Synapse formation and function is modulated by the amyloid precursor protein

Abstract

The amyloid precursor protein (APP) is critical in the pathogenesis of Alzheimer's disease. The question of its normal biological function in neurons, in which it is predominantly located at synapses, is still unclear. Using autaptic cultures of hippocampal neurons, we demonstrate that hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor-mediated EPSCs. The size of the readily releasable synaptic vesicle pool was also increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP, whereas the amplitude of miniature synaptic currents was not found to be altered. Together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunohistochemical analysis revealing an increase of synaptophysin-positive puncta per cultured APP knock-out neuron. In conclusion, lack of APP affects synapse formation and transmission in cultured hippocampal neurons.

Figure 1.

Evoked neurotransmitter release in WT and APP KO neurons. A, Representative AMPA EPSC traces from wild-type and APP knock-out neurons. B, Representative NMDA EPSC traces from wild-type and APP knock-out neurons. C, Mean AMPA and NMDA EPSC amplitudes in neurons from wild-type (n = 24 and n = 35, respectively) and APP knock-out (n = 23 and n = 33, respectively) mice (∗p < 0.05).

Figure 2.

Spontaneous neurotransmitter release in WT and APP KO neurons. A, B, Representative mEPSC traces from wild-type and APP knock-out neurons in the presence of 200 nm TTX. C, Left, mEPSC frequency in neurons from wild-type (n = 25) and APP knock-out (n = 23) mice. Middle, mEPSC amplitude in neurons from wild-type (n = 25) and APP knock-out (n = 23) mice. Right, Estimated EPSC vesicle number per evoked EPSC in wild-type (n = 10) and APP knock-out (n = 11) neurons (∗p < 0.05).

Figure 3.

Estimated RRP, RRP refilling, and release probability in WT and APP KO neurons. A, Estimated RRP vesicles for wild-type (n = 10) and APP knock-out (n = 11) mice. Inset shows representative traces. Calibration: 1 s, 0.5 nA. ∗∗ p < 0.005. B, Average ratio of the second hypertonic sucrose response to the initial sucrose response measured 4 s before wild-type (n = 14) and APP knock-out (n = 18) mice. Inset shows sample traces. Calibration: 1 s, 0.5 nA). C, Analysis of MK-801 blocking rate in wild-type (n = 13) and APP knock-out (n = 10) mice. The average NMDA EPSC amplitude normalized to the first response in the presence of MK-801 (5 μm) is plotted against evoked EPSC stimulus number.

Figure 4.

Prolonged repetitive and high-frequency stimulation of WT and APP KO neurons. A, Sample recordings of the first 10 responses in a 10 Hz train. B, Average EPSC amplitudes of wild-type (n = 19) or APP knock-out (n = 16) neurons during 10 Hz train normalized to the size of first response. C, Sample recordings of five responses in a 50 Hz train. D, Average EPSC amplitudes of wild-type (n = 22) and APP knock-out (n = 23) cells during 50 Hz train normalized to the size of first response.

Figure 5.

Evoked and spontaneous neurotransmitter release in neurons after incubation with DAPT, Aβ₄₂, or conditioned medium (CM). A, AMPA EPSC amplitude in neurons from control wild-type (n = 15) compared with wild-type neurons treated with DAPT (n = 15) or control APP knock-out neurons (n = 15) compared with APP knock-out neurons treated with either Aβ₄₂ (n = 15) or CM from wild-type cultures (n = 15). B, mEPSC frequency in control wild-type neurons (n = 15) compared with wild-type neurons treated with DAPT (n = 15) or control APP knock-out neurons (n = 15) compared with APP knock-out neurons treated with either Aβ₄₂ (n = 15) or CM from wild-type cultures (n = 15) (∗p < 0.05).

Figure 6.

Structural analysis of synapses in microisland cultures from WT and APP KO neurons. A, Immunostains of autaptic neurons. Aa, Ab, Staining for the dendritic marker MAP-2 to assess the size of wild-type and APP knock-out neurons. Ac, Ad, Staining of the same culture for synaptophysin to assess the number of synapses. Ae, Af, Merged picture of MAP-2 and synaptophysin staining. Scale bar, 25 μm. B, Left, Total dendritic length of wild-type (n = 55) and APP knock-out (n = 62) neurons (left). Right, Average number of synapses of wild-type (n = 55) and APP knock-out (n = 62) neurons (∗∗∗p < 0.0005).

Figure 7.

Synaptophysin staining of sections derived from wild-type and APP knock-out mice. A, Synaptophysin-positive presynaptic terminals in mouse hippocampus. Aa, Ab, Low-magnification view of wild-type and APP knock-out hippocampus from 20-d-old mice: 1, stratum granulosum; 2, stratum moleculare; 3, stratum lacunosum; 4, stratum radiatum; 5, stratum pyramidale; 6, stratum oriens. Scale bar, 100 μm. Ac, Ad, Synaptophysin staining of the stratum radiatum from 20-d-old wild-type and APP knock-out mice. Ae, Af, Synaptophysin-positive presynaptic terminals in the stratum moleculare of 20-d-old wild-type and APP knock-out mice. Scale bar, 25 μm. B, Average staining intensity in the stratum radiatum of wild-type (n = 18 areas, 9 sections) and APP knock-out (n = 18 areas, 9 sections) mice at the age of 20 d and 11 months, respectively. C, Average staining intensity in the stratum moleculare of wild-type (n = 18 areas, 9 sections) and APP knock-out (n = 18 areas, 9 sections) mice at the age of 20 d and 11 months, respectively (∗∗p < 0.005). D, Representative Western blot analysis of synaptophysin (SYN; bottom band) and β-actin (top band) expression in hippocampal brain sections of wild-type (left lane) and APP knock-out (right lane) mice.