Article Figures & Data
Figures
- Figure 1.
Age-dependent pathological, memory and gene expression changes in APPSw,Ind mice. A, Age-dependent amyloid pathology in the hippocampus of APPSw,Ind (APP) mice. Brain sections were stained with an anti-Aβ 6E10 antibody. M, months; Hip, hippocampus; Cx, cortex. Scale bars: Hp, 250 μm; Cx, 20 μm. B, Biochemical analysis of APP and APP C-terminal fragment (CTF; “Saeko” antibody) and α-secretase-derived αAPPs fragment (1736 antibody) in hippocampus of WT, APPSw,Ind and presenilin-1 (PS1) conditional knock-out mouse (PS1cKO). C, Age-dependent spatial memory deficits in APPSw,Ind mice analyzed as number of target platform crossings and percentage time in the target quadrant in the probe test in the MWM. Data are mean ± SEM (n = 7–8 mice/group); *p < 0.05, **p < 0.001. D, E, Mice were trained for 5 d in the water maze (+) or treated identically without training (−) before analysis of c-fos and Bdnf IV mRNAs by qRT-PCR in different brain regions. Levels of mRNA were normalized to Gapdh. Values represent mean ± SEM (n = 4–5 mice/group); *p < 0.01, **p < 0.001, ***p < 0.0001 compared with controls. #p < 0.01, ##p < 0.001 compared with nontrained. F, Expression of activity-dependent genes in trained APPSw,Ind hippocampus at 2–18 months. Values represent gene changes relative to trained nontransgenic controls. Data represent mean ± SEM (n = 4–6 mice/group); *p < 0.05, **p < 0.001, ***p < 0.0001 compared with trained controls. Statistical analyses were determined by two-way ANOVA followed by Scheffé's S post hoc test.
- Figure 2.
Hippocampal transcriptome changes in spatial trained APPSw,Ind mice. A, Experimental design of the groups used for gene profiling analyses (top) and Venn diagram (bottom) showing the number of genes differentially expressed in the hippocampus of APPSw,Ind mice versus control mice in the microarray analysis. B, ClueGO analysis of the whole gene microarray results showing the most significant functional gene network (k score > 0.5) altered in the hippocampus of spatial memory trained APPSw,Ind mice compared with trained WT mice. Biological pathways are visualized as colored nodes linked with related groups based on their κ score level (≥0.3). The node size reflects the enrichment significance of the term and functionally related groups are linked. Not grouped terms are shown in white. C, Heat map of the normalized gene data showing differential expression of CREB target genes in the hippocampus of naive (four top lines) and spatial trained (three bottom lines) APPSw,Ind mice versus WT mice. Blue and red indicate genes downregulated or upregulated in APPSw,Ind mice compared with WT mice. (D, E) Expression of genes associated with neurotransmission and synaptic plasticity quantified by qRT-PCR in the hippocampus of spatial trained WT and APPSw,Ind mice at 6 months (D) and 2 months (E). Values represent fold gene changes ± SEM (n = 4–5 mice/group). Values were normalized to the geometric mean of Ppia, Hprt, and β-actin. Bdnf refers to Bdnf IV; *p < 0.05, **p < 0.001 (D, E), compared with WT control or naive. Statistical analyses were determined by one-way ANOVA followed by Bonferroni post hoc test.
- Figure 3.
Reduced Crtc1 dephosphorylation, nuclear translocation and activation in APPSw,Ind mice. A, Biochemical analyses of Crtc1, pCrtc1 (Ser151), CREB, and pCREB (Ser133) in hippocampus of naive and memory trained control (WT) and APPSw,Ind mice. Values represent fold changes ± s.e.m (n = 4 mice/group); *p < 0.05, **p < 0.002, and #p < 0.05 compared with naive and WT mice, respectively. B, Reduced Crtc1 and unchanged CBP, CREB, and pCREB in purified nuclear brain extracts of trained APPSw,Ind mice. Data are the mean ± SEM (n = 3–4 mice/group); *p < 0.05 compared with controls. C, Confocal images showing localization of Crtc1 (green) and MAP2 (red) in DG, CA1, and CA3 hippocampus in naive and spatial trained mice. Nuclear translocation of Crtc1, as revealed by colocalization with Hoechst (blue; arrowheads) is more evident in CA3 hippocampal neurons of WT mice after spatial training, and reduced in trained APPSw,Ind mice. CA3: Green Crtc1 staining in the left side of the images represents terminal axons from DG granular cells (mossy fibers), whereas dendritic MAP2 staining (red) is detected as punctuate staining due to its transversal position in the coronal sections. Images (20×, zoom 0.5) are representative of n = 5–6 mice/group. Scale bar, 40 μm. D, Expression of CREB target genes in 10 DIV cultured neurons expressing scramble or Crtc1 shRNAs treated with vehicle (−) or FSK/KCl (+). Data are normalized to Gapdh and represent the mean ± SEM (n = 3); #p < 0.0001, *p < 0.05, **p < 0.01 compared with vehicle-treated or FSK/KCl-treated control neurons. E, Protein levels of Crtc1-dependent genes in noninfected (NI) or scramble (Scr)- or Crtc1 shRNA-infected neurons (10 DIV; n = 4–5 cultures per group); *p < 0.05, **p < 0.01 compared with scramble-FSK/KCl. Values are normalized to β-tubulin. F, ChiP analysis shows activity-dependent recruitment of Crtc1 to specific gene promoters. IgG, Irrelevant antibody. Data represent the mean ± SEM of three independent experiments; *p < 0.05, **p < 0.005, compared with IgG FSK/KCl IP. G, Expression of Arc (green) is evident in neurons expressing high Crtc1 levels (red; arrowheads) compared with neurons with very low Crtc1 levels (arrows) in CA3 hippocampus of WT trained mice. Scale bar, 20 μm. Statistical analysis was determined by one- or two-way ANOVA followed by Student-Newman–Keuls post hoc test.
- Figure 4.
Adeno-associated viral-mediated Crtc1 overexpression prevents early Aβ-induced transcriptional and memory deficits. A, Long-term Crtc1-myc expression in the mouse dorsal hippocampus. Overexpression of Crtc1-myc (green) in CA3 pyramidal neurons (NeuN, red) three weeks after stereotaxic intrahippocampal AAV-Crtc1-myc injection. Injection point is indicated in red in the brain diagram. Insets, Magnified images of the selected regions (square) showing Crtc1-myc localization in the neuronal nucleus (left inset) or cytoplasm (right inset). Scale bar, 50 μm. B, Increased Crtc1-myc and total Crtc1 mRNAs normalized to Gapdh in AAV-Crtc1-myc-injected mice. Data are the mean ± SEM (n = 4–5 mice/group); *p < 0.05, **p < 0.001, compared with AAV-GFP-injected control mice. C, Crtc1-myc protein levels in injected mice. Data are the mean ± SEM (n = 4 mice/group); **p < 0.001 compared with AAV-GFP-injected control mice. D, Overexpression of AAV-Crtc1 rescues spatial learning (top panel) and long-term memory (middle and bottom panels) deficits in 6-month-old APPSw,Ind mice. Data indicate percentage of time in the target quadrant or number of target platform crossings compared with the average of time or number of crossings in the three other quadrants, respectively. Data are the mean ± SEM (n = 8 mice/group); *p < 0.002, **p < 0.0001, compared with controls or the average of other quadrants as determined by two-way ANOVA. E, Crtc1-dependent gene expression normalized to the geometric mean of Gapdh, Hprt1, and Tbp in hippocampus of AAV-injected mice. Data represents the mean ± SEM (n = 4–5 mice/group); *p < 0.05 compared with WT-GFP; #p < 0.05 compared with APP-GFP mice. Statistical analyses were determined by one- or two-way ANOVA followed by Student-Newman–Keuls post hoc test.
- Figure 5.
CRTC1 levels and transcriptional changes in human brain at intermediate AD pathological stages. A, Levels of Arc, NR4A2, CRTC1, and CYR61 transcripts in the human hippocampus at Braak 0 (Control; n = 16), I–II (n = 22), III–IV (n = 14), and V–VI (n = 16) stages. Arc is significantly reduced at early (I–II) and intermediate (III–IV) Braak stages compared with controls (F(3,64) = 4.7, p < 0.005), whereas NR4A2 is reduced at intermediate stages. Gene changes in log2 scale relative to controls are normalized to the geometric mean of PPIA, GAPDH, and β-actin. Values represent mean ± SEM; *p < 0.05, **p < 0.02, compared with controls. B, Western blotting and quantification of total and phosphorylated (Ser151) CRTC1 (pCRTC1) in human hippocampus at different AD stages. Values represent mean fold change ± SEM (n = 5–12 per group); *p < 0.05 compared with control as determined by one-way ANOVA followed by Scheffé's S post hoc test.
Tables
- Table 1.
Genes and functional groups of the relevant gene network deregulated in the hippocampus of trained APPSw,Ind mice
Group GO term Associated Genes (%) Corrected p value Group/term genes 1 Learning 15.38 0.0004 Apbb1, Atp1a2, Chst10, Gabra5, Gria1, Hif1a, Mecp2, Neto1, Neurod2, Ntan1, Ptn, Vdac1 2 Regulation of neurological system 25.00 0.0002 Camk2a, Hras1, Mecp2, Mgll, Neto1, Neurod2, Ppp3ca, Prkce, Prkcz, Rnf10, Serpine2, Slc1a3, Snca, Ywhag 3 Long-term depression 15.28 0.00001 Adcy9, Calm1, Calm2, Camk2a, Camk2g, Gna11, Gnao1, Gria1, Gucy1b3, Hras1, Itpr1, Jun, Map3k4, Mapk1, Ppp2ca, Ppp2cb, Prkcb, Prkcc, Tubb3 4 Oxidative phosphorylation 10.74 0.00001 1110020P15Rik, Atp1a1, Atp1a2, Atp5o, Atp6v0a1, Atp6v0d1, Atp6v0e2, Atp6v1c1, Atp6v1d, Atp6v1g2, Cox15, Cox4i1, Cox7a2l, Cox7c, Cplx1, Ndufb2, Ndufb8, Ndufc2, Sdhb, Syt1, Vamp2 5 Long-term potentiation 18.84 0.001 Abl1, Adcy9, Atp1a1, Atp1a2, Calm1, Calm2, Camk2a, Camk2g, Crk, Gna11, Gnao1, Gng10, Gria1, Gucy1b3, Hras1, Itpr1, Jun, Map3k4, Mapk1, Ppp1ca, Ppp2ca, Ppp2cb, Ppp2r2c, Ppp2r5a, Ppp3ca, Ppp3cb, Prkcb, Prkcc, Rpl3, Skp1a, Slc12a2, Tubb3, Vamp2, Ywhag Significant nongrouped terms None Alzheimer's disease 10.53 0.0002 1110020P15Rik, Apbb1, Apoe, Atp5o, Calm1, Calm2, Capn2, Cox4i1, Cox7a2l, Cox7c, Itpr1, Lpl, Mapk1, Ndufb2, Ndufb8, Ndufc2, Ppp3ca, Ppp3cb, Sdhb, Snca None Spliceosome 11.43 0.0007 Cwc15, Dhx15, Hnrnpk, Hspa8, Lsm4, Lsm5, Ncbp2, Nhp2l1, Prpf19, Sf3a3, Sf3b5, Sfrs7, Snrpb, Snrpb2, Snrpd3, Syf2 None Glutamatergic synapse 10.85 0.004 Adcy9, Dlgap1, Glul, Gnao1, Gng10, Gria1, Itpr1, Mapk1, Ppp3ca, Ppp3cb, Prkcb, Prkcc, Slc1a3, Slc38a1 None Regulation of insulin secretion 26.32 0.0143 Hif1a, Nnat, Pfkm, Ppp3cb, Prkce None GTP binding 14.81 0.017 Arf1, Arf3, Arf5, Arl3, Gnao1, Hras1, Rraga, Rragb None Metencephalon development 13.12 0.039 Hspa5, Kat2a, Ldb1, Mecp2, Neurod2, Pfdn1, Sdf4, Serpine2 None GABAergic synapse 10.90 0.041 Adcy9, Gabarap, Gabarapl1, Gabra5, Glul, Gnao1, Gng10, Prkcb, Prkcc, Slc38a1 Genes are grouped according to their biological function as determined by GO analysis of the mouse microarray data using ClueGO v1.4. Associated genes indicates the percentage of changed genes of the total of genes of the term.
- Table 2.
Potential CREB target genes differentially expressed in the hippocampus of trained APPSw,Ind
Biological pathway/gene Gene GenBank Log fold-change p Biological function Metabolism Lactate dehydrogenase A Ldha NM_010699 −5.08 0.0001 Conversion of L-lactate to pyruvate ATP synthase subunit c1 Atp5g1 NM_007506 −4.00 0.001 ATP synthesis Glu oxaloacet transaminase1 Got1 NM_010324 −3.71 0.001 Aspartate aminotransferase activity Enolase 2/γ-enolase Eno2 NM_013509 −3.03 0.028 Glycolysis Na+/K + ATPase α1 Atp1a1 NM_144900 −2.37 0.01 Na+/K+ transport, ATP synthesis Isopentenyl-diphosphate δ-isomer1 Idi1 NM_145360 −1.86 0.008 Isoprenoid biosynthetic pathway Solute carrier family 38 Slc38a1 NM_134086 −1.38 0.011 Glutamine transporter Uncoupling protein 2 Ucp2 NM_011671 −1.12 0.041 Mitochondria proton uncoupling Pyruvate dehydrogenase kinase 4 Pdk4 NM_013743 1.78 0.019 Pyruvate metabolism N(α)-acetyltransferase 50 Naa50 NM_028108 1.12 0.025 Acetyltransferase activity Neurotransmission, plasticity, ves traff Neuritin/cpg 15 Nrn1 NM_153529 −4.34 0.001 Neuritogenesis, synaptic plasticity Secretogranin II Scg2 NM_009129 −3.76 0.007 Vesicle release, neuromodulation Glutamate receptor GluA1 Gria1 NM_008165 −3.00 0.005 Neurotransmission, memory Syntaxin 4A Stx4a NM_009294 −2.28 0.004 Docking of synaptic vesicles Synaptotagmin IV Syt4 NM_009308 −1.99 0.004 Exocitosis of synaptic vesicles Chromogranin A Chga NM_007693 −1.50 0.033 Vesicle release, neuromodulation RAB2A Rab2a NM_021518 −1.42 0.002 Protein transport from ER to Golgi Syntaxin 18 Stx18 NM_026959 −1.40 0.042 SNAP receptor Leucin rich repeat TM Neuronal 1 Lrtm1 NM_028880 −1.36 0.049 Synapse formation, axon traficking Cell adhesion/cytoskeleton Claudin 5 Cldn5 NM_013805 −2.92 0.02 Component of tigh junctions Neurofilament, light peptide Nefl NM_010910 −2.60 0.0001 Neurofilament member Fibronectin 1 Fn1 NM_010233 −2.38 0.042 Cell adhesion and migration Brain angiogenesis inh 1-ass.prot 2 Baiap2 NM_130862 −2.25 0.01 Actin cytoskeleton, neuritogenesis Angio-associated migratory protein Aamp NM_146110 −1.69 0.003 Cell migration Chondroitin sulfate proteoglycan 5 Cspg5 NM_013884 −1.64 0.013 Dendritic branching and synapses Calsyntenin 3 Clstn3 NM_153508 −1.46 0.005 Cell adhesion, associative learning Myelin oligodendrocyte glycoprotein Mog NM_010814 −1.18 0.002 Maintenance myelin sheath Neurocan Ncan NM_007789 1.14 0.03 Neuronal adhesion, neurite growth Cell signaling Protein phosph 1, reg sub 11 Ppp1r11 NM_029632 −2.29 0.02 Inhibitor of PP1 TrkB Ntrk2 NM_008745 −2.16 0.02 BDNF, NT-3/4/5 receptor P tyrosine phosphatase 4a1 Ptp4a1 NM_011200 −2.15 0.03 Protein tyrosine phosphatase Transducer of ErbB-2.1 Tob1 NM_009427 −1.93 0.02 Antiproliferation, learning/memory Regulator of G-protein signaling 4 Rgs4 NM_009062 −1.83 0.02 Regulates GTPase activity Cyclin-dependent kinase 5 Cdk5 NM_007668 −1.32 0.002 Neurodegen., associative memory Disabled homolog1 Dab1 NM_010014 −1.13 0.03 Adapter molecule, neural develop. Cyclin-dependent kinase 16 Cdk16 NM_011049 −1.09 0.03 Neurite outgrowth, neuron migration Transcriptional regulation Histone cluster 1, H2bj Hist1h2bj NM_178198 −3.58 0.001 Compaction of chromatin Activating Transcriptional factor 4 Atf4 NM_009716 −3.29 0.004 Transcription activator, binds to CRE Jun oncogene Jun NM_010591 −2.64 0.002 Transcription factor Inhibitor of DNA binding 2 Id2 NM_010496 −2.3 0.032 Inhibitor of transcription factors Nuclear receptor sub 4, 2 Nr4a2 NM_0113613 −1.80 0.007 Transcription factor Nuclear receptor sub 4, 1 Nr4a1 NM_010444 −1.74 0.028 Transcription factor Polymerase (RNA) II polypeptide K Polr2k NM_023127 1.013 0.042 RNA polymerase Translation/cell survival Poly(A)binding protein, cytoplas. 1 Pabpc1 NM_008774 −3.75 0.003 Poly(A) translation, initiation Translation initiation factor 3, sub D Eif3d NM_018749 −1.92 0.009 Component of the eIF-3 complex Programmed cell death 7 Pdcd7 NM_016688 −1.43 0.025 Promotes apoptosis Protein Degradation Myeloid leukemia factor 2 Mlf2 NM_145385 −3.03 0.0001 Protein degradation tagging activity Ubiquitin-conjugated enzyme E2G 1 Ube2g1 NM_025985 −1.31 0.041 Attachment of ubiquitin to proteins Unclassified Abhydrolase domain containing 11 Abhd11 NM_145215 −2.38 0.001 Deleted Williams-Bernes syndrome Relative mRNA levels in the hippocampus of APPSw,Ind mice compared to nontransgenic control mice after spatial training in the MWM. Relevant genes from microarray hybridizations are listed in each column, with log2-fold changes indicating relative decrease (<1) or increase (>1) of mRNA levels in APPSw,Ind mice compared with controls. Genes are grouped according to their biological pathway and relative gene expression changes. A gene could be assigned to more than one biological function term. Only genes with p < 0.05 are given.
Braak stage n Sex Age PMD (h) RIN Control 16 6F/10M 49.9 ± 7..8 7.1 ± 3.6 6.22 ± 1.2 I–II 22 4F/18M 69.6 ± 10.2 6.0 ± 3.6 6.26 ± 0.9 III–IV 14 8F/6M 78.6 ± 6.3 5.0 ± 3.9 6.49 ± 1.0 V–VI 16 8F/8M 79.8 ± 7.2 7.4 ± 5.0 6.27 ± 0.9 Data are represented as mean ± SD. F, Female; M, male; PMD, postmortem delay; h, hours; RIN, RNA integrity number.
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