Lrp4 in astrocytes modulates glutamatergic transmission

Abstract

Neurotransmission requires precise control of neurotransmitter release from axon terminals. This process is regulated by glial cells; however, the underlying mechanisms are not fully understood. We found that glutamate release in the brain was impaired in mice lacking low-density lipoprotein receptor-related protein 4 (Lrp4), a protein that is critical for neuromuscular junction formation. Electrophysiological studies revealed compromised release probability in astrocyte-specific Lrp4 knockout mice. Lrp4 mutant astrocytes suppressed glutamatergic transmission by enhancing the release of ATP, whose level was elevated in the hippocampus of Lrp4 mutant mice. Consequently, the mutant mice were impaired in locomotor activity and spatial memory and were resistant to seizure induction. These impairments could be ameliorated by blocking the adenosine A1 receptor. The results reveal a critical role for Lrp4, in response to agrin, in modulating astrocytic ATP release and synaptic transmission. Our findings provide insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity.

Figure 1. Reduced EPSC frequency and impaired synaptic plasticity in GFAP-Lrp4^−/− mice

(a) Lrp4^f/f mice were crossed with GFAP::Cre mice; resulting GFAP::Cre;Lrp4^f/+ mice were crossed with Lrp4^f/+ mice to generate GFAP::Cre;Lrp4^f/f (GFAP-Lrp4^−/−) and Lrp4^f/f (Control) mice. (b) Lrp4 was not detectable in hippocampus of one-month-old GFAP-Lrp4^−/− mouse. 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 pairs of mice. 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₍₂₎ = 128.3, p < 0.0001. (d) Recording diagrams. Pyramidal neurons were recorded in whole-cell configuration. Blue color denotes Lrp4 mutant. (e) Representative traces of sEPSCs in CA1 pyramidal neurons from control and GFAP-Lrp4^−/− mice. Scale bar: 2 s, 10 pA. (f, g) Cumulative probability plots of sEPSC inter-event intervals and histograms of sEPSC frequency (f) and amplitude (g). n = 15 neurons of 4 mice for both genotypes. For (f), student’s t test, t₍₂₈₎ = 2.538, p = 0.017; for (g), student’s t test, t₍₂₈₎ = 0.9343, p = 0.3581. (h) Representative traces of mEPSCs in CA1 pyramidal neurons from control and GFAP-Lrp4^−/− mice. Scale bar: 2 s, 10 pA. (i, j) Cumulative probability plots of mEPSC inter-event intervals and histograms of mEPSC frequency (i) and amplitude (j). n = 29 neurons, 5 control mice; n = 22 neurons, 5 GFAP-Lrp4^−/− mice. For (i), student’s t test, t₍₄₉₎ = 2.12, p = 0.0391; for (j), student’s t test, t₍₄₉₎ = 0.6891, p = 0.494. (k) Depressed I/O curves in the hippocampus of GFAP-Lrp4^−/− mice. fEPSPs were recorded by stimulating SC-CA1 pathway with gradual increasing intensities. n = 9 slices from 4 mice for both genotypes. Two-way ANOVA, F_{(1, 112)} = 20.35, p < 0.0001. (l) Impaired LTP at SC-CA1 synapses in the hippocampus of GFAP-Lrp4^−/− mice. Normalized fEPSP slopes were plotted every 1 min. Arrow denotes LTP induction. Shown on the right were representative traces taken before (a) and 50 min after high frequency stimulation (b). Scale bar: 2 ms, 0.2 mV. (m) Quantitative analysis of LTP level in (l). n = 9 slices from 4 mice for both genotypes. Student’s t test, t₍₁₆₎ = 2.879, p = 0.0109. Data in (c and k) were presented as mean ± s.e.m; data in (f, g, i, j and m) were presented as median with interquartile range, whiskers are the minimum and maximum; *, p < 0.05; **, p <0.01.

Figure 2. Reduced excitatory vesicle release probability in the absence of Lrp4

(a) Representative superimposed sweeps with three different inter-stimulus intervals of pair-pulse stimulations from control and GFAP-Lrp4^−/− mice. Scale bar, 50 ms, 100 pA. (b) Paired-pulse ratios plotted against inter-stimulus intervals. n = 11 neurons, 3 mice for both control and GFAP-Lrp4^−/− genotypes. Two-way ANOVA, F_(1,60) = 13.55, p = 0.0005 (c) Representative traces of NMDAR currents in the presence of MK-801 (40 µm). Scale bar, 20 ms, 40 pA. (d) Normalized NMDAR currents plotted against stimulus number. The first pulse given in the presence of MK-801 was considered as 100%. (e) Increased τ values in GFAP-Lrp4^−/− mice. n = 7 neurons, 3 mice for both genotypes. Student’s t test, t₍₁₂₎ = 3.594, p = 0.0037. (f) Representative 10 successive individual sweeps of EPSCs evoked by minimal stimulation. Scale bar, 5 ms, 10 pA. (g, h and i) Reduced release probability, reduced synaptic efficacy, and no change in synaptic potency. n = 13 neurons, 4 control mice; n = 14 neurons, 4 GFAP-Lrp4^−/− mice. For (g), student’s t test, t₍₂₅₎ = 2.682, p = 0.0128; for (h), student’s t test, t₍₂₅₎ = 2.615, p = 0.0149; for (i), student’s t test, t₍₂₅₎ = 0.1254, p = 0.9012. Data in (b) were presented as mean ± s.e.m; data in (e and g-i) were presented as median with interquartile range, whiskers are the minimum and maximum; *, p < 0.05; **, p < 0.01.

Figure 3. Astrocytic Lrp4 is critical for glutamatergic transmission

(a) Enriched β-gal activity in SLM and ML layers. X-gal staining was carried out with coronal brain sections of muscle rescued Lrp4-LacZ homozygous mice. Left, whole brain section; right, enlarged dotted area of the left image. SO: Stratum oriens; SP: Stratum pyramidale; SR: Stratum radiatum; SLM: stratum lacunosum- moleculare; ML: molecular layer. Scale bars, 2 mm (left), 0.2 mm (right). Shown were representative images of more than three independent experiments with similar results. (b) Co-localization of β-gal with astrocytic marker GFAP. Sections were stained with antibodies against β-gal and GFAP or neuronal marker NeuN. Arrow heads, cells positive for β-gal and GFAP; arrows, cells positive for NeuN, but not β-gal. Scale bar, 20 µm (left), 5 µm (right). Shown were representative images of three independent experiments with similar results. (c) Reduced Lrp4 level in the hippocampus in GFAP-CreER;Lrp4^−/− mice, two weeks after tamoxifen injection. Shown were representative blots of three independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (d) Quantitative analysis of data in (c). n = 4 pairs of mice. 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₍₃₎ = 3.727, p = 0.0337. (e) Recording diagrams. Pyramidal neurons were recorded in whole-cell configuration. Blue color denotes Lrp4 mutant; red color indicate control. (f) Representative sEPCS traces of CA1 pyramidal neurons of control and GFAP-CreER;Lrp4^−/− mice. Scale bar: 1 s, 10 pA. (g) Reduced sEPSC frequency in CA1 pyramidal neurons of GFAP:: CreER;Lrp4^−/− mice. n = 13 neurons, 4 control mice; n = 14 neurons, 4 GFAP-CreER;Lrp4^−/− mice. Student’s t test, t₍₂₅₎ = 2.145, p = 0.0419. (h) sEPSC amplitude was comparable between control and GFAP-CreER;Lrp4^−/− hippocampus. n = 13 neurons, 4 control mice; n = 14 neurons, 4 GFAP-CreER;Lrp4^−/− mice. Student’s t test, t₍₂₅₎ = 0.3502, p = 0.7291. (i) Representative traces of mEPSCs in CA1 pyramidal neurons from Control and GFAP-CreER;Lrp4^−/− mice. Scale bar: 2 s, 10 pA. (j, k) Reduction in mEPSC frequency (j), but not amplitude (k). n = 13 neurons, 3 control mice; n = 12 neurons, 3 GFAP-CreER;Lrp4^−/− mice. For (j), student’s t test, t₍₂₃₎ = 2.368, p = 0.0266; for (k), t₍₂₃₎ = 0.2528, p = 0.8027. (l) Paired-pulse ratios plotted against inter-stimulus intervals. n = 10 neurons, 4 control mice; n = 11 neurons, 4 GFAP-CreER;Lrp4^−/− mice. Two-way ANOVA, F_(1,57) = 16.15, p = 0.0002. (m) Increased τ values in GFAP-CreER;Lrp4^−/− mice. n = 7 neurons, 4 control mice; n = 8 neurons, 4 GFAP-CreER;Lrp4^−/− mice. Student’s t test, t₍₁₃₎ = 2.32, p = 0.0373. (n) Depressed I/O curves in the hippocampus of GFAP-CreER;Lrp4^−/− mice. fEPSPs were recorded by stimulating SC-CA1 pathway with gradual increasing intensities. n = 8 slices from 4 control mice; n = 7 from 4 GFAP-CreER;Lrp4^−/− mice. Two-way ANOVA, F_{(1, 91)} = 14.52, p = 0.0003. (o) Impaired LTP at SC-CA1 synapses in the hippocampus of GFAP-CreER;Lrp4^−/− mice. Normalized fEPSP slopes were plotted every 1 min, Arrow denotes LTP induction. Shown on the right were representative traces taken before (a) and 50 min after high frequency stimulation (b). Scale bar: 4 ms, 0.4 mV. (p) Quantitative analysis of LTP level in (o). n = 8 slices from 4 control mice; n = 7 from 4 GFAP-CreER;Lrp4^−/− mice. Student’s t test, t₍₁₃₎ = 2.354, p = 0.035. Data in (d, g, h, j, k, m and p) were presented as median with interquartile range, whiskers are the minimum and maximum; data in (l and n) were presented as mean ± s.e.m; *, p < 0.05; **, p < 0.01.

Figure 4. Astrocytic Lrp4 affects glutamatergic transmission in a non-contact way

(a) Experimental design for co-culture. Neurons and astrocytes were isolated from control (red) or mutant (blue; GFAP-Lrp4^−/−) mice and cultured in separate dishes. After maturation, one cover slip of neurons was co-cultured with three cover slips of astrocytes in different combinations. (b) Representative sEPSC traces of neurons co-cultured with astrocytes. Genotypes of neurons and astrocytes are color-coded: red, control; blue, mutant. Scale bar: 2 s, 10 pA. (c, d) Quantitative analysis of sEPSC frequency (c) and amplitude (d). 3 times of cell culture; n = 11 control neurons co-cultured with control astrocytes; n = 9 control neurons co-cultured with mutant astrocytes; n = 8 mutant neurons co-cultured with control astrocytes; n = 8 mutant neurons co-cultured with mutant astrocytes. For (c), one-way ANOVA, F_(3,32) = 5.652, p = 0.0032. Control neuron + control astrocyte vs control neuron + mutant astrocyte, student’s t test, t₍₁₈₎ = 2.532, p = 0.0209; control neuron + control astrocyte vs mutant neuron + control astrocyte, student’s t test, t₍₁₇₎ = 0.6137, p = 0.5475; mutant neuron + control astrocyte vs mutant neuron + mutant astrocyte, student’s t test, t₍₁₄₎ = 3.573, p = 0.0031. For (d), one-way ANOVA, F_(3,32) = 0.8197, p = 0.4926. Data in (c and d) were presented as median with interquartile range, whiskers are the minimum and maximum; n.s., not significant; *, p < 0.05; **, p < 0.01.

Figure 5. Modulation of astrocytic ATP release by Lrp4

(a) Effects of chemicals on sEPSCs of CA1 pyramidal neurons in Control and GFAP-Lrp4^−/− mice. ΔsEPSC frequency was calculated by (f2 - f1)/f1, where f1 and f2 were the frequencies of sEPSCs recorded before and after drug treatment, respectively. The number of recorded neurons and mice were as follows: 8 neurons of 3 control mice and 9 neurons of 3 GFAP-Lrp4^−/− mice for DL-AP5 (100 µM). Student’s t test, t₍₁₅₎ = 0.2202, p = 0.8287; 7 neurons of 3 mice of each genotype for ATP (10 µM). Student’s t test, t₍₁₂₎ = 2.944, p = 0.0123; 8 neurons of 4 mice of each genotype for ARL67156 (100 µM). Student’s t test, t₍₁₄₎ = 2.675, p = 0.0181; 8 neurons of 3 mice of each genotype for AOPCP (300 µM). Student’s t test, t₍₁₄₎ = 2.483, p = 0.0263; 9 neurons of 3 mice of each genotype for Suramin (10 µM). Student’s t test, t₍₁₆₎ = 0.4528, p = 0.6567; 7 neurons of 3 control mice and 8 neurons of 3 GFAP-Lrp4^−/− mice for DPCPX (800 nM). Student’s t test, t₍₁₃₎ = 2.451, p = 0.0291; 8 neurons of 3 control mice and 7 neurons of 3 GFAP-Lrp4^−/− mice for SCH58261 (5 µM). Student’s t test, t₍₁₃₎ = 0.8303, p = 0.4213. (b) Increased ATP levels in the dialysate from GFAP-Lrp4^−/− and tamoxifen-treated GFAP-CreER;Lrp4^−/− hippocampus and condition medium of mutant astrocytes, but not neurons. Cells were cultured from control and GFAP-Lrp4^−/− (mutant) mice. n = 5 samples of dialysate from 5 control and 5 GFAP-Lrp4^−/− mice, Student’s t test, t₍₈₎ = 3.33, p = 0.0104; n = 4 samples of dialysate from 4 control and 4 GFAP-CreER;Lrp4^−/− mice; Student’s t test, t₍₆₎ = 3.565, p = 0.0119; n = 8 dishes (4 mice) for astrocytes, Student’s t test, t₍₁₄₎ = 2.276, p = 0.0391; n = 5 dishes (4 mice) for neurons, Student’s t test, t₍₈₎ = 0.7401, p = 0.4804 (c) Western blotting showing similar level of ATP5a in cultured astrocytes of control and mutant genotype. Shown were representative blots of two independent experiments with similar results. Full-length blots/gels are presented in Supplementary Figure 12. (d) Quantitative analysis of data in (c). n = 4 dishes from 2 times of astrocyte culture for each genotype. ATP5a band density was normalized by the loading control β-actin; values of control mice were taken as 100%. Paired student’s t test, t₍₃₎ = 2.923, p = 0.0613. (e) Similar levels of total ATP in mutant hippocampal astrocytes. Cultured astrocytes were homogenized. Supernatants after centrifugation were subjected to ATP measurement. n = 8 dishes from 2 times of culture for both genotypes. Paired student’s t test, t₍₃₎ = 0.4237, p = 0.6845. (f) Increased adenosine levels in the dialysate from GFAP-Lrp4^−/− and tamoxifen-treated GFAP-CreER;Lrp4^−/− hippocampus and condition medium of mutant astrocytes, but not neurons. n = 5 samples of dialysate from 5 control and 5 GFAP-Lrp4^−/− mice, Student’s t test, t₍₈₎ = 3.394, p = 0.0094; n = 4 samples of dialysate from 4 control and 4 GFAP-CreER;Lrp4^−/− mice, Student’s t test, t₍₆₎ = 2.816, p = 0.0305; n = 6 dishes (4 mice) for astrocytes, Student’s t test, t₍₁₀₎ = 2.562, p = 0.0283; n = 4 dishes (4 mice) for neurons, Student’s t test, t₍₆₎ = 0.4766, p = 0.6505. Data in (a, b, e and f) were presented as median with interquartile range, whiskers are the minimum and maximum; data in (d) were presented as mean ± s.e.m; n.s., not significant; *, p < 0.05.

Figure 6. Agrin regulates astrocytic ATP release

(a, b) Agrin expression in brain regions and neurons. Total RNA of indicated regions (a), and from cultured astrocytes and neurons (b) was subjected to real-time quantitative PCR. GAPDH was used as the internal control. Expression in spinal cord (a) and astrocytes (b) was set as 100%. n = 4 times for (a); n = 3 times for (b). (c) MuSK expression in astrocytes. Total RNA was isolated from cultured astrocytes, neurons and muscle cells. Expression level in muscle cells was set as 100%. n = 3 times. (d) Agrin activated MuSK in astrocytes. Astrocytes were cultured from control and GFAP-Lrp4^−/− (mutant) mice and treated with agrin (100 ng/ml) for different times. 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. (e) Quantitative analysis of data in (d). p-tyrosine intensity was normalized by that of MuSK. n = 3 times. For control 0 vs 10 min groups, paired student’s t test, t₍₂₎ = 5.667, p = 0.0298; for mutant 0 vs 10 min groups, paired student’s t test, t₍₂₎ = 0.4547, p = 0.6939. (f) Lrp4-depedent reduction of ATP release by agrin. Astrocytes of different genotypes were treated with vehicle or agrin (100 ng/ml) for 24 h. ATP in the condition medium was measured. n = 8 dishes of cultured astrocytes, 3 times. For control + vehicle vs control + agrin groups, student’s t test, t₍₁₄₎ = 5.217, p = 0.0001;for control + vehicle vs mutant + vehicle groups, student’s t test, t₍₁₄₎ = 2.902, p = 0.0116; for mutant + vehicle vs mutant + agrin groups, student’s t test, t₍₁₄₎ = 0.7957, p = 0.4395. (g) Agrin increased sEPSC frequency in CA1 pyramidal neurons of control but not GFAP-CreER;Lrp4^−/− mice and this effect was blocked by DPCPX. n = 8 neurons, 3 control mice for vehicle; n = 10 neurons, 3 control mice for agrin; n = 9 neurons, 3 control mice for DPCPX; n = 10 neurons, 3 control mice for DPCPX + agrin; n = 9 neurons, 3 GFAP-CreER;Lrp4^−/− mice for vehicle; n = 9 neurons, 3 GFAP-Lrp4^−/− mice for agrin. For control + vehicle vs control + agrin groups, student’s t test, t₍₁₆₎ = 2.168, p = 0.0456; for control + vehicle vs control + DPCPX groups, student’s t test, t₍₁₅₎ = 3.412, p = 0.0039; for control + vehicle vs control + agrin groups, student’s t test, t₍₁₆₎ = 2.168, p = 0.0456; for control + DPCPX vs control + DPCPX + agrin groups, student’s t test, t₍₁₇₎ = 0.5006, p = 0.6231; for GFAP-CreER;Lrp4^−/− + vehicle vs GFAP-CreER;Lrp4^−/− + agrin groups, student’s t test, t₍₁₆₎ = 1.059, p = 0.3052. Data in (a, f and g) were presented as median with interquartile range, whiskers are the minimum and maximum; data in (b, c and e) were presented as mean ± s.e.m; n.s., not significant; *, p < 0.05;**, p < 0.01.

Figure 7. Ablation of Lrp4 caused abnormal behavior

(a) Representative traces of first 5 min in the open field test. (b) Reduced total distance traveled by mutant mice in 30 min. Mice were pretreated without or with DPCPX 30 min before test. n = 9 mice for each genotype without DPCPX; n = 11 mice for each genotype with DPCPX. For control vs GFAP-Lrp4^−/− groups, student’s t test, t₍₁₆₎ = 2.417, p = 0.0279; for GFAP-Lrp4^−/− vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₁₈₎ = 5.543, p < 0.0001; for control + DPCPX vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₂₀₎ = 1.089, p = 0.2891. (c) Reduced time mutant mice spent in the center. n = 9 mice for each genotype without DPCPX; n = 11 mice for each genotype with DPCPX. For control vs GFAP-Lrp4^−/− groups, student’s t test, t₍₁₆₎ = 2.178, p = 0.0447; for GFAP-Lrp4^−/− vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₁₈₎ = 2.411, p = 0.0268; for control + DPCPX vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₂₀₎ = 0.8403, p = 0.4107. (d) Reduced center entry by GFAP-Lrp4^−/− mice. n = 9 mice for each genotype without DPCPX; n = 11 mice for each genotype with DPCPX. For control vs GFAP-Lrp4^−/− groups, student’s t test, t₍₁₆₎ = 2.246, p = 0.0391; for GFAP-Lrp4^−/− vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₁₈₎ = 3.258, p = 0.0044; for control + DPCPX vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₂₀₎ = 0.1557, p = 0.8779. (e) Representative traces in the probe test of Morris water maze. (f) No difference in latency to the platform during the hidden platform task between the two genotypes. Mice were pretreated without or with DPCPX 30 min before behavioral test. n = 9 mice for each genotype without DPCPX; n = 12 mice for each genotype with DPCPX. Two-way ANOVA, F_(3,190) = 1.757, p = 0.1568. (g) Reduced number of plateform crossings. n = 9 mice for each genotype without DPCPX; n = 12 mice for each genotype with DPCPX. For control vs GFAP-Lrp4^−/− groups, student’s t test, t₍₁₆₎ = 2.49, p = 0.0241; for GFAP-Lrp4^−/− vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₁₉₎ = 2.513, p = 0.0211; for control + DPCPX vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₂₂₎ = 0.9893, p = 0.3333. (h) Reduced time spent in the N30 area. n = 9 mice for each genotype without DPCPX; n = 12 mice for each genotype with DPCPX. For control vs GFAP-Lrp4^−/− groups, student’s t test, t₍₁₆₎ = 2.318, p = 0.034; for GFAP-Lrp4^−/− vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₁₉₎ = 4.625, p = 0.0002; for control + DPCPX vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₂₂₎ = 0.1478, p = 0.8839. (i) Representative time courses of seizure development by repeated pilocarpine injection. Mice of two genotypes were subjected to pilocarpine injection every 30 min and scored for seizure stage. (j) Increased number of pilocarpine injection needed to reach stage-5 seizure for mutant mice. n = 10 mice for each genotype. Student’s t test, t₍₁₈₎ = 2.834, p = 0.011. (k) Increased latency of mutant mice to generalized convulsive seizure in response to PTZ. Mice were pretreated without or with DPCPX 30 min before PTZ injection. Latency to generalized convulsive seizure was recorded. n = 8 mice for each genotype without DPCPX; n = 6 mice for each genotype with DPCPX. For control vs GFAP-Lrp4^−/− groups, student’s t test, t₍₁₄₎ = 2.561, p = 0.0226; for control + DPCPX vs GFAP-Lrp4^−/− + DPCPX groups, student’s t test, t₍₁₀₎ = 0.798, p = 0.4434. Data in (b–d, g, h, j and k) were presented as median with interquartile range, whiskers are the minimum and maximum; data in (f) were presented as mean ± s.e.m; n.s., not significant; *, p < 0.05;**, p < 0.01.