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. 2010 Jul 14;30(28):9402-10.
doi: 10.1523/JNEUROSCI.2154-10.2010.

beta-Amyloid disrupts activity-dependent gene transcription required for memory through the CREB coactivator CRTC1

Judit España  1 , Jorge Valero, Alfredo J Miñano-Molina, Roser Masgrau, Elsa Martín, Cristina Guardia-Laguarta, Alberto Lleó, Lydia Giménez-Llort, José Rodríguez-Alvarez, Carlos A Saura
Affiliations

beta-Amyloid disrupts activity-dependent gene transcription required for memory through the CREB coactivator CRTC1

Judit España et al. J Neurosci. .

Abstract

Activity-dependent gene expression mediating changes of synaptic efficacy is important for memory storage, but the mechanisms underlying gene transcriptional changes in age-related memory disorders are poorly understood. In this study, we report that gene transcription mediated by the cAMP-response element binding protein (CREB)-regulated transcription coactivator CRTC1 is impaired in neurons and brain from an Alzheimer's disease (AD) transgenic mouse expressing the human beta-amyloid precursor protein (APP(Sw,Ind)). Suppression of CRTC1-dependent gene transcription by beta-amyloid (Abeta) in response to cAMP and Ca(2+) signals is mediated by reduced calcium influx and disruption of PP2B/calcineurin-dependent CRTC1 dephosphorylation at Ser151. Consistently, expression of CRTC1 or active CRTC1 S151A and calcineurin mutants reverse the deficits on CRTC1 transcriptional activity in APP(Sw,Ind) neurons. Inhibition of calcium influx by pharmacological blockade of L-type voltage-gated calcium channels (VGCCs), but not by blocking NMDA or AMPA receptors, mimics the decrease on CRTC1 transcriptional activity observed in APP(Sw,Ind) neurons, whereas agonists of L-type VGCCs reverse efficiently these deficits. Consistent with a role of CRTC1 on Abeta-induced synaptic and memory dysfunction, we demonstrate a selective reduction of CRTC1-dependent genes related to memory (Bdnf, c-fos, and Nr4a2) coinciding with hippocampal-dependent spatial memory deficits in APP(Sw,Ind) mice. These findings suggest that CRTC1 plays a key role in coupling synaptic activity to gene transcription required for hippocampal-dependent memory, and that Abeta could disrupt cognition by affecting CRTC1 function.

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Figures

Figure 1.
Figure 1.
Impaired CRTC1-dependent transcription in primary neurons from APPSw,Ind mice. A, Cortical (left) or hippocampal (right) neurons were transfected at 7 DIV with a CRE-luciferase (0.5 μg) and TK-Renilla (0.25 μg) plasmids for 24 h and then treated with vehicle, forskolin (FSK, 20 μm), KCl (30 mm), or FSK/KCl in the presence of cyclosporine (CsA, 5 μm), FK-506 (5 μm), or tetrodotoxin (TTX, 1 μm). B, Cortical neurons were transfected with CRE-luciferase (0.5 μg), TK-Renilla (0.25 μg), and vector, CRTC1, CRTC2, p300, CBP, CREB, or CREB R314A (0.5 μg) for 24 h before FSK/KCl treatment. C, Cortical neurons were untreated (−) or infected at 0 DIV for 7 d with lentiviral CRTC1 or nonsilencing (NS) scramble shRNAs. Western blotting shows that endogenous CRTC1 is efficiently downregulated by CRTC1 shRNA in cortical neurons. CRE-mediated transcription was analyzed as described in A after vehicle or FSK/KCl treatment for 4 h. + and ++ represent 1 and 2 transducing viral units per cell, respectively. D, Top, Real-time RT-PCR analysis performed in control neurons expressing scramble (NS, nonsilencing) or CRTC1 shRNAs reveal that induction of c-fos, Bdnf IV, and Nr4a2 but not Cyr61 are significantly downregulated by CRTC1 shRNA in response to KCl/FSK treatment. Bottom, Quantitative real-time RT-PCR analysis shows differential expression of endogenous CRTC1 target genes in response to FSK/KCl in APPSw,Ind neurons. Values of each gene are normalized to GAPDH and represent percentage of FSK/KCl-treated WT neurons. Bdnf refers to Bdnf IV. E, ChIP assays demonstrate recruitment of CRTC1 to CRE responsive c-fos and Nr4a2 promoters but not to Cyr61 promoter in response to FSK/KCl. IgG indicates immunoprecipitation with an irrelevant antibody. Input lysate is shown as control. Data represent the mean ± SEM of three independent transfections or treatments performed by triplicate. *p < 0.05, **p < 0.01, compared to WT or NS.
Figure 2.
Figure 2.
Aβ oligomers impair CRTC1-dependent signaling. A, CRE transcriptional activity in primary cortical neurons treated with vehicle (−), DAPT (125 nm), or anti-Aβ antibody (Ab20.1; 1 μg) for 48 h. B, Biochemical (6E10 antibody) and electron microscopy analysis of Aβ species present in soluble monomeric Aβ1-42 and ADDL preparations. C, CRE-luciferase activity in cortical neurons treated for 48 h with vehicle (−), soluble ADDLs (1-30 μm), aggregated Aβ42-1 (20 μm), Aβ1-42 monomers (1 or 20 μm), or media from control (WT) or APPSw,Ind neurons. Data represent the mean ± SEM of three independent transfections performed by triplicate. *p < 0.05, compared to WT medium; **p < 0.01, compared to FSK/KCI-treated neurons.
Figure 3.
Figure 3.
Aβ impairs CRTC1/CREB-dependent transcription by affecting CRTC1 dephosphorylation. A, CRE transcriptional activity in cortical neurons transfected with CRE-luciferase (0.5 μg), TK-Renilla (0.25 μg), and empty or calcineurin ΔCnA plasmids (0.5 μg). When indicated, neurons were treated with vehicle or STS (10 nm). B, Western blot images and quantitative analyses of CRTC1, CREB, and pCREB (Ser133) in total and nuclear lysates from control (WT) and APPSw,Ind neurons (n ≥ 3). The lower migrating band corresponding to dephosphorylated CRTC1 (top blot, upper graph) and nuclear CRTC1 levels (bottom blot and graph) are significantly decreased in APPSw,Ind neurons stimulated with FSK/KCl for 30 min. In these conditions, pCREB is similarly increased in WT and APPSw,Ind neurons (middle graph). C, Western blot showing that the anti-pSer151 CRTC1 antibody recognizes mouse pCRTC1 but not the CRTC1 S151A mutant expressed in HEK 293T cells. D, Western blot analysis of phosphorylated CRTC1 (Ser151) in hippocampal lysates from WT and APPSw,Ind mice at 6 months of age (n = 6–8 per genotype). E, Expression of CRTC1 and CRTC1 S151A reverses CRE-transcriptional deficits in APPSw,Ind neurons. Data represent the mean ± SEM of three independent transfections performed in duplicate. *p < 0.05, **p < 0.001, ***p < 0.0001.
Figure 4.
Figure 4.
Aβ interferes with calcium-induced CRTC1 activation. A, Decreased calcineurin activity in cultured hippocampal (Hip) or cortical (CX) neurons and brain from APPSw,Ind mice (n = 3 independent cultures and brains). B, Western analysis showing expression of calcineurin in total lysates from WT and APPSw,Ind cortical neurons. C, Intracellular Ca2+ responses in basal and FSK/KCl conditions. Representative calcium images (top) and traces (bottom left) and the mean of peak amplitudes (bottom right) of control and APPSw,Ind neurons are shown. Data represent the mean ± SEM of three independent cultures per genotype (n ≥ 15 cells per culture). D, L-type VGCC blockers nimodipine (5 μm) and verapamil (100 μm) mimic and occlude the effect of Aβ on CRE-transcriptional activity in cortical neurons. E, L-type VGCC agonists Bay K-8644 (10 μm), FPL 64176 (5 μm), and nefiracetam (5 μm) increase and reverse CREB-transcriptional deficits in APPSw,Ind cortical neurons. CRE-luciferase activity was determined as described in Figure 1. *p < 0.05, **p < 0.01.
Figure 5.
Figure 5.
Reduced CRTC1-dependent CREB target genes in the hippocampus of cognitive impaired APPSw,Ind mice. A, APPSw,Ind transgenic mice display learning and spatial memory deficits in the Morris water maze. Six-month-old littermate APPSw,Ind and nontransgenic control mice (n = 6 per genotype) were trained in the MWM for 5 d. APPSw,Ind mice learnt the task but they required significantly longer latencies to locate the platform (two-way ANOVA; latencies: genotype effect, F(1,50) = 19.9; day effect, F(4,50) = 31.6; p < 0.0001). In the probe trial, APPSw,Ind transgenic mice spent significantly less time searching and crossing the target quadrant (TQ) platform location than nontransgenic controls. Data represent the mean ± SEM. *p < 0.05, compared to the same day during training or the rest of quadrants during the probe trial. OP, Opposite platform; AR, adjacent right platform; AL, adjacent left platform. B, Quantitative analysis of hippocampal mRNA of CREB target genes by real-time RT-PCR. Values were normalized to GAPDH. n = 4–5 per genotype. C, PCR analysis showing differential expression of CRTC1 target genes in the hippocampus of APPSw,Ind mice compared to controls. Bdnf refers to Bdnf IV. D, Western blot images showing reduction of c-fos and Bdnf but not Egr1 in the hippocampus of APPSw,Ind mice. E, Quantitative analysis of c-fos, Bdnf, and Egr1 protein levels in hippocampus of 6-month-old WT and APPSw,Ind mice. Data represent the mean ± SEM. *p < 0.05.
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