Lrp4 in hippocampal astrocytes serves as a negative feedback factor in seizures

Abstract

Background: Epilepsy is characterized by the typical symptom of seizure, and anti-seizure medications are the main therapeutic method in clinical, but the effects of these therapy have not been satisfactory. To find a better treatment, it makes sense to further explore the regulatory mechanisms of seizures at genetic level. Lrp4 regionally expresses in mice hippocampus where is key to limbic epileptogenesis. It is well known that neurons release a high level of glutamate during seizures, and it has been reported that Lrp4 in astrocytes down-regulates glutamate released from neurons. However, it is still unclear whether there is a relationship between Lrp4 expression level and seizures, and whether Lrp4 plays a role in seizures.

Results: We found that seizures induced by pilocarpine decreased Lrp4 expression level and increased miR-351-5p expression level in mice hippocampus. Glutamate reduced Lrp4 expression and enhanced miR-351-5p expression in cultured hippocampal astrocytes, and these effects can be partially attenuated by AP5. Furthermore, miR-351-5p inhibitor lessened the reduction of Lrp4 expression in glutamate treated hippocampal astrocytes. Local reduction of Lrp4 in hippocampus by sh Lrp4 lentivirus injection in hippocampus increased the threshold of seizures in pilocarpine or pentylenetetrazol (PTZ) injected mice.

Conclusions: These results indicated that high released glutamate induced by seizures down-regulated astrocytic Lrp4 through increasing miR-351-5p in hippocampal astrocytes via activating astrocytic NMDA receptor, and locally reduction of Lrp4 in hippocampus increased the threshold of seizures. Lrp4 in hippocampal astrocytes appears to serve as a negative feedback factor in seizures. This provides a new potential therapeutic target for seizures regulation.

Keywords: Astrocytic NMDA receptor; Epilepsy; Low-density lipoprotein receptor-related protein 4; The threshold of seizure; microRNA.

Fig. 1

Seizures induced by pilocarpine decreased Lrp4 expression in mice hippocampus. a Relative mRNA level of Lrp4 in the hippocampus of pilocarpine injected mice, which were collected at different time point (0 h, 2 h, 4 h, 12 h) after injection. Comparing to control mice (vehicle injection), Lrp4 mRNA decreased after injection (2 h, 4 h, 12 h). b X-gal staining the brain slice of pilocarpine injected Lrp4-lacZ/+ mice. β-Gal activity decreased in both stratum lacunosum-moleculare layer (LMol) and molecules layer (Mol) of hippocampus. c Lrp4 protein level in the hippocampus of pilocarpine injected mice, which were collected at different time point (0 h, 2 h, 4 h, 12 h) after injection, Actin as an internal control. d Quantification of Lrp4 protein level in c, Lrp4 protein decreased after injection (4 h, 12 h). For each experiment, three separate experiments were performed in duplicate (**p < 0.01)

Fig. 2

Glutamate decreased Lrp4 expression through activating NMDA receptor in cultured hippocampal astrocytes. a Relative mRNA of Lrp4 in cultured hippocampal astrocytes after glutamate treatment, which were collected at different time point (0 h, 2 h, 4 h, 12 h) after treatment. Lrp4 mRNA decreased after glutamate treatment (2 h, 4 h, 12 h). b Protein level of Lrp4 in cultured hippocampal astrocytes after glutamate treatment, which were collected at different time point (0 h, 2 h, 4 h, 12 h) after treatment, Actin as an internal control. Lrp4 protein decreased after glutamate treatment (4 h, 12 h). c Quantification of Lrp4 protein level in b. d Relative mRNA level of Lrp4 in primary cultured astrocytes which were treated with glutamate (0.5 mM) only or glutamate (0.5 mM) plus AP5 (50 mM). After 12 h, Lrp4 mRNA decreased about 75% after glutamate treatment, but decreased about 20% after glutamate plus AP5 treatment. e Lrp4 protein level in cultured astrocytes which were treated with glutamate (0.5 mM) only or glutamate (0.5 mM) plus AP5 (50 mM). Actin as an internal control in Western blotting. f Quantification of Lrp4 protein level in e, Lrp4 protein decreased about 70% after glutamate treatment, but decreased about 25% after glutamate plus AP5 treatment. For each experiment, three separate experiments were performed in duplicate (**p < 0.01)

Fig. 3

Seizures increased miR-351-5p in vivo and glutamate increased miR-351-5p in vitro through activating NMDA receptor. a Relative level of microRNA in hippocampus, injecting pilocarpine into mice. 4 h after injection, the relative level of mmu-miR-351-5p in hippocampus was increased. Two separate experiments were performed in triplicate (**p < 0.01). b Time course of the mmu-miR-351-5p relative level in hippocampus after injection, mmu-miR-351-5p increased at each time point (2 h, 4 h, 12 h) after injection. c Time course of the mmu-miR-351-5p relative level in hippocampus after glutamate treatment. mmu-miR-351-5p increased at each time point (2 h, 4 h, 12 h) after treating. d Relative level of mmu-miR-351-5p expression in hippocampal astrocytes which were treated with glutamate (0.5 mM) only or glutamate (0.5 mM) plus AP5 (50 mM). After 4 h treating, the elevation of mmu-miR-351-5p induced by glutamate plus AP5 treating is significantly lower than that induced by glutamate treating. For each experiment, three separate experiments were performed in duplicate (**p < 0.01)

Fig. 4

miR-351-5p inhibitor attenuated the reduction of Lrp4 expression by glutamate treating in cultured hippocampal astrocytes. a Relative level of mmu-miR-351-5p in hippocampal astrocytes which were transfected respectively by miR-351-5p mimics, miR-351-5p m NC (mimics negative control), miR-351-5p inhibitor, miR-351-5p I NC (inhibitors negative control). miR-351-5p mimics enhanced the miR-351-5p level and miR-351-5p inhibitors weakened the miR-351-5p level. b Relative level of Lrp4 mRNA in primary cultured hippocampal astrocytes which were transfected with miR-351-5p mimics at different concentration (0 nM, 25 nM, 50 nM, 100 nM). Lrp4 mRNA level decreased at the test concentration of the mimics (25 nM, 50 nM, 100 nM). c Lrp4 protein level in cultured astrocytes after transfecting miR-351-5p mimics by Western blotting. Action as an internal control. d Quantification of Lrp4 protein level from panel C, miR-351-5p mimics cut down Lrp4 protein expression. e mRNA level of Lrp4 in astrocytes after glutamate treatment only or glutamate treatment plus miR-351-5p inhibitors transfection. mRNA level of Lrp4 in astrocytes decreased about 70% by glutamate treating, and decreased about 25% by glutamate plus miR-351-5p inhibitors treating. f Lrp4 protein level in cultured astrocytes after glutamate treatment only or glutamate treatment plus miR-351-5p inhibitors transfection, Actin as an internal control. g Quantification of Lrp4 protein level from f, Lrp4 protein in astrocytes decreased about 70% by glutamate treating and decreased about 25% by glutamate plus miR-351-5p inhibitors treating. For each experiment, three separate experiments were performed in duplicate (**p < 0.01)

Fig. 5

The reduction of Lrp4 expression in hippocampus increased the threshold of seizures. a Schematic diagram of the site of stereotactic injection of lentivirus. The expression of GFP showed that virus was precisely injected into lacunosum-moleculare layer (LMol) and molecules layer (Mol) of the hippocampus in mice. b Lrp4 protein level of the hippocampus in the mice, which were trans-infected by sh control lentivirus (sh pll3.7 vector injection), sh Lrp4 scramble lentivirus, sh Lrp4 lentivirus, Actin as an internal control in Western blotting. c Quantification of Lrp4 protein level in b. Lrp4 protein expression decreased about 70% in sh Lrp4 lentivirus trans-infected mice. Three separate experiments were performed in duplicate (**p < 0.01). d Representative time courses of seizure development by repeated pilocarpine injection. Different virus trans-infected mice were subjected to pilocarpine injection every 30 min and seizure stages were scored at the same time. e Increased number of pilocarpine injections were needed to reach stage 5 seizure for sh Lrp4 lentivirus trans-infected mice (10 injections), comparing with sh control lentivirus (5 injections) and sh Lrp4 scramble lentivirus (5 injections) trans-infected mice. (n = 10 mice for each group; **p < 0.01). f Increased latency of sh Lrp4 lentivirus trans-infected mice to generalized convulsive seizure in response to PTZ. (n = 10 mice for each group; **p < 0.01)