Npn-2 Expression in Adult Hippocampus

Previous studies of Npn-2 signaling mainly focused on its functions during development. To understand its functions in adult animals, we first examined Npn-2 and its ligand Sema3F expression levels in rat hippocampus during different developmental stages. qPCR and western blot results showed that Npn-2 and Sema3F maintained their expression in adult hippocampus at both mRNA (Fig. 1A, B) and protein levels (Fig. 1C–F). Immunofluorescent staining with Npn-2 (green) antibody and DAPI (blue) revealed that Npn-2 in adult hippocampus is mainly distributed in the neuropil areas, specifically in dentate gyrus granule cell inner molecular layer and mossy fibers (Fig. 1G). Given its expression and distribution patterns in adult hippocampus, Npn-2 might play a role in the mature central nervous system.

Fig. 1

Expression of Npn-2 in adult rat hippocampus. A, B The mRNA levels of Npn-2 and its ligand Sema3F in rat hippocampus at different developmental stages (P0, P7, P14, P28, P42, and P70) were measured by real-time quantitative PCR. Npn-2 and Sema3F mRNA maintained their expression in adult hippocampus. C, D The protein levels of Sema3F and Npn-2 in rat hippocampus at different developmental stages (P0, P7, P14, P28, P42, and P70) were assessed by western blot analysis. Npn-2 and Sema3F retained their expression in adult hippocampus. E, F Quantitation of C and D, respectively. G Immunofluorescent detection of Npn-2 expression in rat hippocampus. Immunofluorescent staining with Npn-2 (green) and DAPI (blue) showed that Npn-2 is expressed in adult hippocampus, mainly in the neuropil, dentate gyrus granule cell inner molecular layer, and mossy fibers. Scale bar, 100 μm

Npn-2 Knockdown Increases Spontaneous MFS

The key feature of MFS is the aberrant axon sprouting and abnormal orientation of dentate gyrus granule neurons, which could be regulated by axon guidance cues. Axon guidance cue receptor Npn-2 was found to distribute in dentate gyrus granule neurons in adult animals. We ask if Npn-2 signaling could potentially modulate MFS.

AAV encoding either control short hairpin RNA (shCtrl) or short hairpin RNA targeting rat Npn-2 (shNpn-2) was injected into the dentate gyrus of adult animals to reduce Npn-2 expression (Fig. 2A–D). Mossy fibers can be visualized with Timm stain. The Timm stain reaction deposits were rarely observed in the IML of control rats while Npn-2 knockdown significantly increased the reaction deposits of these animals (Fig. 2E, F). Synaptoporin (SPO) is known to specifically label mossy fiber synapses [24], thus, immunofluorescent staining with SPO was performed as a second method to verify the sprouting of mossy fibers. The result showed an increased number of SPO-positive puncta in the IML of Npn-2 knockdown dentate gyrus (Fig. 2H, I), suggesting that Npn-2 knockdown increased spontaneous MFS.

Fig. 2

Increased spontaneous MFS in Npn-2 knockdown animals. A In vivo Npn-2 knockdown validated by immunofluorescent staining. Fourteen days after intrahippocampal injection of shCtrl AAV or shNpn-2 AAV, immunofluorescent staining with a Npn-2 antibody showed that Npn-2 expression in knockdown animals was significantly reduced compared with the controls. Arrows indicate Npn-2 (red). Scale bar: 100 μm. B Quantitation of immunofluorescence signal in A. n = 3, *P = 0.0128. C In vivo Npn-2 knockdown validated by western blot. Fourteen days after intrahippocampal injection with shCtrl AAV or shNpn-2 AAV, western blot results showed that endogenous Npn-2 in hippocampus was significantly reduced. D Quantitation of Npn-2 in C. n = 3, *P = 0.0484. E Spontaneous MFS in Npn-2 knockdown rats assessed by the Timm stain. Representative Timm stain images of rat brain coronal sections from either shCtrl- or shNpn-2 AAV-injected groups. Significantly increased MFS was detected in the shNpn-2 AAV-injected DG inner molecular layer (E2, E2') when compared with shCtrl AAV-injected group (E1, E1'). No obvious difference in infrapyramidal tract (IPT) between shNpn-2 group and shCtrl group was observed (E1'' vs E2''). E1', E2', E1'', and E2'' are higher magnification views of E1 and E2, respectively. E1, E2 Scale bar, 400 µm. E1', E2' Scale bar, 100 µm. E1'', E2'' Scale bar, 200 µm. F Quantitation of the Timm score in E. n = 6, *P = 0.0493. G Quantitation ratio of IPT length to MT length in E, n = 6, P > 0.05. H Spontaneous MFS in Npn-2 knockdown rats measured by immunofluorescent staining. Representative immunofluorescent images of rat brain coronal sections from either shCtrl- or shNpn-2 AAV-injected groups, labeled with synaptoporin (SPO) antibody and visualized under a laser scanning confocal microscope. A significantly increased SPO-positive puncta (red signal) number was detected in the shNpn-2-injected DG inner molecular layer when compared with shCtrl-injected group. Scale bar, 20 µm. I Quantitation of SPO punctate number in H, n = 3, *P = 0.0389. J Increased dendrite spine density in shNpn-2 granule neurons. Representative immunofluorescent images of rat brain coronal sections from either shCtrl- or shNpn-2 AAV-injected groups, labeled with GFP antibody, and visualized under a laser scanning confocal microscope. Significantly increased dendritic spine density was detected in shNpn-2-injected granule cells when compared with shCtrl-injected group. Scale bar, 10 µm. K Quantitation of spine density in J, ****P < 0.0001. Unpaired t-test. Error bars represent SEM

Sprouted mossy fibers synapse with dendritic spines of granule cells, thus forming excitatory neural circuits [25]. Embryonic Npn-2 knockout led to increased dendritic spine density [11]. We are curious about the functions of Npn-2 signaling in spine remodeling in adulthood. Immunofluorescent staining with GFP showed increased spine density in Npn-2 knockdown dentate granule neurons compared with control neurons (Fig. 2J, K).

Sema3F/Npn-2 signaling was reported to involve in axon pruning [26] during development. We are wondering if Npn-2 knockdown in adult brains could affect already pruned axons. Thus, we checked the ratio of infrapyramidal tract (IPT) length to main tract (MT) length in the adult hippocampus using the Timm stain. No obvious IPT defects were observed in Npn-2 knockdown animals (Fig. 2E, G).

Reduction of Npn-2 Increases MFS in Epileptic Rats

Mutant animal results suggested that Sema3F and its receptor Npn-2 play important roles in animal models of epilepsy [11, 13]. We are wondering if the expression level of Sema3F and Npn-2 could change during epilepsy. To this end, we generated a pilocarpine-induced rat model of epilepsy. Hippocampal Sema3F mRNA and protein level was decreased in epileptic rats (Fig. 3A–C). On the other hand, the Npn-2 protein level was slightly increased in the hippocampus of epileptic rats (Fig. 3D, F), which could be the result of compensation for the reduced level of Sema3F. These data indicated that Sema3F and Npn-2 probably play roles in the pilocarpine-induced rat model of epilepsy.

Fig. 3

Increased MFS in Npn-2 knockdown epileptic rats. Sema3F mRNA level was decreased 7 days, 14 days, and 28 days after pilocarpine injection, measured by real-time PCR, n = 3, *P < 0.05. Sema3F protein level was decreased 14 days and 28 days after pilocarpine injection, measured by western blot. C Quantitation of Sema3F protein levels in B, n = 3, **P < 0.01. D Npn-2 mRNA level was transiently decreased 1 day after pilocarpine injection and recovered at 7 days, 14 days, and 28 days, measured by real-time PCR, n = 3, *P < 0.05. E Npn-2 protein level was increased 14 days and 28 days after pilocarpine injection, measured by western blot. F Quantitation of Npn-2 protein levels in E, n = 3, *P < 0.05. G Validation of Npn-2 knockdown and human Npn-2 expression in vivo. Western blot using an antibody recognizing both rat and human forms of Npn-2 showed that shNpn-2 AAV effectively reduced endogenous Npn-2 expression in vivo, which was rescued by hNpn-2 expression. H Quantitation of Npn-2 protein levels in G. n = 3, *P < 0.05, **P < 0.01. I MFS in epileptic rats at 14 days after pilocarpine injection. Representative Timm stain of rat brain coronal sections in shCtrl group and shNpn-2 group at 14 days after pilocarpine injection. Significantly increased MFS was observed in Npn-2 knockdown animals (I2, I2') compared with control animals (I1, I1'). I1'–I2' are higher magnification views of I1–I2, respectively. I1–I2 Scale bar, 400 µm. I1'–I2' Scale bar, 100 µm. J Quantitation of Timm’s score in I, unpaired t-test, and error bars represent SEM. n = 4, *P = 0.0272. K MFS in epileptic rats at 21 days after pilocarpine injection. Representative Timm stain of coronal brain sections in shCtrl group and shNpn-2 group at 21 days after pilocarpine intraperitoneal injection. Increased MFS was observed in shNpn-2 group (K2, K2') compared with shCtrl group (K1, K1'), which was rescued by hNpn-2 expression (K3, K3'). K1'–K3' are higher magnification views of K1–K3, respectively. K1–K3 Scale bar, 400 µm. K1'–K3' Scale bar, 100 µm. L Quantitation of Timm score in K, n = 4, *P = 0.0232. One-way ANOVA, post hoc Tukey test. Error bars represent SEM

MFS is proposed to relate to the recurrent excitation in the hippocampus during epileptogenesis [6, 27]. We found that Npn-2 knockdown could lead to spontaneous MSF. It is plausible that Npn-2 signaling is also involved in MFS during epilepsy. We then investigated the function of Npn-2 signaling in MFS in epileptic rats. Intrahippocampal injection of shCtrl AAV, shNpn-2 AAV, or shNpn-2 AAV plus hNpn-2 lentivirus was performed to regulate the level of Npn-2 (Fig. 3G, H). Fourteen days after intrahippocampal injection, a pilocarpine-induced rat model of epilepsy was established. Mossy fiber terminals were visualized at 14 days and 21 days after pilocarpine injection. Mossy fibers from Npn-2 knockdown animals had significantly more aberrant sprouting than control animals (for 14 days, Fig. 3I, J; for 21 days, Fig. 3K, L). The Timm stain at 21 days showed that hNpn-2 expression rescued the MFS phenotype (Fig. 3K, L). These data demonstrated that Npn-2 knockdown increased MFS of pilocarpine-induced rat model of epilepsy.

Npn-2 Knockdown in Adult Hippocampus Increases Seizure Activity

Embryonic ablation of Npn-2 increased seizure activity in kainic acid-induced and pentylenetetrazol (PTZ) kindling seizure models [11, 14]. Given that Npn-2 expression persists in adult brains, especially in the hippocampus and the expression of Sema3F and Npn-2 change in mTLE patients and adult animal models of epilepsy [15–17], we ask whether it plays any role in epilepsy during adulthood.

Pilocarpine was injected to induce SE after 14 days and rats were then continuously surveilled for 21 days. Two out of 18 rats failed to develop SE in the shCtrl group. Three of 18 rats in the shCtrl group and 5 out of 20 rats in the shNpn-2 group died during the SRS stage (mortality rate: 16.7% in the shCtrl group and 25% in the shNpn-2 group). Behaviors of 13 rats in the shCtrl group and 15 rats in the shNpn-2 group were analyzed. Thirty-eight percent of rats in the shCtrl group while 73% in the shNpn-2 knockdown group developed SRS during the 21-day surveillance period (Fig. 4A). The mean duration per seizure of Npn-2 knockdown rats was 29.59 s, which was significantly more than the 21.84 s of control rats (Fig. 4B). There was no significant difference in SRS stage (Fig. 4C), SRS latency (Fig. 4D), and SE latency (Fig. 4E) between shCtrl group and shNpn-2 group. The data indicated that reduction of Npn-2 increased seizure activity in pilocarpine-induced rat model.

Fig. 4

Npn-2 knockdown increases seizure activity. A Overview of SRS. No SRS was found with normal saline (NS) injection in both shCtrl and shNpn-2 groups. In pilocarpine injection groups, 5 out of 13 (38%) rats in shCtrl group and 11 out of 15 (73%) rats in shNpn-2 group developed SRS during the 21-day surveillance period after pilocarpine injection. Five out of 8 (62%) rats developed SRS in shNpn-2 plus hNpn-2 group. B Seizure duration. The mean duration per seizure in Npn-2 knockdown group was 29.59 s, which was significantly more than the control group (21.84 s, *P = 0.0307). hNpn-2 expression decreased seizure duration to control level (20.32 s, P > 0.05). C SRS stage. No significant difference in SRS stage was observed among shCtrl, shNpn-2, and hNpn-2 rescue groups (4.800 vs 4.577 vs 4.680, P = 0.4354). D SRS latency. There was no significant difference in SRS latency among shCtrl group, shNpn-2 group, and hNpn-2 rescue group (15.20 days vs 15.00 days vs 13.60 days, P = 0.4602). E SE latency. The SE latency of rats in shCtrl group, shNpn-2 group, and hNpn-2 rescue group were similar to each other (15.06 min vs 14.82 min vs 18.10 min, P = 0.1259). One-way ANOVA, post hoc Tukey test. Error bars represent SEM

In human Npn-2 (hNpn-2) rescue group, 2 rats died during SRS (mortality rate: 20%). Behaviors of 8 rats were analyzed. Fewer rats in the rescue group (62.5%) developed SRS when compared with shNpn-2 group (Fig. 4A). The mean SRS duration in the rescue group was similar to that in shCtrl group (Fig. 4B). These data suggested that the expression of hNpn-2 ameliorated the increased seizure activity caused by Npn-2 knockdown, suggesting the specificity of Npn-2 to modulate seizure activity in adult.

Npn-2 Knockdown in Adult Has No Effect on GABAergic Interneurons Survival

Previous studies mainly focused on the effect of Npn-2 signaling in the development stages and their data have shown that embryonic Npn-2 knockout increased seizure activity by reducing the number of GABAergic interneurons [11]. We examined whether Npn-2 knockdown in adult animals affects interneuron survival.

Immunofluorescent staining with NeuN showed no loss of neurons in general (Figure S2A and S2B), and labeling with GAD-65/57 showed no obvious GABAergic interneuron loss in Npn-2 knockdown rats (Figure S2C and S2D). These results suggested that Npn-2 knockdown in adult hippocampus influences seizure activity without affecting GABAergic inhibitory interneurons, which was partly different from that during development.

Npn-2 Signaling Controls Axon Collateral Formation

Given the 3-step cellular mechanism of MFS, namely axon branching, reverse projection, and fasciculation [7], we tested the role of Npn-2 signaling in axon collateral formation and outgrowth using an in vitro assay.

Dissociated hippocampal cultures were transfected with shCtrl, shNpn-2, or shNpn-2 plus hNpn-2 plasmids at 0 day in vitro (DIV 0). Npn-2 knockdown by shNpn-2 and hNpn-2 expression were validated by in vitro experiments (Fig. 5A–D, Figure S1). Forty-eight hours after transfection, primary cultures were treated with a control medium or 5 nM AP-Sema3F for 24 h. Axons and their collaterals were labeled with GFP and Tau-1, a biomarker for axons (Fig. 5E, Figure S3). Sema3F treatment reduced the number and length of axon collaterals as well as the main axon length of shCtrl-transfected neurons but not Npn-2 knockdown neurons. These phenotypes were rescued by hNpn-2 expression (Fig. 5F–H). The results suggested that Sema3F modulates axon collateral formation and elongation through Npn-2, which may serve as its cellular mechanism to regulate MFS.

Fig. 5

Npn-2 controls axon collateral formation. A Npn-2 knockdown by shNpn-2 validated in primary neuron cultures by western blot using an antibody for rat Npn-2. B Quantitation of Npn-2 in A, n = 3, ***P = 0.0003. C hNpn-2 expression validated by western blot. Expression of hNpn-2 in neurons was detected by western blot using an anti-Flag antibody, showing that the expression of human full-length Npn-2 cannot be affected by the rat shNpn-2 used. D Quantitation of Npn-2 in C, n = 3, P > 0.05. E Neonatal rat primary hippocampal neurons were transfected with shCtrl, shNpn-2, or shNpn-2 plus hNpn-2 plasmids at DIV 0 and treated with either control medium or Sema3F (5 nM) at 48 h after transfection for 24 h. Axons and their collaterals were detected by immunofluorescent staining with GFP and Tau-1. Arrows indicate main axons and stars indicate axon collaterals. Scale bar, 40 μm. F, G Quantitation of axon collateral number and length in E. Sema3F treatment reduced the number and length of axon collaterals in neurons transfected with shCtrl but not in neurons with shNpn-2 transfection, which was rescued by hNpn-2 co-transfection. *P < 0.05, **P < 0.01. H Quantitation of main axon length in E. Main axon elongation in neurons transfected with shCtrl was inhibited upon Sema3F treatment, but not in neurons with shNpn-2 transfection, which was rescued by hNpn-2 expression. **P < 0.01, ***P < 0.001. One-way ANOVA, post hoc Tukey test. Error bars represent SEM

Npn-2 Signaling Regulates CRMP2 Phosphorylation

CRMP2 is a classical downstream molecule in secreted semaphorin signaling [25, 26]. CRMP2 exhibits neurite outgrowth-promoting function, which is regulated by its phosphorylation state [28]. Our previous data have shown that the phosphorylation of CRMP2 at serine 522 (S522), threonine 514 (T514), or threonine 555 (T555) was downregulated in the hippocampus of pilocarpine-induced rat model of epilepsy at 1 day, 7 days, and 14 days after pilocarpine injection [29], suggesting that CRMP2 may play some roles in epilepsy. Another semaphorin, Sema3A, is able to increase CRMP2 phosphorylation [30]. Here, in vitro and in vivo assays were performed to examine if Sema3F/Npn-2 signaling regulates CRMP2 phosphorylation.

Rats with shCtrl AAV, shNpn-2 AAV, or shNpn-2 AAV plus hNpn-2 lentivirus injection were sacrificed 14 days after injection for western blot. Npn-2 knockdown reduced CRMP2 phosphorylation levels at S522, T514, or T555 sites, but not the total CRMP2 amount. The reduction of CRMP2 phosphorylation was rescued by hNpn-2 expression (Fig. 6A–E).

Fig. 6

Sema3F/Npn-2 signaling regulates CRMP2 phosphorylation. A Reduction of CRMP2 phosphorylation in Npn-2 knockdown tissues. Adult rat hippocampi injected with various viruses as indicated were subjected to western blot with different phospho-CRMP2 antibodies. The levels of p-S522, p-T514, and p-T555-CRMP2 were significantly reduced in Npn-2 knockdown animals compared with the control group while the expression of CRMP2 was not changed, and the knockdown effects were rescued by the expression of hNpn-2. B–E Quantitation of A. n = 4, *P < 0.05, ***P < 0.001. F Semaphorin induced CRMP2 phosphorylation in primary cultures. Primary cultured neurons were treated with 5 nM AP, AP-Sema3F, or AP-Sema3A for 12 h. CRMP2 phosphorylation levels were detected by various phospho-CRMP2 antibodies as indicated using western blot. Sema3F treatment upregulated the levels of p-S522, p-T514, and p-T555-CRMP2 while made no effect on CRMP2 expression. G–J Quantitation of F. n = 4, *P < 0.05, **P < 0.01. One-way ANOVA, post hoc Tukey test. Error bars represent SEM. K Time course of Sema3F regulating CRMP2 phosphorylation. Primary cultured neurons were treated with 5 nM AP-Sema3F for 5 min, 30 min, 60 min, 6 h, 12 h, and 24 h. Levels of CRMP2 phosphorylation were detected by various phospho-CRMP2 antibodies as indicated using western blot. The levels of p-S522-CRMP2, p-T514-CRMP2, and p-T555-CRMP2 increased over time and peaked at around 12 h, while CRMP2 expression was not significantly changed. L Quantitation of K (n = 3). M Sema3F that regulates CRMP2 phosphorylation is dose-dependent. Primary cultured neurons were treated with AP-Sema3F at various doses as indicated for 12 h. CRMP2 phosphorylation levels were detected by phospho-CRMP2 antibodies as indicated using western blot. The levels of p-S522-CRMP2, p-T514-CRMP2, and p-T555-CRMP2 were increased over increased doses and peaked around 0.5 nM, while the expression of CRMP2 was not significantly changed. N Quantitation of M (n = 3)

Primary neuronal cultures were treated with 5 nM AP, AP-Sema3F, or AP-Sema3A for 12 h. Western blot results showed that CRMP2 can be phosphorylated by Sema3F while levels of total CRMP2 remained unchanged (Fig. 6F–J). Time-course and dose-dependent experiments showed that CRMP2 phosphorylation was increased over time (peaked at 12 h) (Fig. 6K, L) and dose (peaked at 0.5 nM) (Fig. 6M, N), demonstrating the specificity of Sema3F/Npn-2 signaling in regulating CRMP2 phosphorylation.

Our data indicated that Sema3F/Npn-2 signaling facilitated CRMP2 phosphorylation, which lays the foundation for this pathway to regulate the function of CRMP2.

CRMP2 Mediates Npn-2 Controlled Axon Collateral Formation

CRMP2 was reported to mediate Sema3F/Npn-2 signaling in axon retraction, axon guidance, axon pruning, and dendritic spine remodeling [26]. In addition, the axon outgrowth promoting function of CRMP2 is mainly determined by its phosphorylation state, which was validated to be regulated by Sema3F/Npn-2 signaling in this study. Here we asked whether Npn-2 signaling regulates axon collateral formation via CRMP2.

Dissociated hippocampal cultures were transfected with shCtrl or CRMP2 shRNA (shCRMP2) plasmids at DIV 0. CRMP2 knockdown by shCRMP2 has been validated in vitro (Fig. 7A, B, Figure S1). Cultures were treated with a control medium or 5 nM AP-Sema3F at 48 h after transfection for 24 h. The axons and their collaterals were labeled with Tau-1 and mCherry antibodies (Fig. 7C, Figure S4). Compared with the control medium, Sema3F treatment decreased axon collateral number and length as well as main axon length in shCtrl-transfected neurons (Fig. 7D–F). In control medium-treated neurons, CRMP2 knockdown reduced axon branch number and length as well as main axon length compared with shCtrl transfection (Fig. 7D–F). Interestingly, upon CRMP2 was knocked down, Sema3F failed to inhibit axon collateral number and length as well as main axon length (Fig. 7D–F). These results demonstrated that CRMP2 mediates Sema3F/Npn-2 controlled axon collateral formation and elongation.

Fig. 7

Npn-2 signaling regulates axon collateral formation through CRMP2. A CRMP2 knockdown by CRMP2 shRNA (shCRMP2) was validated in primary neurons by western blot. B Quantitation of CRMP2 in n = 3, **P = 0.0045. (C) CRMP2-mediated Sema3F controlled collateral formation. Neonatal rat primary hippocampal neurons were transfected with shCtrl or shCRMP2 plasmids at DIV 0. Forty-eight hours after transfection, the cultures were treated with a control medium or 5 nM AP-Sema3F for 24 h. Main axons and their collaterals were detected by immunofluorescent staining with mCherry and Tau-1. Arrows indicate main axons and stars indicate axon collaterals. Scale bar, 40 µm. D, E Quantitation of axon collateral number and length in C. CRMP2 knockdown led to a decrease in the number and length of axon collaterals. Sema3F treatment reduced the number and length of axon branches in neurons transfected with shCtrl but not in neurons with shCRMP2 transfection. *P < 0.05, **P < 0.01. F Quantitation of main axon length in C. CRMP2 knockdown led to a decrease in main axon length. Sema3F treatment reduced the length of main axons in neurons transfected with shCtrl but not in neurons with shCRMP2 transfection. **P < 0.01. One-way ANOVA, post hoc Tukey test. Error bars represent SEM

Npn-2 Modulates MFS Through CRMP2

We have demonstrated that Sema3F/Npn-2 signaling modulates axon collateral formation through CRMP2. Since axon branching is the first step of MFS, we then test if CRMP2 mediates Npn-2 signaling in MFS in epileptic animals.

Rats were injected with shCtrl AAV, shCRMP2 AAV, shNpn-2 AAV, or shNpn-2 AAV plus shCRMP2 AAV 14 days before pilocarpine-induced rat model of epilepsy establishment. Epileptic rats were sacrificed at 21 days after pilocarpine injection to visualize hippocampal mossy fiber terminals using the Timm stain. CRMP2 knockdown epileptic animals showed significantly reduced MFS compared with control epileptic animals (Fig. 8A, B). Furthermore, the reduction of CRMP2 rescued the increased MFS in Npn-2 knockdown epileptic animals (Fig. 8C, D). These results clearly showed that CRMP2 is mediating Npn-2 signaling in regulating MFS.

Fig. 8

Npn-2 modulates MFS through CRMP2. A CRMP2 knockdown reduced MFS in epilepsy animals. Representative Timm stain of rat brain coronal sections in various AAV-injected groups as indicated at 21 days after pilocarpine injection. CRMP2 knockdown in epileptic rats (A2, A2') reduced MFS compared with the control group (A1, A1'). A1'–A2' are higher magnification views of A1–A2, respectively. A1–A2 Scale bars: 400 µm. A1'–A2' Scale bars: 100 μm. B Quantitation of Timm score in A, n = 4, *P = 0.0401. C CRMP2 mediated Npn-2 signaling in MFS in epilepsy animals. Representative Timm stain of rat brain coronal sections in various AAV-injected groups as indicated 21 days after pilocarpine injection. Reduction of CRMP2 (C2, C2') rescued the increased MFS caused by Npn-2 knockdown (C1, C1'). C1'–C2' are higher magnification views of C1–C2, respectively. C1–C2 Scale bars: 400 µm. C1'–C2' Scale bars: 100 μm. D Quantitation of Timm score in C, n = 4, **P = 0.0069. Unpaired t-test. Error bars represent SEM

CRMP2 Mediates Npn-2 Function in Regulating Seizure Activity

Given that CRMP2 plays significant role in Npn-2 mediated MFS and MFS is closely related to seizure activity, we next asked whether Npn-2 signaling regulates seizure activty through CRMP2.

To this end, rats with shCRMP2 AAV or shNpn-2 AAV plus shCRMP2 AAV were intraperitoneally injected with pilocarpine and surveilled for 21 days. The efficacy of shCRMP2 AAV was validated in vivo (Fig. 9A, B). Two of 10 rats in the shCRMP2 group died during SRS (mortality rate: 20%). Behavior data of 8 rats in the shCRMP2 group and 10 rats in shNpn-2 plus shCRMP2 group were analyzed.

Fig. 9

CRMP2 mediates Npn-2 function in regulating seizure activity. A CRMP2 knockdown validation in vivo. Fourteen days after intrahippocampal injection of shCtrl AAV or shCRMP2 AAV, rats were sacrificed for western blot analysis, and an effective knockdown of CRMP2 was observed. B Quantitation of CRMP2 in A, n = 3, **P = 0.0039. C Overview of SRS. Five out of 13 (38%) rats in shCtrl group and 4 out of 8 (50%) rats in shCRMP2 group developed SRS during the 21-day surveillance period post pilocarpine injection. Four out of 10 (40%) in shNpn-2 plus shCRMP2 group, while 11 out of 14 (73%) rats in shNpn-2 alone group developed SRS. D Mean duration per seizure. Seizure duration in shNpn-2 plus shCRMP2 group was 20.23 s, which was significantly less than the 29.59 s in shNpn-2 group (*P = 0.0459). E SRS stage. There was no significant difference in SRS stage among shCtrl group, shCRMP2 group, shNpn-2 group, and shNpn-2 plus shCRMP2 group (4.800 vs 4.950 vs 4.577 vs 4.718, P = 0.2579). F SRS latency. No significant difference in SRS latency was found among shCtrl group, shCRMP2 group, shNpn-2 group, and shNpn-2 plus shCRMP2 group (15.20 days vs 15.00 days vs 15.00 days vs 14.50 days, P = 0.9867). G SE latency. The SE latency of rats in among shCtrl group, shCRMP2 group, shNpn-2 group, and shNpn-2 plus shCRMP2 group was similar to each other (15.06 min vs 15.00 min vs 14.82 min vs 16.7 min, P = 0.7306). One-way ANOVA, post hoc Tukey test. Error bars represent SEM

Fewer rats in shNpn-2 plus shCRMP2 group (40%) developed SRS during 21-day surveillance compared with that in the shNpn-2 group (73%) (Fig. 9C). The mean duration per seizure in shNpn-2 plus shCRMP2 group was 20.23 s, which was significantly less than the 29.59 s in the shNpn-2 group (Fig. 9D). There was no significant difference in SRS stage (Fig. 9E), SRS latency (Fig. 9F), and SE latency (Fig. 9G) among groups. These data showed that increased seizure activity in Npn-2 knockdown rats can be rescued by the reduction of CRMP2 expression, suggesting that CRMP2 mediates Npn-2 signaling in regulating seizure activity.