Disrupted in Schizophrenia 1 reverse ectopic migration of neural precursors in mouse hilus after pilocarpine-induced status epilepticus

doi:10.21203/rs.3.rs-2587049/v1

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 vitroand 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, 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.

Status epilepticus

DISC1

Migration

Neural precursors

Hilus

DISC1 could partly reverse the ectopic migration of neural precursors to the hilus via EphB2 pathway after SE;
Both Reelin-ApoER2/EphB2 and Reelin-Integrin β1/Integrin α5 axes may participate in the modulation of neurogenesis after SE;
DISC1 may play a protective role in the neurogenesis response to SE.

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[12–14], 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[17–19]. 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 can 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.

Animals

Males, 4–6 weeks, 20 ± 2 g, C57BL/6 mice were used in this study. They were housed in a standardized controlled environment (light/dark cycle: 12/12 hours, room temperature (RT): 22 ± 4°C, humidity: 60%) and had free access to food and water. All animal care and procedure were according to the requirements of the Chinese Animal Ethics Committee and approved by Kunming Medical University.

Pilocarpine‑induced SE

SE was induced as described previously [22].

Antibodies

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

Table 1

The antibodies used in this experiment
Antibodies	Source	Identifier
Mouse monoclonal anti- DISC1	Santa Cruz	SC-365591
Rabbit polyclonal anti- DISC1	Thermo Fisher	40-6900
Rabbit polyclonal anti-Doublecortin	Cell Signaling Technology	#4604
Mouse monoclonal anti-Neun	Cell Signaling Technology	#94403
Mouse monoclonal anti-Reelin	Merck	#MAB5364
Rabbit monoclonal anti- ApoER2	Abcam	Ab108208
Rat monoclonal anti- EphB2	R&D	MAB4671
Rabbit monoclonal anti- EphB2	Cell Signaling Technology	#83029
Rat monoclonal anti- Integrin β1	R&D	MAB2405
Rat monoclonal anti- Integrin α5	R&D	MAB1754
Rabbit monoclonal anti- Integrin β1	Cell Signaling Technology	#34971
Rabbit monoclonal anti- Integrin α5	Cell Signaling Technology	#98204
Mouse monoclonal anti-β-Actin	Cell Signaling Technology	#3700
Goat anti-mouse IgG (H + L) HRP	Affinity	S0001
Goat anti- Rabbit IgG (H + L) HRP	Cell Signaling Technology	#5366
Alexa Flour 594 Goat Anti-Rabbit	Proteintech
Alexa Flour 488 Goat Anti-Rabbit	Proteintech
Alexa Flour 594 Goat Anti-Mice	Proteintech
Alexa Flour 488 Goat Anti-Mice	Proteintech
Alexa Flour 488 Goat Anti-Rat	Bioss
Goat anti-mouse secondary antibody	Proteintech
Goat anti-rabbit secondary antibody	Proteintech

DISC1 single gene intervention

Seven days after SE, mice were randomly divided into AAV-DISC1 group (HBAAV2/9-Ta1-m-Disc1-3xflag-Null, 1.8*10^12 vg/mL, Shanghai Han Heng Technology Co., LTD) and negative AAV group (HBAAV2/9-Ta1-Null, 1.8*10^12 vg/mL, Shanghai Han Heng Technology Co., LTD). Additionally, three mice were randomly injected with HBAAV2/9-Ta1-ZsGreen (1.7*10^12 vg/ mL, Shanghai Han Heng Technology Co., LTD) to observe the injection location and AAV transfection effect. Mice were anesthetized by 7.2% chloral hydrate (3 ml /kg, i.p.) and fixed on the stereotaxic apparatus (RWD Life Science, Shenzhen, China). The AAV (2 µl) was injected into bilateral hippocampi (coordinates: AP = 2.0 mm posterior to bregma, ML = ± 1.6 mm lateral to bregma, DV = 2.5 mm). The needle should keep still for 5 mins after injection ended which could help AAV spread and infect efficiently. Samples were collected 3 and 6 weeks after the AAV injection.

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 4 ℃ overnight: mouse anti-DISC1 (1:200), rabbit anti-DISC1 (1:200), rabbit anti-DCX (1:200), mouse anti-Neun (1:200), mouse anti-Reelin (1:200), Rat anti-EphB2 (1:150), rabbit anti-EphB2 (1:200), rat anti-integrin β1(1:200), rat anti-integrin α5 (1:200). Secondary antibodies used: goat anti-rabbit (1:300), goat anti-mouse (1:300), goat anti-rat (1:300). Fluoromount-G solution containing 40,6-diamidino-2-phenylindole (DAPI, Sigma, USA) was used to counterstain sections. The DG region was captured using a positive fluorescence BX53 Olympus microscope and analyzed via the ImageJ software package (Media Cybernetics, Inc., Rockville, MD, USA).

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).

Western blot

Hippocampus tissue was resolved with 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Blocking with 5% skim milk for 2 h, at RT, the membranes were incubated overnight (4 ℃) with the following primary antibodies: mouse anti-DISC1(1:1000, Santa Cruz), mouse anti-Reelin (1:2000), rabbit anti-ApoER2 (1:1000), anti-EphB2 (1:2000), rabbit anti- integrin β1 (1:2000), rabbit anti-integrin α5 (1:2000). Subsequently, the membranes were then rinsed and incubated with secondary antibodies as follows: goat anti-mouse secondary antibody conjugated with peroxidase (1:2000) and goat anti-rabbit secondary antibody conjugated with peroxidase (1:2000) for 2 h at RT. Following 3 washes with TBST (with 0.1% Tween-20) for 10 min, the membranes were visualized by ECL reagent (Millipore, USA). The densitometer of protein bands was analyzed by ImageJ.

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 mins, 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. 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[23, 24]. 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-1W vs SE-1W, p < 0.05; CON-2W vs SE-2W, p < 0.01; CON-4W vs SE-4W, p < 0.01), but also ectopic migrated from the SGZ to the hilus (CON-1W vs SE-1W, p < 0.0001; CON-2W vs SE-2W, p < 0.01; CON-4W vs SE-4W, p < 0.001, CON-8W vs SE-8W, p < 0.001) (Figs. 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-1W vs SE-1W, p < 0.001; CON-8W vs SE-8W, p < 0.05) and the SGZ post-SE (CON-1W vs SE-1W, p < 0.001; CON-2W vs SE-2W, p < 0.001) (Figs. 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 [25]. 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 Figs. 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 (Figs. 2h-k), but Integrin β1 and Integrin α5 showed an increase at 1-week post-SE (no statistical difference) (Figs. 2l, m).

To investigate the relationship between DISC1 and Reelin, double-label immunofluorescence was performed. As expected, DISC1 and Reelin co-located in the hilus (Fig. 3a, Supplementary Fig. 2), and DISC+/Reelin + cells were significantly down-expressed at 1, 2, 4, and 8 weeks after SE (p < 0.01) (Fig. 3c). Moreover, compared to the CON groups, we found the DISC1+/EphB2 + cells decreased at 4 and 8 weeks in the SE groups (p < 0.05) (Figs. 3b, d, Supplementary Fig. 3). Additionally, DISC1+/ Integrin β1 + cells in the hilus were decreased at 1-and 4-weeks after SE (Figs. 4a, c, Supplementary Fig. 4). Meanwhile, the number of DISC1 and Integrin α5 co-expressed cells in the hilus was decreased at SE-8W group (Figs. 4b, d, Supplementary Fig. 5). Accordingly, DISC1 may regulate the ectopic migration of neurons after SE via the Reelin/ ApoER2/ EphB2 / Integrin β1 / Integrin α5 axis.

Upregulation of DISC1 reversed ectopic migration of neural precursors to the hilus

To evaluate the effect of DISC1 on the ectopic migration of neural precursors, we upregulated the expression of DISC1 by injecting adeno-associated virus (AAV) carrying HBAAV2/9-Ta1-m-Disc1-3xflag-Null or HBAAV2/9-Ta1-Null into DG 7 days following pilocarpine‑induced SE (Fig. 5a). The injection location and infection efficiency of AAV were confirmed (Fig. 5b). DISC1 was efficiently up-regulated in the hilus (AAV-3W vs AAV-DISC1-3W, p < 0.001; AAV-6W vs AAV-DISC1-6W, p < 0.05) and SGZ (AAV-3W vs AAV-DISC1-3W, p < 0.0001; AAV-6W vs AAV-DISC1-6W, p < 0.01) at 3 weeks and 6 weeks (Figs. 5c, d, e, Table S4). As shown in Figs. 5C, F, in the hilus, the ectopic migration of DCX + cells were observed more in the AAV group than in the AAV-DISC1 group (AAV-3W vs AAV-DISC1-3W, p < 0.05; AAV-6W vs AAV-DISC1-6W, p < 0.01). However, DISC1 up-regulation barely influences DCX + cells that migrated to the SGZ (Figs. 5c, g, Table S5). These results suggest that DISC1 partly and specifically rescues the ectopic migration of neural precursors in the hilus.

DISC1 regulated Reelin/ApoER2/EphB2 axis positively while Reelin/Integrin β1 or Reelin/Integrin α5 negatively

Western blot and RT-PCR were used to test the efficiency of DISC1 up-regulation in the whole hippocampus (Figs. 6a, b, h). With DISC1 up-regulation, Reelin, ApoER2, and EphB2 increased (Figs. 6a, c-e, i-k), while Integrin β1 and Integrin α5 decreased (Figs. 6a, f, g, l, m). These results indicated that DISC1 might have a positive regulatory effect on Reelin/ApoER2/EphB2 axis while negative on Integrin β1 and Integrin α5.

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 Figs. 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, Figs. 7b, f, Supplementary Fig. 7), whereas the number of DISC1+/Integrin β1 + cells or DISC1+/Integrin α5 + cells was barely altered (Figs. 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.

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 balanced 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, 26]. In vitro, Wu et al. found that DISC1 deficiency reduced the proliferation and migration of fetal NSCs, and vice versa [27]. Additionally, Namba et al. treated adult mice with amantadine (an 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 [26]. 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 [28–30]. 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, 31]. 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, 32]. As a signal for migration halt, Reelin has been found decreased in both epileptic patients and animal models [33]. In intractable TLE, Haas et al. found that the severity of granule cell dispersion (GCD) was negatively correlated with Reelin mRNA expression [34]. 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 [35]. 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 [36]. 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 [37] and regulates synaptic plasticity, promoting axon growth and regeneration, and enhancing dendritic filopodia motility [38, 39]. 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 [40, 41]. 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 [41]. 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 could regulate ectopic migration of newborn neurons in the hilus after SE by regulating EphB2.

Integrin proteins are transmembrane heterodimers including α and β subunits [42], which were involved in axonal guidance, synaptogenesis, astrocyte maturation and migration by binding laminin and other extracellular matrix proteins [43, 44]. 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 [45]. The integrin α5 subunit binds to the integrin β1 subunit and regulates the growth and development of neurites [46]. Moreover, Reelin activates integrin a5β1 among those migrating neurons via the intracellular ApoER2/VLDLR-Dab1-Crk/CrkL-C3G-Rap1 pathways [17]. Additionally, integrin proteins also increase in pilocarpine-induced epilepsy models, suggesting that they participate in the process of epilepsy [47, 48]. 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 [49]. 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 up-regulation 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.

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 can partly reverse the ectopic migration of newborn neurons via EphB2.

Supplemental Information Supplemental Information includes nine figures and five tables.

Author contributions Qian Wu: conceived and designed experiments. Lu Chen, Jing Xu, Lin Zhu, and Puying Xu: performed experiments and analyzed the data. Lu Chen, Jing Xu, and Lin Zhu: wrote the paper and final approval of the manuscript. Lu Chen, Puying Xu, and Lvhua Chang: prepared the figures. Qian Wu, Yanbing Han and Lu Chen: responded funding. Qian Wu, Yanbing Han, and Lvhua Chang: critically reviewed and commented on the manuscript.

Funding This work was supported by the National Natural Science Foundation of China (82160260, 81601134 to Q.W, 82160261 to Y. H. ), the Yunnan Health Training Project of High-Level Talents (H-2018056 to Q.W., L-2019019 to Y. H.), the Young and Middle-aged Academic and Technical Leaders Reserve Talents Project of Yunnan Province Science and Technology Department (202205AC160019 to Q.W.), Yunnan Applied Basic Research Projects (202201AY070001-083 to Q.W.), and the Scientific Research Fund Project of the Education Department of Yunnan Province (2022Y218 to L.C.).

Availability of Data and Materials All data generated or analyzed in this study are available in the published article.

Code Availability Not applicable.

Acknowledgements Not applicable.

Ethics approval All animal-based experiments were according to the requirements of the Chinese Animal Ethics Committee and approved by Kunming Medical University.

Consent to participate Not applicable.
Consent for Publication All authors consent to publication of this manuscript.
Competing interests The authors declare no competing interests.

Beghi, E. (2020) The Epidemiology of Epilepsy. Neuroepidemiology 54(2): 185-191.
Yang, C., et al. (2022) Cadherins and the pathogenesis of epilepsy. Cell Biochem Funct 40(4): 336-348.
Jessberger, S. and J.M. Parent. (2015) Epilepsy and Adult Neurogenesis. Cold Spring Harb Perspect Biol 7(12).
Zhao, C.S. and L. Overstreet-Wadiche. (2008) Integration of adult generated neurons during epileptogenesis. Epilepsia 49 Suppl 5: 3-12.
Hester, M.S. and S.C. Danzer. (2013) Accumulation of abnormal adult-generated hippocampal granule cells predicts seizure frequency and severity. J Neurosci 33(21): 8926-36.
Parent, J.M. and M.M. Kron. (2011) Neurogenesis and epilepsy. Epilepsia 51(Supplement s5): 45-45.
Scharfman, H.E. (2019) The Dentate Gyrus and Temporal Lobe Epilepsy: An "Exciting" Era. Epilepsy Curr 19(4): 249-255.
Althaus, A.L., et al. (2015) Intrinsic neurophysiological properties of hilar ectopic and normotopic dentate granule cells in human temporal lobe epilepsy and a rat model. J Neurophysiol 113(4): 1184-94.
Duan, X., et al. (2008) Development of neural stem cell in the adult brain. Curr Opin Neurobiol 18(1): 108-15.
Ye, F., et al. (2017) DISC1 Regulates Neurogenesis via Modulating Kinetochore Attachment of Ndel1/Nde1 during Mitosis. Neuron 96(5): 1041-1054 e5.
Bradshaw, N.J. and D.J. Porteous. (2012) DISC1-binding proteins in neural development, signalling and schizophrenia. Neuropharmacology 62(3): 1230-41.
Lee, H., et al. (2015) DISC1-mediated dysregulation of adult hippocampal neurogenesis in rats. Front Syst Neurosci 9: 93.
Duan, X., et al. (2007) Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell 130(6): 1146-58.
Fournier, N.M., et al. (2010) The effect of amygdala kindling on hippocampal neurogenesis coincides with decreased reelin and DISC1 expression in the adult dentate gyrus. Hippocampus 20(5): 659-71.
Fournier, N.M., et al. (2009) Decreased levels of disrupted-in-schizophrenia 1 (DISC1) are associated with expansion of the dentate granule cell layer in normal and kindled rats. Neurosci Lett 455(2): 134-9.
Jossin, Y. (2020) Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation. Biomolecules 10(6).
Sekine, K., et al. (2012) Reelin controls neuronal positioning by promoting cell-matrix adhesion via inside-out activation of integrin alpha5beta1. Neuron 76(2): 353-69.
Elisabeth Bouché1, et al. (2013) Reelin induces EphB activation. Cell Research 23(4): 473-490.
Hirota, Y., et al. (2018) ApoER2 Controls Not Only Neuronal Migration in the Intermediate Zone But Also Termination of Migration in the Developing Cerebral Cortex. Cereb Cortex 28(1): 223-235.
Stanfield BB and C. WM. (1979) The development of the hippocampus and dentate gyrus in normal and reeler mice. The Journal of comparative neurology 185(3): 423–459.
Zhao, S., et al. (2004) Reelin is a positional signal for the lamination of dentate granule cells. Development 131(20): 5117-25.
Chen, L., et al. (2022) Disrupted in Schizophrenia 1 Regulates Ectopic Neurogenesis in the Mouse Hilus After Pilocarpine-induced Status Epilepticus. Neuroscience 494: 69-81.
Dennis, C.V., et al. (2016) Human adult neurogenesis across the ages: An immunohistochemical study. Neuropathol Appl Neurobiol 42(7): 621-638.
Shahsavani, M., et al. (2018) An in vitro model of lissencephaly: expanding the role of DCX during neurogenesis. Mol Psychiatry 23(7): 1674-1684.
Gong, C., et al. (2007) Reelin regulates neuronal progenitor migration in intact and epileptic hippocampus. J Neurosci 27(8): 1803-11.
Namba, T., et al. (2011) NMDA receptor regulates migration of newly generated neurons in the adult hippocampus via Disrupted-In-Schizophrenia 1 (DISC1). J Neurochem 118(1): 34-44.
Wu, Q., et al. (2017) DISC1 Regulates the Proliferation and Migration of Mouse Neural Stem/Progenitor Cells through Pax5, Sox2, Dll1 and Neurog2. Front Cell Neurosci 11: 261.
Althaus, A.L., et al. (2019) Altered Synaptic Drive onto Birthdated Dentate Granule Cells in Experimental Temporal Lobe Epilepsy. J Neurosci 39(38): 7604-7614.
Scharfman, H., et al. (2007) Ectopic granule cells of the rat dentate gyrus. Dev Neurosci 29(1-2): 14-27.
Dai, Y., et al. (2021) In Vivo Microelectrode Arrays for Detecting Multi-Region Epileptic Activities in the Hippocampus in the Latent Period of Rat Model of Temporal Lobe Epilepsy. Micromachines (Basel) 12(6).
Feng BH, et al. (2021) Expression of Disrupted-in-schizophrenia 1 protein and related inflammatory factors inflammatory factors in refractory temporal lobe epilepsy in children. J Biol Regul Homeost Agents 35: 221–226.
Bosch, C., et al. (2016) Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells. Cereb Cortex 26(11): 4282-4298.
Machado, R.A., et al. (2019) Reelin, tau phosphorylation and psychiatric complications in patients with hippocampal sclerosis and structural abnormalities in temporal lobe epilepsy. Epilepsy Behav 96: 192-199.
Haas CA, et al. (2002) Role for reelin in the development of granule cell dispersion in temporal lobe epilepsy. The Journal of neuroscience : the official journal of the Society for Neuroscience 22(14): 5797–5802.
Bradshaw, N.J., et al. (2020) Disrupted in Schizophrenia 1 regulates the processing of reelin in the perinatal cortex. Schizophr Res 215: 506-513.
Senturk, A., et al. (2011) Ephrin Bs are essential components of the Reelin pathway to regulate neuronal migration. Nature 472(7343): 356-60.
Bouvier, D., et al. (2008) Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain. J Neurochem 106(2): 682-95.
Grunwald IC, et al. (2001) Kinase-Independent Requirement of EphB2 Receptors in Hippocampal Synaptic Plasticity. Neuron 32(6): 1027–1040.
Davy, A. and P. Soriano. (2005) Ephrin signaling in vivo: look both ways. Dev Dyn 232(1): 1-10.
Chumley MJ, et al. (2007) EphB Receptors Regulate Stem/Progenitor Cell Proliferation, Migration, and Polarity during Hippocampal Neurogenesis. J Neurosci 27(49): 13481–13490.
Catchpole, T. and M. Henkemeyer. (2011) EphB2 tyrosine kinase-dependent forward signaling in migration of neuronal progenitors that populate and form a distinct region of the dentate niche. J Neurosci 31(32): 11472-83.
Campbell, I.D. and M.J. Humphries. (2011) Integrin structure, activation, and interactions. Cold Spring Harb Perspect Biol 3(3).
Tharmalingam, S., et al. (2016) The calcium-sensing receptor and integrins modulate cerebellar granule cell precursor differentiation and migration. Dev Neurobiol 76(4): 375-89.
Lilja, J. and J. Ivaska. (2018) Integrin activity in neuronal connectivity. J Cell Sci 131(12).
Brooker, S.M., et al. (2016) beta1-integrin restricts astrocytic differentiation of adult hippocampal neural stem cells. Glia 64(7): 1235-51.
Izumi, Y., et al. (2017) Integrin alpha5beta1 expression on dopaminergic neurons is involved in dopaminergic neurite outgrowth on striatal neurons. Sci Rep 7: 42111.
Fasen, K., et al. (2003) Distribution of alpha and beta integrin subunits in the adult rat hippocampus after pilocarpine-induced neuronal cell loss, axonal reorganization and reactive astrogliosis. Acta Neuropathol 106(4): 319-22.
Jaudon, F., et al. (2021) Integrin adhesion in brain assembly: From molecular structure to neuropsychiatric disorders. Eur J Neurosci 53(12): 3831-3850.
Wu, X., et al. (2017) Atomic force microscopy investigations of fibronectin and alpha5beta1-integrin signaling in neuroplasticity and seizure susceptibility in experimental epilepsy. Epilepsy Res 138: 71-80.

SupplementaryFig.1.tif
Supplementary Fig. 1 Expression of DISC1 and DCX in the hilus. After SE, DCX+ cells increased in the SGZ and migrated ectopically to the hilus. No co-localization of DISC1 and DCX was observed. Green fluorescence labels DISC1, red fluorescence labels DCX, and blue fluorescence labels the nucleus. The white arrows show ectopically migrated DCX+ cells, scale bar = 20 μm.
SupplementaryFig.2.tif
Supplementary Fig. 2 Co-expression of DISC1 and Reelin in the hilus. DISC1 co-localized with Reelin in the SGZ and the hilus. The white arrows show DISC1+/ Reelin + cells, red fluorescence labels DISC1, green fluorescence labels Reelin, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.3.tif
Supplementary Fig. 3 Co-expression of DISC1 and EphB2 in the hilus. DISC1 co-localized with EphB2 in the SGZ and the hilus. The white arrows show DISC1+/ EphB2 + cells, red fluorescence labels DISC1, green fluorescence labels EphB2, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.4.tif
Supplementary Fig. 4 Co-expression of DISC1 and Integrin β1 in the hilus. DISC1 co-localized with Integrin β1 in the GCL, SGZ, and the hilus. The white arrows show DISC1+/ Integrin β1+ cells, red fluorescence labels DISC1, green fluorescence labels Integrin β1, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.5.tif
Supplementary Fig. 5 Co-expression of DISC1 and Integrin α5 in the hilus. DISC1 co-localized with Integrin α5 in the GCL, SGZ, and the hilus. The white arrows show DISC1+/ Integrin α5+ cells, red fluorescence labels DISC1, green fluorescence labels Integrin α5, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.6.tif
Supplementary Fig. 6 Co-expression of DISC1 and Reelin in the hilus after DISC1 up-regulation. DISC1 co-localized with Reelin in the GCL, SGZ, and the hilus. The white arrows show DISC1+/ Reelin+ cells, red fluorescence labels DISC1, green fluorescence labels Reelin, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.7.tif
Supplementary Fig. 7 Co-expression of DISC1 and EphB2 in the hilus after DISC1 up-regulation. DISC1 co-localized with EphB2 in the GCL, SGZ, and the hilus. The white arrows show DISC1+/ EphB2+ cells, red fluorescence labels DISC1, green fluorescence labels EphB2, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.8.tif
Supplementary Fig. 8 Co-expression of DISC1 and Integrin β1 in the hilus after DISC1 up-regulation. DISC1 co-localized with Integrin β1 in the GCL, SGZ, and the hilus. The white arrows show DISC1+/ Integrin β1+ cells, red fluorescence labels DISC1, green fluorescence labels Integrin β1, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryFig.9.tif
Supplementary Fig. 9 Co-expression of DISC1 and Integrin α5 in the hilus after DISC1 up-regulation. DISC1 co-localized with Integrin α5 in the GCL, SGZ, and the hilus. The white arrows show DISC1+/ Integrin α5+ cells, red fluorescence labels DISC1, green fluorescence labels Integrin α5, and blue fluorescence labels the nucleus, scale bar = 20 μm.
SupplementaryTables.docx

Disrupted in Schizophrenia 1 reverse ectopic migration of neural precursors in mouse hilus after pilocarpine-induced status epilepticus

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