Model of the regulation of synaptic functions by Reelin. Aβ activates the striatal-enriched protein tyrosine phosphatase, leading to dephosphorylation of tyrosine residues on NMDA receptors. Dephosphorylation of the NR2B subunit correlates with increased NMDA receptor endocytosis and suppression of its synaptic function (9). Reelin activates Src family tyrosine kinases (SFK) and enhances tyrosine phosphorylation of NR2A and NR2B subunits. Reelin signaling may prevent Aβ-induced NMDA receptor endocytosis and SFK activation by Reelin restores NMDA receptor activity. We propose that Reelin signaling through the ApoE receptors Apoer2 and Vldlr counteracts the suppressive effect of Aβ at the synapse and that this is important for the maintenance of normal synaptic function.

Opposing effects of Aβ and Reelin on NMDA receptor tyrosine phosphorylation. (A) High doses of Aβ peptides prevent NMDA receptor phosphorylation in response to Reelin treatment. Acute WT hippocampal slices were incubated in the presence (R) or absence of Reelin (C) and Aβ_25–35. NR2A and NR2B were immunoprecipitated from the homogenized lysates of the slices and analyzed for tyrosine phosphorylation. Total amounts of immunoprecipitated NR2A and NR2B are shown (NR2A, NR2B); one independent experiment is shown (n = 3). Tyrosine phosphorylation of NR2A and NR2B in the presence of Aβ and Reelin was not significantly different from control (lane 1) by paired t test. (B) Reelin enhances LTP in the absence of Aβ, but fails to rescue LTP suppression induced by high concentrations of Aβ_25–35. (Upper) In WT hippocampal slices, Reelin (closed circles) enhances and Aβ_25–35 (at 1 μM, open triangles) suppresses TBS-induced LTP compared with non-treated control slices (open squares). Reelin fails to restore LTP in the presence of high concentrations of Aβ (1 μM). Application of Reelin alone (closed circles) induces an immediate shift in baseline currents as a result of its direct effect on NMDA and AMPA receptor activity (21). (Lower) Statistical analysis of average LTP responses 30 to 35 min after TBS. Control LTP (open bar), 129.30% ± 14.16%, n = 2; Reelin (gray bar), 165.65% ± 14.11%, n = 2; high-dose Αβ_25–35 (black closed bar), 99.85% ± 19.19%, n = 4; Reelin with high-dose Αβ_25–35, 97.56% ± 6.83%, n = 4. ANOVA was performed, followed by Bonferroni post-test. Control compared with Reelin, P < 0.05; control compared with high-dose Aβ_25–35, P < 0.05; high-dose Aβ_25–35 compared with Reelin and Aβ_25–35 together, P > 0.05. Asterisk denotes significance.

Reelin rescues Aβ-induced LTP suppression in CA1. (A) Suppression of LTP by Aβ_1–42 is rescued by Reelin in WT hippocampal slices. (Upper) LTP was induced by theta burst stimulation (TBS) consisting of 5 trains of 4 pulses at 100 Hz with an inter-burst interval of 20 s at 0 min. Open squares show responses from control slices receiving no treatment (n = 5). Solid black circles show responses from slices treated with 400 nM Aβ_1–42 for 40 to 60 min before TBS. Solid gray triangles show responses of slices from the same animals treated with Aβ_1–42 in the presence of 5 nM Reelin. Representative fEPSP traces before and 30 min after TBS for the different conditions are shown (Top). (Scale bar: 0.2 mV, 20 ms.) (Lower) Statistical analysis of average LTP responses between 30 and 35 min after TBS. Aβ_1–42 significantly attenuates LTP in the absence of Reelin (from 152.99% ± 9.85% to 112.89% ± 6.35%, n = 5; P < 0.05). LTP reduction by Aβ_1–42 is prevented in the presence of 5 nM Reelin and no longer significantly different from controls (147.76% ± 9.30%, n = 5; P > 0.05). Asterisk denotes significance on one-way ANOVA followed by Bonferroni post-test. (Inset, Right, Top) Representative immunoblot of synthetic Aβ_1–42 oligomers used throughout the study. Monomer, dimers, trimers, and tetramers are present. (Bottom) Immunoblot of primary cortical neuronal lysates shows induction of Dab1 tyrosine phosphorylation by different concentrations of purified Reelin used throughout the study. (B) Suppression of LTP by Aβ_25–35 is rescued by Reelin in WT hippocampal slices. (Upper) LTP was induced by TBS consisting of 5 trains of 4 pulses at 100 Hz with an inter-burst interval of 20 s at 0 min. Open squares show responses from control slices receiving no treatment (n = 8). Solid black circles show responses from slices treated with 100 nM Aβ_25–35 for 40 to 60 min before TBS. Solid gray triangles show responses of slices from the same animals treated with Aβ_25–35 in the presence of 5 nM Reelin. Representative fEPSP traces before and 30 min after TBS for the different conditions are shown (Top). (Scale bar: 0.2 mV, 20 ms.) (Lower) Statistical analysis of average LTP responses between 30 and 35 min after TBS. Aβ_25–35 significantly attenuates LTP in the absence of Reelin (from 141.63% ± 11.75% to 109.21% ± 3.09%, n = 8; P < 0.05). LTP reduction by Aβ_25–35 is prevented in the presence of 5 nM Reelin and no longer significantly different from controls (145.26% ± 7.80%, n = 8; P > 0.05). Asterisk denotes significance on one-way ANOVA followed by post-tests. High resolution traces are shown in the SI.

Reelin prevents LTP suppression induced by cortical AD brain extract. (A) Human cortical brain extracts from a normal subject (Con, control) and a clinically and histopathologically confirmed AD case were prepared as described in Materials and Methods and by Shankar et al. (11). Aβ monomers, oligomers, and high molecular weight complexes (approximately 80 kDa) were immunoprecipitated and separated by LDS PAGE. Monomers and oligomers (dimers, trimers, and tetramers) derived from synthetic Aβ_1–42 are shown (Left). Control brain extract (Middle) contains monomeric Aβ, but no detectable oligomers and only a trace amount of higher-order aggregates (Upper). By contrast, AD brain extract contains comparable amounts of monomeric Aβ, but Aβ dimers and higher-order multimers were also present. (B) (Top) AD brain extract (50 μL per mL perfusate, closed circles) potently suppresses LTP, compared with equivalent extracts from control brain (50 μL per mL perfusate, open squares). Reelin almost completely prevents LTP suppression by AD brain extract (gray triangles). (Lower) Statistical analysis of average LTP responses between 30 and 35 min after TBS. Control brain extract (open bar), 133.99% ± 7.14%, n = 4; AD brain extract (closed bar), 101.86% ± 5.42%, n = 8; AD brain extract plus Reelin (gray bar), 128.3% ± 8.42%, n = 8. (ANOVA followed by Bonferroni post-test.) Control compared with AD extract, P < 0.05; AD extract compared with AD extract plus Reelin, P < 0.05. Control extract compared with AD extract plus Reelin, P > 0.05. Asterisk denotes significance. (C) (Top) AD brain extract failed to suppresses NMDA receptor-independent LTP in the presence of NMDA receptor antagonist D-AP5 (50 μM, closed circles) compared with control brain extract (open squares), and Reelin did not alter NMDA receptor-independent LTP in the presence of AD brain extract (gray triangles). (Lower) Statistical analysis of average LTP response. Control (open bar), 160.49% ± 14.19%, n = 7; AD extract (closed bar), 115.79% ± 6.53%, n = 12; AD extract plus Reelin (gray bar), 156.84% ± 12.33%, n = 11. (ANOVA followed by Bonferroni post-test.) Control versus AD extract, P < 0.05; AD extract versus AD extract plus Reelin, P < 0.05. Control extract versus AD extract plus Reelin, P > 0.05. Asterisk denotes significance. High resolution traces are shown in the SI.

Reelin rescues Aβ-induced suppression of NMDA currents in mouse hippocampus by activating SFKs. (A) Reelin prevents Aβ_25–35-induced suppression of NMDA currents. (Upper) Normalized average amplitudes of evoked NMDA peak currents at +30 mV in single-cell recordings. Open squares show responses from control slices treated with 100 nM Aβ_25–35 in the absence of Reelin; solid gray circles show responses in the presence of Aβ and 5 nM Reelin. Gray bar indicates the timing of Aβ application. Reelin was applied 25 to 30 min before Aβ perfusion. After stabilization, baseline levels were readjusted (by reducing the gain of the amplifier) to “pre-Reelin” levels and monitored for at least 10 min before Aβ application. The time of Aβ application is 0 min. (Top) Traces show representative whole-cell recordings 30 min before and after Reelin and Aβ treatment. (Scale bar, 20 pA, 100 ms.) (Lower) Statistical analysis comparing extent of NMDA excitatory postsynaptic current (EPSC) amplitude suppression 30 min after Aβ application in the absence and presence of Reelin. Aβ significantly suppresses NMDA currents (64% ± 7.70% of control, n = 6; P < 0.01, 2-way ANOVA). Reelin prevents this effect (101.07% ± 11.98%, n = 6; P > 0.05, 2-way ANOVA followed by post-test). Asterisks denote significance. (B) SFK activation is required for the prevention of Aβ_1–42-induced NMDA receptor suppression by Reelin. (Upper) Normalized average amplitudes of evoked NMDA peak currents at +30 mV in single-cell recordings. Solid gray triangles show responses from control slices treated with 5 nM Reelin. Open squares show responses from slices treated with PP2 (10 μM) and Reelin. (Top) Traces show representative whole-cell recordings 10 min before and 30 min after Aβ_1–42 application. (Scale bar, 50 pA, 100 ms.) (Lower) Statistical analysis of NMDA receptor-dependent EPSC amplitude suppression. No significant change (94.85% ± 18.84% of control) in NMDA currents was seen when 100 nM Aβ_1–42 was applied in the presence of 5 nM Reelin. When the SFK inhibitor PP2 (10 μM) was added to the perfusate, application of Aβ_1–42 to slices from the same animals caused a significant suppression of the NMDA currents (58.62% ± 1.05% of control, n = 4; P < 0.05, unpaired t test). (C) The function-blocking CR50 antibody prevents Reelin rescue of LTP suppression by Aβ. TBS LTP was induced as described in Fig. 1. In the presence of Reelin alone, LTP is enhanced (16). Aβ_25–35 oligomers diminish LTP, and this is almost completely prevented by Reelin. Preincubation of Reelin with a threefold molar access of the function blocking CR50 antibody (30) prevents this. CR50 alone has no effect. High resolution traces are shown in the SI.