP and empty vector, APP, APP M671V, or shRNA against APP as indicated. To rescue the RNAi effect, a shRNA-resistant form of APP (human APP) was also expressed with APP shRNA. Postsynaptic marker PSD-95 was immunostained 3 days after transfection. G–H. Quantification of puncta density of PSD-95 (10 neurons/group, *p<0.05; **p<0.01). I–N. Hippocampal neurons (DIV21) were tranfected with expression vectors encoding EGFP and empty vector, APP, APP-M671V, APP-shRNA, APP-shRNA plus rescue construct as indicated. 3 days after transfection, neurons were immunostained against presynaptic marker Basson. O–P. Quantification of puncta density of Basson (10 neurons/group, *p<0.05; **p<0.01).

Figure 4

APP regulates the expression of GluR2 subunits of AMPA receptors. A–E. Cultured hippocampal neurons (DIV21) were transfected with expression vectors encoding EGFP and empty vector, APP, APP-shRNA, or APP-shRNA plus rescue construct as indicated. Immunocytochemistry of surface GluR2 (sGluR2) was conducted under non-permeablized conditions. F–G. Quantification of integrated intensity of surface GluR2 (10 neurons/group, *p<0.05). H–L. Cultured neurons were transfected with expression vectors encoding EGFP and empty vector, APP, APP-shRNA, or APP-shRNA plus rescue construct as indicated. Immunocytochemistry of total GluR2 (tGluR2) was conducted under permeablized conditions. M and N. Quantification of integrated intensity of total GluR2 (10 neurons/group, *p<0.05; **p<0.01). O–R and T–W. Cultured neurons (DIV14) were transfected with expression vectors encoding EGFP and individual deletion constructs of APP (ΔE1, ΔE1E2, or β-CTF). Surface and total GluR2 (sGluR2 and tGluR2, respectively) were immunostained as indicated. S and X. Quantification of integrated intensity of surface and total GluR2 (10 neurons/group, *p<0.05; **p<0.01; ***p<0.001).

Figure 5

Effect of APP on the expression of GluR1. A–E. Cultured hippocampal neurons (DIV21) were transfected with expression vectors encoding EGFP and empty vector, APP, APP-shRNA, or APP-shRNA plus rescue construct as indicated. Immunocytochemistry of surface GluR1 was conducted under non-permeablized condition. F–G. Quantification of integrated intensity of surface GluR1 (10 neurons/group). H–L. Cultured neurons were transfected with expression vectors encoding EGFP and empty vector, APP, APP-shRNA, or APP-shRNA plus rescue construct as indicated. Immunocytochemistry of total GluR1 was conducted under permeablized condition. M–N. Quantification of integrated intensity of total GluR1 (10 neurons/group).

Figure 6

Spine density and length in APP-deficient mice. A, Representative micrograph of a hippocampal CA1 neuron illustrating dendritic regions analyzed. B, Representative dendritic segments of CA1 pyramidal neurons from 12-month-old wild-type (WT), heterozygote (HT), and APP knock-out (KO) animals (N=6 mice/genotype). Scale bar, 5 µm. C, Mean spine density from WT and APP KO mice in CA1 regions (28 neurons/genotype, *p<0.05; **p<0.01). D. Cumulative distribution plot of hippocampal spine length from WT and APP-deficient mice (230–250 spines/genotype, Kolmogorov-Smirnov Test, p<0.001). E. Representative dendritic segments of cortical layer II/III pyramidal neurons from 12-month-old wild-type (WT), heterozygote (HT), and APP knock-out (KO) animals (N=6 mice/genotype). Scale bar, 5 µm. CTX, cortex F. Mean spine density from WT, HT, and KO mice (28 neurons/genotype, *p<0.05; **p<0.01, ***p<0.001). G. Cumulative distribution plot of cortical layer II/III pyramidal neurons spine length from WT and APP-deficient mice (230–250 spines/genotype, Kolmogorov-Smirnov Test, p<0.001).

Figure 7

Spine density and length in APP-transgenic mice. A. Representative dendritic segments of hippocampal CA1 region (left) and cortical layer II/III pyramidal neurons (right) from 12-month-old WT and APP transgenic (TG) mice (N=4 mice/genotype). Scale bars, 5 µm B. Mean spine density from WT and APP TG animals in hippocampal CA1 region (28 neurons/genotype, *p<0.05). C. Cumulative distribution plot of hippocampal CA1 region spine length in 12 month old WT and APP- transgenic mice (230–250 spines/genotype, Kolmogorov-Smirnov Test, p<0.001). D. Mean spine density from 12 month old WT and APP TG animals in cortical layer II–III pyramidal neurons (28 neurons/genotype, *p<0.05). E. Cumulative distribution plot of cortical layer II/III III pyramidal neurons spine length in 12 month old WT and APP- transgenic mice (230–250 spines/genotype, Kolmogorov-Smirnov Test, p<0.001). F. Representative dendritic segments of hippocampal CA1 region (left) and cortical layer II/III pyramidal neurons (right) from 1-month-old WT and APP transgenic (TG) mice (N=4 mice/genotype). Scale bars, 5 µm. G. Mean spine density in hippocampal CA1 pyramidal neurons from 1 month old WT and APP-transgenic mice (25 neurons/genotype, **p < 0.01). H. Cumulative distribution plot of hippocampal CA1 region spine length in 1 month old WT and APP-transgenic mice (230–250 spines/genotype, Kolmogorov-Smirnov Test, p<0.001). I. Mean spine density from 1 month old WT and APP TG animals in cortical layer II–III pyramidal neurons (28 neurons/genotype, *p<0.05). J. Cumulative distribution plot of cortical layer II/III III pyramidal neurons spine length in 1 month old WT and APP- transgenic mice (230–250 spines/genotype, Kolmogorov-Smirnov Test, p<0.001).

Figure 8

NMR analysis of amino acid transmitters in APP knockout and transgenic mice brains. A and D, Metabolic flux of ¹³C label into individual isotopmers of metabolites in whole brain tissue extracted in 6% perchloric acid (Mann Whitney test, n=4/genotype, *p<0.05). Glu, glutamate; Gln, glutamine; Lac, lactate; Asp, aspartate; GABA, gamma-amino butyric acid. B and E, Percentage of incorporation of ¹³C into isotopmers of glutamate, lactate, GABA, glutamine and aspartate detected by [¹H-decoupled]-¹³C NMR. C and F, Total amount of metabolites in whole brain tissue as shown by [¹³C-decoupled]-¹H NMR.