hippocampal lrp4 expression and morphological ... and cortex. (a) lrp4 expression in the hippocampus...

Supplementary Figure 1

Hippocampal Lrp4 expression and morphological characterization of GFAP-Lrp4–/– hippocampus and cortex.

(a) Lrp4 expression in the hippocampus at different stages. Shown were representative blots of two independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (b) Quantitative analysis of Lrp4 expression in a. n =3 mice. Lrp4 band density was normalized by the loading control β-actin; values of postnatal day 0 in (a) were taken as 100%. (c) Cortical sections of control (top) and GFAP-Lrp4–/– (bottom) mice were stained for neuron marker NeuN. Enlarged images of areas in dotted square were shown on the right. Scale bar: left, 250 μm; right, 50 μm. (d) Quantification of NeuN+ cells in different cortical layers. n = 8 slices of 3 mice for both genotypes. Two-way ANOVA, F(1,70) = 3.736, p = 0.0573. (e) Dorsal hippocampal sections of control (top) and GFAP-Lrp4–/– (bottom) mice were stained for neuron marker NeuN. Images in dotted areas (marked as a’ in CA1 and a’’ in CA3) were enlarged and shown on the right. Scale bar: left, 200 μm, right, 20 μm. (f) Quantification of NeuN+ cells in CA1 and CA3 regions in dorsal hippocampus. n = 10 slices, 3 mice for both genotypes. Two-way ANOVA, F(1,36) = 0.1503, p = 0.7005. Data in (b) were presented as mean ± s.e.m; data in (d, f) were presented as median with interquartile range, whiskers are the minimum and maximum; p > 0.05.

Nature Neuroscience: doi:10.1038/nn.4326


Synaptic protein expression and dendritic morphology of CA1 pyramidal neurons in GFAP-Lrp4–/– mice.

(a) Hippocampal tissues were collected from one-month-old control and GFAP-Lrp4–/– mice and homogenized for western blotting. Shown were representative blots of three independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (b) Quantitative analysis of data in (a). n =5 mice of each genotype for Lrp4, Synaptophysin, GluR1, GluR2 and GABAα1; n = 4 mice of each genotype for PSD95 and Gephyin; n = 3 mice of each genotype for GluN1, GluN2A and GluN2B. Band densities of interested proteins were normalized by the loading control β-actin; values of control mice were taken as 100%. Paired student’s t test; Lrp4, t(4) = 28.05, p < 0.0001; PSD95, t(3) = 0.4635, p = 0.6746; Gephyrin, t(3) = 1.54, p = 0.2212; Synaptophysin, t(4) = 0.3952, p = 0.7128; GluR1, t(4) = 0.2008, p = 0.8507; GluR2, t(4) = 0.8446, p = 0.4459; GluN1, t(2) = 0.003932, p = 0.9972; GluN2A, t(2) = 0.3696, p = 0.7472; GluN2B, t(2) = 0.6231, p = 0.5968; GABARα1, t(4) = 2.008, p = 0.1151. (c) Representative traced CA1 pyramidal neurons were superimposed with concentric circles in Sholl analysis. The radius interval between circles was 20 µm per step, ranging from 10 µm to 290 µm from the center of the neuronal soma. (d) Quantitative analysis of data in (c). n = 15 neurons from 3 mice per genotype. Two-way ANOVA, F(1,363) = 0.1266, p = 0.7222.. Data in (b) were presented as median with interquartile range, whiskers are the minimum and maximum; data in (d) were presented as mean ± s.e.m; p > 0.05.



Electrophysiological characterization of CA1 pyramidal neurons from GFAP-Lrp4–/– mice.

(a) Representative traces of spikes in CA1 pyramidal neurons evoked by injecting depolarizing currents of 50 pA. Scale bar: 100 ms, 20 mV. (b) Firing rate plotted against increasing injected currents. n = 9 neurons, 3 control mice; n = 8 neurons, 3 GFAP-Lrp4–/– mice. Two-way ANOVA, F(1,150) = 0.006, p = 0.9396. (c, d) Spike threshold evoked by 50-pA currents and input resistance. n = 9 neurons, 3 control mice; n = 8 neurons, 3 GFAP-Lrp4–/– mice. For (c), student’s t test; t(15) = 0.2535, p = 0.8033; for (d), student’s t test, t(15) = 0.7621, p = 0.4578. (e) Representative traces of EPSCs evoked at holding potentials (–60 mV to + 60 mV at a step of 20 mV). Scale bar: 20 ms, 60 pA. (f) Current-voltage plot of peak currents mediated by AMPAR. n = 11 neurons of 3 mice for both genotypes. Two-way ANOVA, F(1,140) = 1.449, p = 0.2307. (g) Current-voltage plot of NMDAR EPSCs measured 50 ms after the peak of AMPAR EPSCs. n = 11 neurons of 3 mice for both genotypes. Two-way ANOVA, F(1,140) = 0.022, p = 0.8825. (h) AMPA/NMDA ratio. The ratio was calculated by dividing the amplitude of AMPAR currents measured at –60 mV by the amplitude of NMDAR currents measured 50 ms after the peak at + 40 mV. n = 11 neurons of 3 mice for both genotypes. Student’s t test, t(20) = 0.4578, p = 0.652. Data in (b, f and g) were presented as mean ± s.e.m; data in (c, d and h) were presented as median with interquartile range, whiskers are the minimum and maximum; p > 0.05.



Characterization of spines of CA1 pyramidal neurons of GFAP-Lrp4–/– mice.

(a) Representative Golgi staining images of primary and second/tertiary (sec/ter) dendrites of CA 1 pyramidal neurons of control and GFAP-Lrp4–/– mice. Scale bar, 10 μm. (b) Quantitative analysis of data in (a). For primary dendrites, n = 46 segments, 46 neurons from 3 mice per genotype, student’s t test, t(90) = 2.055, p = 0.0427; For sec/ter dendrites, n = 80 segments, 46 neurons from 3 control mice, n = 76 segments, 42 neurons from GFAP-Lrp4–/– mice, student’s t test, t(154) = 3.195, p = 0.0017. (c) Representative EM images of CA1 synapses from control and GFAP-Lrp4–/– mice. Arrows, PSD; arrowheads, vesicles. Scale bar: 200 nm. (d) Quantitative analysis of PSD length. n = 63 synapses, 3 control mice; n = 67 synapses, 3 GFAP-Lrp4–/– mice. Student’s t test, t(128) = 1.28, p = 0.2029. (e) Quantitative analysis of synaptic vesicle density. n = 61 synapses, 3 control mice; n = 64 synapses, 3 GFAP-Lrp4–/– mice. Student’s t test, t(123) = 0.3366, p = 0.737. (f) Quantitative analysis of vesicle diameter. n = 249 vesicles, 3 control mice; n = 263 vesicles, 3 GFAP-Lrp4–/– mice. Student’s t test, t(510) = 0.7743, p = 0.4391. (g) Quantitative analysis of astrocyte-synaptic membrane distance. n = 164, 3 control mice; n = 172, 3 GFAP-Lrp4–/– mice. Student’s t test, t(334) = 1.339, p = 0.1815. Data in (b, d, e, f and g) were presented as median with interquartile range, whiskers are the minimum and maximum. *, p < 0.05; **, p < 0.01.



Lrp4 expression in astrocytes and characterization of GFAP::CreER mice.

(a) Lrp4 was detectable in homogenate of control, but not GFAP-Lrp4–/–, astrocytes in culture. Astrocytes were isolated from P2-3 mice, cultured in vitro for 7 days and lysed for western analysis. Shown were representative blots of more than three independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (b) Top, GFAP::CreER mice were crossed with reporter mice (tdTomato) to generate GFAP::CreER;tdTomato mice. Bottom, GFAP::CreER;tdTomato at P30 were administered by gavage one time with tamoxifen (100 mg/kg) and sacrificed at P35. (c) Co-localization of tdTomato with astrocytic marker GFAP. Sections were stained with antibodies against NeuN and GFAP. Arrowheads denote colocalization between tdTomato and GFAP. Scale bar: top, 200 μm; bottom, 30 μm. (d) Representative Golgi staining images of primary and second/tertiary (sec/ter) dendrites in CA 1 pyramidal neurons of control and GFAP-CreER;Lrp4–/– mice. Scale bar, 10 μm. (e) Quantitative analysis of data in (d). For primary dendrites, n = 47 segments, 47 neurons from 3 control mice; n = 40 segments, 40 neurons from 3 GFAP-CreER;Lrp4–/– mice, student’s t test, t(85) = 1.011, p = 0.3151. For sec/ter dendrites, n = 74 segments, 47 neurons from 3 control mice; n = 77 segments, 40 neurons from 3 GFAP-CreER;Lrp4–/– mice, student’s t test, t(149) = 1.434, p = 0.1537. Data in e were presented as median with interquartile range, whiskers are the minimum and maximum; p > 0.05.



Similar sEPSCs and PPR between control and Camk2a-Lrp4–/– hippocampal pyramidal neurons.

(a) Cre activity was expressed in neurons in Camk2α::Cre mice. Shown were a hippocampal slice of Camk2α::Cre;tdTomato mice stained with neuronal marker NeuN (green) and astrocytic marker GFAP (blue) on the left. Images in the dotted areas (marked as a’ in CA1 and a’’ in CA3) were enlarged and shown on the right. Scale bar: left: 200 μm; right: 30 μm. (b) Lrp4 level in the hippocampus was comparable between one-month-old control and Camk2α -Lrp4–/– mice. Shown were representative blots of three independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (c) Quantitative analysis of data in (b). n = 6 hippocampi from 3 mice for each genotype. Lrp4 band density was normalized by the loading control β-actin; values of control mice were taken as 100%. Paired student’s t test, t(5) = 1.297, p = 0.2512. (d), Diagrams showing recording of pyramidal neuron in whole-cell patch configuration. Note that blue color denotes Lrp4 mutant and red color denotes control neurons. (e) Representative sEPCS traces of CA1 pyramidal neurons of control and Camk2α-Lrp4–/– mice. Scale bar: 2 s, 10 pA. (f, g) No difference in sEPSC frequency (f) and amplitude (g). n = 10 neurons of 3 mice for both genotypes. For (f), student’s t test, t(18) = 0.6552, p = 0.5206; for (g), student’s t test, t(18) = 0.3296, p = 0.7455. (h) No difference in paired-pulse ratios between control and mutant neurons. n = 6 neurons, 3 control mice; n = 8 neurons, 3 Camk2α-Lrp4–/– mice. Two-way ANOVA, F(1,35) = 0.02, p = 0.8843. Data in (c and h) were presented as mean ± s.e.m; data in (f and g) were presented as median with interquartile range, whiskers are the minimum and maximum; p > 0.05.



Characterization of processes and numbers of GFAP-Lrp4–/– astrocytes in vivo and in vitro.

(a) Cortical sections of control (top) and GFAP-Lrp4–/– (bottom) mice were stained for GFAP. Enlarged images of dotted area were shown on the right. SR: Stratum radiatum; SLM: stratum lacunosum- moleculare; ML: molecular layer. Scale bar: left, 150 μm; right, 30 μm. (b) Representative traced astrocytes were superimposed with concentric circles in Sholl analysis. The radius interval between circles was 5 µm per step, ranging from 10 µm to 50 µm from the center of the astrocytic soma. (c) Quantification of GFAP+ cells in SC region and Molecular Layers (ML). n = 6 slices of 3 control mice; n = 7 slices of 3 GFAP-Lrp4–/– mice. Two-way ANOVA, F(1,22) = 2.236, p = 0.149. (d) Quantitative analysis of total process length of astrocytes. n = 32 astrocytes, 3 control mice; n = 31 astrocytes, 3 GFAP-Lrp4–/– mice. Student’s t test, t(61) = 1.339, p = 0.1856. (e) Quantitative analysis of soma size of astrocytes. n = 70 astrocytes, 3 control mice; n = 65 astrocytes, 3 GFAP-Lrp4–/– mice. Student’s t test, t(133) = 0.6318, p = 0.5286. (f) Quantitative analysis of process intersections. n = 32 astrocytes, 3 control mice; n = 31 astrocytes, 3 GFAP-Lrp4–/– mice. Two-way ANOVA, F(1,486) = 2.814, p = 0.0941. (g) Representative images of neurons. Neurons were stained with MAP2 and DAPI, as indicated in green and blue, respectively. Scale bar: 50 μm.


(h) Quantitative analysis of MAP2+ cells. n = 8 coverslips for both genotypes. Student’s t test, t(14) = 0.2747, p = 0.7876. (i) Representative images of astrocytes. Astrocytes were stained with GFAP and DAPI, as indicated in green and blue, respectively. Scale bar: 20 μm. (j) Quantitative analysis of GFAP+ cells. n = 8 coverslip for control group; n = 9 coverslip for GFAP-Lrp4–/– group. Student’s t test, t(15) = 0.074, p = 0.9419. Data in (c-e, h and j) were presented as median with interquartile range, whiskers are the minimum and maximum; data in (f) were presented as mean ± s.e.m; p > 0.05.



Effects of pharmacological chemicals on sEPSC frequency. Hippocampal slices of control mice were treated with different concentrations of indicated chemicals. sEPSCs were recorded in the CA1 pyramidal neurons. sEPSC frequency prior to treatment of each chemical was taken as 100% (Baseline). n = 6 and 8 for 10 µM and 100 µM, respectively for DL-AP5 (a), paired student’s t test, baseline vs 10 µM, t(5) = 2.311, p = 0.0688; baseline vs 100 µM, t(7) = 2.87, p = 0.024; n = 6 for both 1 µM and 10 µM, respectively for ATP (b), paired student’s t test, baseline vs 1 µM, t(6) = 2.26, p = 0.0645; baseline vs 10 µM, t(6) = 7.926, p = 0.0002; n = 6 and 8 for 10 µM and 300 µM, respectively for AOPCP (c), paired student’s t test, baseline vs 10 µM, t(5) = 1.943, p = 0.1097; baseline vs 300 µM, t(7) = 3.115, p = 0.017; n = 6 and 10 for 1 µM and 10 µM, respectively for Suramin (d), paired student’s t test, baseline vs 1 µM, t(5) = 1.604, p = 0.1697; baseline vs 10 µM, t(8) = 3.072, p = 0.0153; n = 5 and 7 for 10 nM and 800 nM, respectively for DPCPX (e), paired student’s t test, baseline vs 10 nM, t(4) = 1.012, p = 0.3688; baseline vs 800 nM, t(6) = 2.927, p = 0.0264; n = 6 and 8 for 1.5 µM and 5 µM, respectively for SCH58261 (f), paired student’s t test, baseline vs 1.5 µM, t(5) = 1.613, p = 0.1677; baseline vs 5 µM, t(7) = 2.556, p = 0.0378. Data were presented as mean ± s.e.m; *, p < 0.05; **, p < 0.01.



No effect of agrin on astrocyte proliferation.

(a) Lrp4 surface expression in astrocytes. Cultured astrocytes were treated with biotin. Biotin-labeled proteins were precipitated with Avidin agarose beads. Cell surface fraction and 10% cell lysates were probed for Lrp4 and TFR (transferin receptor protein). Shown were representative blots of two independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (b) Agrin activation of MuSK in muscle cells. C2C12 myotubes were treated without or with agrin (100 ng/ml) for 20 min. MuSK was isolated by immunoprecipitation and probed with p-tyrosine antibody to detect phosphorylated MuSK. Shown were representative blots of three independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (c) Quantitative analysis of data in (b). n = 3 times for control group, n = 4 times for agrin group. p-Tyrosine band density was normalized by total MuSK; values of control mice were taken as 100%. Student’s t test, t(5) = 3.137, p = 0.0257. (d) C2C12 myotubes were stimulated with or without agrin (100 ng/ml) for 16 h. AChR clusters were stained by R-BTX. Arrows, AChR clusters. Scale bar: 50 μm. (e) Quantitative analysis of data in (d). n = 8 images for each group. Student’s t test, t(14) = 6.966, p < 0.0001.


(f) Effect of agrin on proliferating astrocytes. Representative images of control (left) and GFAP-Lrp4–/–

astrocytes (right) with or without agrin treatment. Astrocytes were stained with Ki67 and GFAP, respectively. Scale bar: 40 μm. (g) Quantitative analysis of Ki67+ cells. n = 12 coverslip for each group. Data were presented as mean ± s.e.m. One-way ANOVA, F(1,44) = 0.188, p = 0.904. Data were presented as mean ± s.e.m; *, p < 0.05; **, p < 0.01.



Characterization of GFAP-Lrp4–/– mice in elevated plus maze, light/dark box, and water maze.

(a) Representative traces of elevated plus maze tests. Scale bar: 5 cm. (b, c, d) No difference in duration in the open arms, number of entries to open arms, latency to open arms. n = 9 mice for each genotype. For (b), student’s t test, t(16) = 0.1493, p = 0.8832; for (c), student’s t test, t(16) = 0.2916, p = 0.7744; for (d), student’s t test, t(16) = 0.8321, p = 0.4176. (e) Representative traces in the light/dark box test. Scale bar: 5 cm. (f, g, h) No difference in duration in light box, number of entries to light box, latency to light box. n = 11 mice for each genotype. For (f), student’s t test, t(20) = 0.8256, p = 0.4188; for (g), student’s t test, t(20) = 0.701, p = 0.4914; for (h), student’s t test, t(20) = 0.6747, p = 0.5076. (i) Quantitative analysis of velocity in Morris water maze of two genotypes. n = 9 mice for each genotype. Student’s t test; t(16) = 0.6239, p = 0.5415. (j) Time-course analysis of duration in the N30 area in probe test. n = 9 mice for each genotype. Repeated two-way ANOVA, for genotype and time interaction: F(5, 80) = 1.032, p = 0.4047. (k) Time-course analysis of platform crossing numbers in probe test. n = 9 mice for each genotype. Repeated two-way ANOVA, for genotype and time interaction: F(5, 80) = 0.1515, p = 0.979. Data were presented as mean ± s.e.m; p > 0.05.



A working model for Lrp4 in astrocytes to modulate glutamatergic transmission.

Lrp4 in astrocytes serves as a receptor for agrin and controls ATP release from astrocytes. ATP is hydrolyzed to adenosine, which suppresses glutamate release by activating A1 purinergic receptors.



Full-length pictures of the blots presented in the main figures and supplementary figures.

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hippocampal lrp4 expression and morphological ... and cortex. (a) lrp4 expression in the hippocampus...

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