Disrupted in Schizophrenia 1 Reverse Ectopic Migration of Neural Precursors in Mouse Hilus After Pilocarpine-Induced Status Epilepticus

Neural precursors in the subgranular zone (SGZ) can be stimulated by status epilepticus (SE) and ectopically migrate to the hilus. These mislocated cells serve as “potential pacemakers” of spontaneous recurrent seizures, and targeting them could potentially reverse the seizure process. Disrupted-in-Schizophrenia 1 (DISC1) regulates hippocampal neurogenesis after seizures both in vitro and in vivo. Our previous study found that DISC1 was colocalized with neural precursors in the hilus after SE. However, its molecular mechanism and pathways contribute to the ectopic migration of neural precursors to the hilus induced by SE awaits exploration. Here, we showed that both Reelin-ApoER2/EphB2 and Reelin-Integrin β1/Integrin α5 axes may participate in the modulation of neurogenesis after SE. Especially, DISC1, as a protective role, might partly reversed the ectopic progenitor migration via EphB2 pathway. Our findings demonstrated that DISC1 played a protective role in the ectopic migration of neural precursors induced by SE insults and DISC1 could be an attractive new target for the treatment of epilepsy.


Introduction
Epilepsy, caused by abnormal discharge of neurons, affects over 70 million people worldwide of all races, social classes, and ages [1,2].Its pathological features include pyramidal cells loss, astrocytes proliferation, mossy fibers sprouting and granulosa cells dispersion in the granule cell layer (GCL) of the dentate gyrus (DG), which is related to abnormal neurogenesis in the hippocampus induced by seizures insults [3].Most newborn neurons generated by seizures, due to excessive proliferation and differentiation, have immature synapses and basal dendrites, which were able to receive the excitatory impulse from the terminal of mossy fibers and eventually become "potential pacemakers" of spontaneous seizures in the chronic phase [4,5].When these "potential pacemakers" misoriented and reverse migration to the hilus or excessive migration to the outer 1/3 layer of GCL, molecular layer (MCL) and even polymorphic layer, excitatory signals will be generalized and propagated, which was supposed to be one hypothesis of epileptogenesis [3,6].Especially, these misplaced newborn neurons in the hilus were recorded to have axons that innervate normal GCs and CA3 pyramidal cells, which possibly synchronized hyperactivities between epileptogenic focus and other regions [7].Moreover, the hilar newborn neurons in epileptic rats showed a higher action potential (AP) firing rate compared to those normally migrated to the GCL, indicating that ectopic migration of newborn neurons to the hilus contributes to hyperexcitability of the epileptic hippocampal network [8].Therefore, identifying its pathophysiological mechanism will help to further understand one pathogenesis of temporal lobe epilepsy (TLE) and may provide potential targets to reverse epilepsy.
Disrupted-in-Schizophrenia 1(DISC1), as an intracellular scaffold protein, is widely expressed in the embryonic brain, whereas in the adult mammalian brain, it is restrictively in the hippocampus and interneurons of the olfactory bulb [9].Numerous studies have shown that DISC1 can regulate synaptic plasticity, neuronal proliferation, migration, and signal transmission by co-work with lissencephaly 2 (LIS2), nuclear distribution factors E homologs like 1 (NDEL1), nuclear distribution factor E homolog 1(NDE1), and Girdin, etc [10,11].Disc1 gene knockout mice and rats presented mislocated newborn neurons with larger soma and increased basal dendrites in the hilus [12][13][14], all these changes resembling morphological features after seizures.Interestingly, in amygdaloid kindling mice, the increase of ectopic granule cells in the hilus was accompanied by a decrease in DISC1 labeling in the subgranular zone (SGZ), GCL, and CA3 [15].According to the evidence aforementioned, the downregulation of DISC1 after epilepsy may be related to the ectopic migration of newborn neurons or granulosa cells to the hilus.
Reelin, as secreted glycoprotein, is proven to exert a critical role in the migration and localization of neuroblasts during brain development [16].Physically, it can promote the migration and integration of newborn GCs via apolipoprotein E receptor 2 (ApoER2) /EphB2, Integrin α5/β1 aix in the hippocampus [17][18][19].Reelin deficiency could result in most GCs failure to migrate to the GCL and over-migrated to the hilus [20,21].Additionally, Carola et al. observed that Reelin and DISC1 were not only decreased in the GCL and SGZ but co-expressed in the hilus of DG, suggesting that reduction of Reelin and DISC1 might contribute to abnormal neurogenesis in kainite (KA) kindled rat model [14].Nevertheless, it is still unclear if and how DISC1 and Reelin pathways co-regulated ectopic migration of new neurons after SE.Therefore, this present study provides evidence that upregulation of DISC1 might rescue ectopic migration of neural progenitor cells to the hilus caused by seizures insults via regulating the expression of EphB2.We further showed that in the hippocampus of pilocarpine-induced SE mice, DISC1 positively regulates the Reelin-ApoER2/ EphB2 axis and negatively regulates Integrin β1 and Integrin α5.Therefore, DISC1 may be an attractive target for epilepsy treatment.

Antibodies
The antibodies used in the whole experiment are shown in Table 1.

Immunofluorescence
Mice were anesthetized and transcardially perfused with 0.9% sterile saline and 4% paraformaldehyde (PFA).Soaked in 4% PFA for 12 h and dehydrated in 10%, 20% and 30% sucrose gradient at 4℃.The coronal brain samples were cut coronally into 10 μm sections using frozen microtome.Immunofluorescence was performed with tissues mounted on charged slides to mark the distribution and expression of proteins.To block nonspecific bindings, sections were blocked by 10% goat serum 2 h, at RT after rinsing with PBS three times.Successively, the sections were incubated with the primary antibodies at

Microscopy and Morphology Analysis
ImageJ software in a random region (20×) was used to quantify the areas of the hilus and cell numbers by two investigators.The statistical methods refer to our published article [22] (n = 3).

Real-Time PCR
Hippocampus samples were rapidly dissected and transferred into liquid nitrogen.Total RNAs were isolated, and reverse transcribed into cDNA using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan).The resulting cDNA was subjected to PCR using TB Green Premix Ex Taq™ II (Takara, Japan) on QuantStudio TM Real-Time PCR System to quantify mRNA expression.The reaction was incubated at 95 ℃ for 10 min, denaturation 45 cycles at 95 ℃ for 15 s and annealing/extension at 60 ℃, 1 min.The process was performed with a real-time PCR system (Mx3005P, Stratagene, USA).β-actin served as endogenous control, and the relative expression levels were determined with the 2 −ΔΔCt method.The primer sequences are listed in Table S1.

Statistical Analysis
GraphPad Prism 8.4.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used to do statistical analysis.All data was normally distributed by testing with Shapiro-Wilk test (p > 0.05).For comparisons between the two groups, an unpaired Student's t-test was performed.And statistical significance was set at p < 0.05.All results are presented as mean ± standard error of the mean (SEM).

Neuronal Precursors Cells Ectopic Migration to the Hilus with DISC1 Decreased in Pilocarpine-Induced SE Mice
Doublecortin (DCX), a marker of neuroblasts, is critical for neuronal migration [24,25].The expression of DCX can be used to identify the migration of neural precursor cells in the DG region.To investigate the ectopic migration of neural precursors cells after SE and its relationship with DISC1, we performed double-label immunofluorescence of DISC1 and DCX in pilocarpine-induced SE mice.Firstly, we found that DISC1 + cells were widely expressed in the DG, CA3 and CA1 regions of the hippocampus (Fig. 1b).Ulteriorly, we observed that DISC1 + and DCX + did not co-locate in the hilus and the SGZ region.Remarkably, in the control group, DCX + cells were confined to the SGZ.After SE, DCX + cells not only significantly increased in the SGZ (CON-1 W vs. SE-1 W, p < 0.05; CON-2 W vs. SE-2 W, p < 0.01; CON-4 W vs. SE-4 W, p < 0.01), but also ectopic migrated from the SGZ to the hilus (CON-1 W vs. SE-1 W, p < 0.0001; CON-2 W vs. SE-2 W, p < 0.01; CON-4 W vs. SE-4 W, p < 0.001, CON-8 W vs. SE-8 W, p < 0.001) (Fig. 1c, f, g, Supplementary Fig. 1, Table S2), indicating that SE induced abnormal migration of neuronal precursors to the hilus hippocampal.Moreover, DISC1 + cells were reduced significantly in the hilus (CON-1 W vs. SE-1 W, p < 0.001; CON-8 W vs. SE-8 W, p < 0.05) and the SGZ post-SE (CON-1 W vs. SE-1 W, p < 0.001; CON-2 W vs. SE-2 W, p < 0.001) (Fig. 1c, d, e, Supplementary Fig. 1, Table S3).Our previous study uncovered that DISC1 might take part in the modulation of neurogenesis in the hilus post-SE [22].Taken together, DISC1 may negatively regulate the ectopic migration of neural precursor cells to the hilus after SE.

SE Insults Altered the Expression of DISC1, Reelin, ApoER2, EphB2, Integrin β1 and Integrin α5
Reelin is a secreted migration guidance signal, the loss of Reelin expression in the adult hippocampus of epilepsy may lead to ectopic migration and aberrant integration of newborn granulosa cells in the DG [26].Accordingly, the Reelin signaling pathway is critical for neuronal migration.To further explore the expression changes of DISC1 and Reelin signaling pathway after SE, we performed Western blot and RT-PCR to assess the proteins and the mRNA expression levels.As shown in Fig. 2a-g, after SE, the expression of DISC1, Reelin, ApoER2, and EphB2 were significantly decreased, while Integrin β1 and Integrin α5 were increased, especially at 1 week.Similarly, the mRNA levels of DISC1, Reelin, ApoER2, and EphB2 were decreased at 1-week, 2-weeks, 4-weeks, and 8-weeks post-SE (Fig. 2h-k), but Integrin β1 and Integrin α5 showed an increase at 1-week post-SE (no statistical difference) (Fig. 2l, m).

Upregulation of DISC1 Rescued the Decreased Co-Expression of DISC1 and EphB2 in the Hilus of SE Mice
To further investigate the influence of DISC1 on the Reelin signaling pathway, double-staining immunofluorescence was adopted to mark the co-expression of DISC1 and Reelin, EphB2, Integrinβ1, and Integrinα5, respectively.As shown in Fig. 7a, e, Supplementary Fig. 6, in the hilus, DISC1+/Reelin + cells increased in the AAV-DISC1 group at 3 weeks after viral vector injection, while the statistical difference was scarcely significant compared to the AAV group.Additionally, the number of DISC1+/EphB2 + cells was higher in the AAV-DISC1 groups (p < 0.05, Fig. 7b, f, Supplementary Fig. 7), whereas the number of DISC1+/ Integrin β1 + cells or DISC1+/Integrin α5 + cells was barely altered (Fig. 7c, d, g, h, Supplementary Figs. 8, 9).These results indicated that DISC1 could inhibit abnormal migration of neurons in the hilus by regulating EphB2.

Discussion
Ectopic migration of newborn neurons to the hilus contributes to the hyperexcitability of the epileptic hippocampal networks.Interfering those ectopic newborn neurons may prohibit the mistaken formation of neural circuits and protect against seizures occurrence.Our results showed that DISC1 negatively regulates the ectopic migration of newborn neurons to the hilus in SE mice.Reelin, ApoER2, EphB2, Integrin β1 and Integrin α5 seemed to regulate the pathophysiological alteration in the hilus with DISC1 after SE.Among them, EphB2, acting as a protector, was proven to participate in modulating ectopic migration in the hilus.In summary, DISC1 might balance the migration of newborn neurons after SE via regulating EphB2.
DISC1 is critical for the proper migration and position of newborn neurons.Knockout of DISC1 in the hippocampus can abnormalize migration and proliferation of neural precursors, stimulate growth of irregular dendrites, speed up premature cell cycle exit and differentiation and increase excitability [12,13,27].In vitro, Wu et al. found that DISC1 deficiency reduced the proliferation and migration of fetal NSCs, and vice versa [28].Additionally, Namba et al. treated adult mice with amantadine (an Fig. 6 Protein and mRNA expression of Reelin, ApoER2, EphB2, Integrin β1 and Integrin α5 with DISC1 up-regulation.a Western blot was used to detect the expression of DISC1, Reelin, ApoER2, EphB2, Integrin β1 and Integrin α5 in the hippocampus, normalized to the β-actin protein.b-g Protein bands were analyzed by using ImageJ software.h-m Quantitative RT-PCR of DISC1, Reelin, ApoER2, EphB2, Integrin β1 and Integrin α5 mRNA expression in the hippocampus, normalized to the β-actin by the 2 −ΔΔCT method.All data are expressed as mean ± SEM; statistical analyses were performed using student's t-test, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ◂ NMDAR receptor antagonist) and found that accompanying down-regulation of DISC1, newborn neurons in DG appeared excessive migration which was then rescued by exogenous supplement of DISC1 [27].Additionally, epileptic insults could induce newborn neurons ectopic migration to the hilus and produce persistent basal dendrites, all of which contribute to higher excitability and spontaneous recurrence seizures [29][30][31].DISC1 seemed to take part in the process of epilepsy.Previous evidence showed that DISC1 expression decreased in the hippocampus of both TLE patients and animal models [15,32].In the present study, we first confirmed that after SE, DISC1 decreased while newborn neurons increased significantly in the SGZ and ectopically migrated to the hilus.Subsequently, we up-regulated DISC1 in the DG could significantly reduce the number of mis-migrated neurons in the hilus.Taken together, the erroneous migration of newborn neurons from SGZ to the hilus caused by SE can be rescued by upregulation of DISC1.
In the embryonic and adult brain, Reelin regulates neuronal migration, dendritic spine formation, dendritic growth and branching, synaptogenesis and synaptic plasticity [16,33].As a signal for migration halt, Reelin has been found decreased in both epileptic patients and animal models [34].In intractable TLE, Haas et al. found that the severity of granule cell dispersion (GCD) was negatively correlated with Reelin mRNA expression [35].Additionally, by crossbreeding heterozygous reeler mice with heterozygous transgenic DISC1 mice, the viability deficit was partially rescued by DISC1 expression.Moreover, both in vitro and in vivo, DISC1 regulated the growth and development of the perinatal cerebral cortex in cell-type and time-specific manners [36].Therefore, DISC1 interacts with Reelin to regulate neuronal migration and colonization.Additionally, Reelin guides neuronal migration by binding to very low-density lipoprotein receptors (VLDLR) and ApoER2 [37].In our study, we demonstrated that DISC1, as the upstream protein, positively regulated both Reelin and ApoER2 in the hilus after SE, which may be associated with neuronal migration in this certain region.
EphB2, as a member of the receptor tyrosine kinases (RTKs) family, expresses in self-renewing neural stem cells in adults [38] and regulates synaptic plasticity, promoting axon growth and regeneration, and enhancing dendritic filopodia motility [39,40].In EphB1 −/− mutant mice, nestin-positive cells decreased by nearly 50% and NSCs with abnormal polarity migrated ectopically to GCL and MCL.When EphB1 and EphB2 were eliminated by compound mutants, the volume of adult DG and the number of mature GCs decreased remarkably, suggesting that EphB2 regulated the proliferation and apoptosis of mature GCs [41,42].It is confirmed that EphB proteins served as receptors of Reelin.The combination of EphBs and Reelin will result in cytoskeletal changes by inducing EphB autophosphorylation [18].Additionally, EphB2 plays a crucial role in the development of lateral suprapyramidal blade (LSB), a specific region of DG, which may be related to neural precursors migration Ephrin-b1, as a ligand, can stimulate the forward signaling of EphB2 and instructed NSCs directly migrated the dorsum of the third matrix to form LSB. The forward signaling pathway is one of key regulators in normalizing Reelin expression in DG [42].Therefore, Reelin and EphB2 seemed playing important roles in neuronal migration in DG.However, the relationship of DISC1, EphB2 and ectopic migration of GCs after SE is scarcely reported up to now.In this study, we found that the number of DISC1+/ EphB2 + cells increased in the hilus after exogenous DISC1 supplement, indicating that DISC1 might regulate ectopic migration of newborn neurons in the hilus after SE by regulating EphB2.
Integrin proteins are transmembrane heterodimers including α and β subunits [43], which were involved in axonal guidance, synaptogenesis, astrocyte maturation and migration by binding laminin and other extracellular matrix proteins [44,45].Integrin β1, the largest subfamily of integrins, is expressed only in Radial glial-like (RGL) NSCs in the DG of adult mice.After integrin β1 knockout, astrocyte differentiation increased and neurogenesis decreased, indicating that integrin β1 plays an essential role in maintaining the neural stem cell niche [46].The integrin α5 subunit binds to the integrin β1 subunit and regulates the growth and development of neurites [47].Moreover, Reelin activates integrin a5/β1 among those migrating neurons via the intracellular ApoER2/VLDLR-Dab1-Crk/CrkL-C3G-Rap1 pathways [17].Additionally, integrin proteins also increase in pilocarpineinduced epilepsy models, suggesting that they participate in the process of epilepsy [48,49].Wu et al. found that monoclonal antibodies against Integrin α5/β1 strongly reversed the increased cell stiffness of dentate gyrus granule cells (DGGC) and play a strong preventive and protective role in the hippocampal kindling mice [50].In brief, integrin α5/β1 may be a new therapeutic target for epilepsy.Similarly, our results uncovered that integrin β1 and integrin α5 were increased post SE while decreased after upregulation of DISC1 in the hippocampus.However, we show that DISC1 intervention had little effect on the number of DISC1+/ integrin β1 + or DISC1+/ integrin α5 + neurons in the hilus.Based on this evidence, we inferred that integrin β1 and integrin α5, as possible co-workers with DISC1, might get involved in pathophysiological response in the hilus after seizures, but they may not take part in the process of neuronal migration.
However, this study has several limitations.Firstly, we did not conduct electrophysiological assessments of ectopic neurons in the DG after SE or after DISC1 intervention, which would reveal the excitability of individual neurons or neuron clusters in the DG.Secondly, we did not utilize EphB2 inhibitors subsequent to the DISC1 rescue experiment.In future studies, employing targeted inhibition of EphB2 in the DG after SE

Conclusion
In conclusion, DISC1, as an upstream molecule of Reelin-EphB2/ApoER2 and integrin β1/integrin α5, participates in regulating neurogenesis or neural development in the hilus after SE.Especially, DISC1 might partly reverse the ectopic migration of newborn neurons via EphB2.

Fig. 1
Fig. 1 Adult-born granule neurons ectopic migration to the hilus after SE. a A schematic of the different divisions of the hippocampus.Green fluorescence labels NeuN, blue fluorescence labels the nucleus, a1, scale bar = 200 μm, a2, scale bar = 100 μm.b The expression distribution of DISC1 in the hippocampus.Green fluorescence labels DISC1, blue fluorescence labels the nucleus, b1, scale bar = 200 μm, b2, scale bar = 100 μm, b3, scale bar = 50 μm.c DCX + cells increased in the SGZ and migrated ectopically to the hilus post-SE.No co-localization of DISC1+/DCX + cells was observed.Green

Fig. 5
Fig. 5 Reverse SE-induced ectopic migration of the neural precursors in the hilus by DISC up-regulation.a Experimental procedure of upregulation of DISC1 in the DG after pilocarpine kindled SE. b Expression of EGFP 21 days after AAV injection.Scale bar = 50 μm.c Expression of DISC1 and DCX in the hilus and SGZ after AAV injection.Green fluorescence labels DISC1, red fluorescence labels DCX, and blue fluorescence labels the nucleus.The white arrows show DCX + cells that ectopically migrated to the hilus, scale bar = 20 μm.d, e Quantitative analysis of DISC1 + cells in the hilus and the SGZ in AAV and AAV-DISC1 groups.f, g Quantitative analysis of DCX + cells in the hilus and the SGZ in AAV and AAV-DISC1 groups.All data are expressed as mean ± SEM; statistical analyses were performed using student's t-test, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ◂

Fig. 7
Fig. 7 Co-expression of DISC1 and Reelin pathway proteins in the hilus after DISC1 up-regulation.a, e Co-expression of DISC1 and Reelin (arrows).The number of DISC1+/Reelin + cells in the hilus increased in the AAV-DISC1 3 W group, despite no statistical difference.Red fluorescence labels DISC1, green fluorescence labels Reelin, and blue fluorescence labels the nucleus.b, f Co-expression of DISC1 and EphB2.The number of DISC1+/EphB2 + cells in the hilus increased at 3, and 6 weeks in the AAV-DISC1 groups.Red fluorescence labels DISC1, green fluorescence labels EphB2, and blue fluo-