Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2

Abstract

Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.

Figure 1.

Characterization and quantification of P0 sternomastoid muscles. A, Double labeling with the anti-Syn antibody and α-BTX of WT, APP^-/-, APLP2^-/-, and APP/APLP2 double knock-out (dKO) animals. Scale bar, 5 μm. B, SV2 and α-BTX double staining of littermate APLP2^-/- and dKO NMJs. The arrowheads in d and f mark the SV2 staining beyond the end plate. C, Quantification of the percentage of AChR-positive end plates covered by SV2 immunoreactivity (average ± SD of 20 end plates per genotype). ^*p < 0.01; t test. D, Western blot analysis of P0 spinal cord proteins using an anti-APP C-terminal antibody (APP) or anti-SV2 antibody (SV2). Anti-tubulin (Tubulin) Western blot was used as the loading control. The images were captured by a confocal microscope and displayed either as individual staining or merged images.

Figure 2.

Triple labeling of P0 WT, APP^-/-, APLP2^-/-, and APP/APLP2 dKO sternomastoid muscles with anti-Syn antibody, anti-NF antibody, and α-BTX. The arrowheads in m, n, and p denote a representative Syn-positive nerve sprouted beyond the end plate. The images were captured by a confocal microscope and displayed either as individual staining or merged images. Scale bar, 20 μm.

Figure 3.

EM analysis of presynaptic terminal structures of P0 sternomastoid muscles. A, B, Representative EM images showing reduced synaptic vesicles in APP/APLP2 dKO terminals compared with littermate APLP2^-/- control terminals of similar sizes. C, Representative electron-dense active zones, which could be identified in both genotypes. D, Quantification of synaptic vesicle densities (number of vesicles per square micrometer of profile area). ^*p < 0.001; Student's t test. E, Quantification of the number of active zones per micrometer of profile length. ^*p < 0.001; Student's t test. F, Number of docked vesicles per active zone. All calculations were done on a per section basis. Each column represents mean ± SE of 40 profiles from two animals. Scale bars: A, B, 1 μm; C, 200 μm.

Figure 4.

Whole-mount NF staining of P0 (a, b), E16.5 (c, d), and E14.5 (e, f) diaphragm of littermate APLP2^-/- control and APP/APLP2 dKO is shown. Nerve terminal sprouting is apparent in E16.5 and P0 but not in E14.5 dKO samples. Scale bar, 400 μm.

Figure 5.

Examination of synaptic distribution of the diaphragm muscle. A, Double labeling with the anti-NF antibody (a, d) and α-BTX (b, e), revealing excessive terminal sprouting and widened AChR-positive end plate band in APP/APLP2 dKO animals. B, Quantification of AChR cluster distribution from the medial edge of the diaphragm (mean ± SD of 3 animals per genotype). ^*p < 0.05; t test. C, Syn and α-BTX double staining, which showed broadened presynaptic distribution. D, Acetylcholine esterase histochemistry, documenting diffused synaptic patterning. The right columns in A and C are the merged images of the first two columns. Scale bars: A, 50 μm; C, D, 100 μm.

Figure 6.

Electrophysiological recordings from P0 sternomastoid muscle fibers of APP/APLP2 double null (dKO) mice and control littermates (APLP2^-/-). A, MEPP recordings. Aa, Reduced MEPP frequency (mean ± SD) in dKO (n = 8) compared with control (n = 7); ^*p < 0.001. Over one-half of the fibers in this analysis did not have detectable MEPPs. Ab, Similar MEPP amplitude in APLP2^-/- control and APP/APLP2 null (dKO) mutant (mean ± SD); p = 0.23. B, EPP recordings. Ba, Percentage of fibers in which an evoked response (EPP or action potential) could not be induced by supramaximal nerve stimulation. Control (APLP2^-/-) fibers, 1 of 41; double null (dKO) fibers, 8 of 31. ^*p < 0.005. Bb, Representative EPP evoked in a sternomastoid fiber by stimulation of the muscle nerve, with μ-conotoxin present in the bath. Top trace, EPP from an APLP2^-/- control; bottom trace, EPP from a littermate mutant (dKO), at 10 times the threshold required to evoke responses in adjacent muscle fibers.