Synaptic function of nicastrin in hippocampal neurons

Abstract

Synaptic dysfunction is widely thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Presenilins, the major gene products involved in familial AD, are essential for short- and long-term synaptic plasticity in mature neurons as well as for the survival of cortical neurons during aging. Presenilin and nicastrin are both indispensable components of the γ-secretase complex, but it remains unknown whether presenilin regulates synaptic function in a γ-secretase-dependent or γ-secretase-independent manner and whether nicastrin plays similar roles in central synapses. In the current study, we address these questions using an electrophysiological approach to analyze nicastrin conditional knockout (cKO) mice in the hippocampal Schaffer collateral pathway. In these mice, we found that, even at 2 mo of age, deletion of nicastrin in excitatory neurons of the postnatal forebrain using Cre recombinase expressed under the control of the αCaMKII promoter led to deficits in presynaptic short-term plasticity including paired-pulse facilitation and frequency facilitation. Depletion of Ca(2+) in the endoplasmic reticulum mimics and occludes the presynaptic facilitation deficits in nicastrin cKO mice, suggesting that disrupted intracellular Ca(2+) homeostasis underlies the presynaptic deficits. In addition, NMDA receptor-mediated responses and long-term potentiation induced by theta-burst stimulation were decreased in nicastrin cKO mice at 3 mo but not at 2 mo of age. Together, these findings show that, similar to presenilins, nicastrin plays essential roles in the regulation of short- and long-term synaptic plasticity, highlighting the importance of γ-secretase in the function of mature synapses.

Fig. 1.

Time course of Nct inactivation in the hippocampus of Nct cKO mice. (A) Western analysis of protein levels of Nct at five different time points. Hippocampal lysates were treated with PNGase-F to deglycosylate Nct. Nct levels are reduced progressively in the Nct cKO hippocampus. (B) Hippocampal lysates from Nct cKO and control mice at age 30, 45, 60, 75, and 90 d were analyzed by immunoblotting. Protein levels were normalized to β-actin and measured by LI-COR quantitative detection system. All data represent means ± SEM. The number of mice used in the experiment is indicated in parentheses.

Fig. 2.

Age-dependent reduction of NMDAR-mediated responses in the hippocampal Schaffer collateral pathway of Nct cKO mice. (A and B) Normal AMPAR-mediated (A) and NMDAR-mediated (B) I/O curves of synaptic transmission in Nct cKO mice at age 2 mo. The FV amplitude is plotted against the initial slope of the evoked fEPSP for the Nct cKO and littermate control mice. Each point represents data averaged across all slices for a narrow bin of FV amplitude. (C and D) Normal AMPAR-mediated (C) but reduced NMDAR-mediated (D) I/O curves of synaptic transmission in Nct cKO mice at age 3 mo. The NMDAR I/O slope in Nct cKO mice (control: y = 0.429×, R² = 0.980; cKO: y = 0.278×, R² = 0.963) is significantly reduced (P < 0.05; Student t test). All data represent means ± SEM. The values in parentheses indicate the number of hippocampal slices (Left) and the number of mice (Right) used in each experiment.

Fig. 3.

Age-dependent impairment of long-term plasticity in the hippocampal Schaffer collateral pathway of Nct cKO mice. (A) Normal LTP induced by 5 TBS in Nct cKO mice (closed circles) compared with controls (open circles) at age 2 mo. Superimposed traces are averages of four consecutive responses 1 min before (thin line) and 50 min after (thick line) TBS induction. (B) Impaired TBS-induced LTP in Nct cKO mice at age 3 mo. Superimposed traces are averages of four consecutive responses 1 min before (thin line) and 50 min after (thick line) TBS induction. The magnitude of LTP during the last 10 min of the recording is significantly reduced in Nct cKO mice (120.8 ± 2.7%) relative to the control (147.3 ± 3.5%) (P < 0.001; Student t test). All data are means ± SEM. The values in parentheses indicate the number of hippocampal slices (Left) and the number of mice (Right) used in each experiment. [Scale bar: 10 ms (x axis) or 1 mV (y axis).]

Fig. 4.

Impaired short-term synaptic plasticity in the hippocampal Schaffer collateral pathway of Nct cKO mice at age 2 mo. (A) Representative traces from control and Nct cKO mice of fEPSPs evoked by two consecutive stimuli with a 60-ms interpulse interval. (B) Average paired-pulse ratios plotted as a function of the interstimulus interval. All data represent means ± SEM (□, P < 0.05; ■, P < 0.01; Student t test). The values in parentheses indicate the number of hippocampal slices (Left) and the number of mice (Right) used in each experiment. [Scale bars: 30 ms (x axis) or 1 mV (y axis).]

Fig. 5.

Depletion of ER calcium mimics and occludes the impaired presynaptic facilitation in Nct cKO mice. (A) Synaptic facilitation elicited by stimulus trains is impaired in a frequency-dependent manner in hippocampal area CA1 of Nct cKO mice at age 2 mo. fEPSP slopes shown are normalized to the slope of the first fEPSP of the stimulus train. (B and C) Effects of TG treatment (2 µM for 1 h) on synaptic facilitation induced by high-frequency stimulus trains in the hippocampal CA1 region of control (B) and Nct cKO (C) mice at age 2 mo. All data are means ± SEM (□, P < 0.05; ■, P < 0.01; Student t test). The values in parentheses indicate the number of hippocampal slices (Left) and the number of mice (Right) used in each experiment.

Fig. 6.

Normal levels of neuronal and synaptic markers in Nct cKO mice. Levels of indicated proteins in control and Nct cKO mice at 2 and 3 mo of age were analyzed by quantitative immunoblotting of cortical lysates using iodinated secondary antibodies and were normalized to GDP dissociation inhibitor as a loading control. Immunoblotting for Nct was performed after treatment with PNGase F to remove N-glycosylation. Data shown are means ± SEM (***P < 0.001; Student t test). The number of mice used in the experiment is indicated in parentheses.