Physiological significance of WDR45, a responsible gene for β-propeller protein associated neurodegeneration (BPAN), in brain development

WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause β-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay with intellectual disability, the neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in excitatory synapses by biochemical fractionation. Since WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by in utero electroporation in mice to recapitulate the pathophysiological conditions of BPAN. Knockdown of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.

Immunohistochemical analysis of Wdr45 in mouse brain. To determine the localization of Wdr45, we performed immunohistochemical analyses of cerebral cortex at embryonic day (E) 15, E17, P0, P7 and P30. At E15 and E17, Wdr45 was broadly detected in neurons in the marginal zone (MZ), cortical plate (CP), subplate (SP) and intermediate zone (IMZ) as well as neuronal progenitors in the ventricular zone (VZ) and subventricular zone (SVZ) ( Fig. 2A,a,b,B). Although the broad distribution pattern was observed until P30, subcellular distribution changed after P7; relative nuclear enrichment of Wdr45 was frequently observed from E15 to P7 while cytoplasmic distribution clearly appeared at P30 ( Fig. 2A,e,C). Notably, Wdr45 was also detected at the ventricular surface of neural progenitors from E15 to P0 (Fig. 2B). In addition, distinct immunoreactivity was observed in the neuropil of cerebral cortex at P30 (Fig. 2D), strongly suggesting enrichment at excitatory synapses. Since Wdr45 changed its distribution during cortical development, we prepared the nuclear and cytoplasmic fractions from cerebra at P7 and P30. While Wdr45 was predominantly detected in the nuclear fraction at P7, it was mainly detected in the cytoplasm at P30 (Fig. 2E). Collectively, Wdr45 was expressed in the nucleus of cortical neurons and the ventricular surface during corticogenesis and its subcellular localization changed from the nucleus to the cytoplasm and then neuropils in developed cerebral cortex. We also confirmed that Wdr45 was predominantly expressed in cortical neurons when compared to astrocytes (Fig. 2F).
Since Wdr45 is expressed in adult mouse central nervous system (CNS), it might play a role in differentiated neurons. We thus examined the expression of Wdr45 in primary cultured hippocampal neurons during differentiation. Wdr45 was mainly observed in the nucleus and soma in immature neurons at 3 days in vitro (div) and 7 div (Fig. 3A,B). Wdr45 was also detected in Tau-1-positive axon and MAP2-positive dendrites (Fig. 3A,B). In 14 div neurons, in addition to the nucleus and cytoplasm, Wdr45 was visualized in dendrites, and partially colocalized with synaptophysin (a presynaptic marker) and PSD-95 (a postsynaptic excitatory neuron marker), but not gephyrin (an inhibitory synaptic marker) ( Fig. 3C-E, lower panels). Together with the biochemical fractionation data (Fig. 1D), we assume that Wdr45 may participate in synaptic structure and/or functions in the hippocampal neurons. www.nature.com/scientificreports/ Histological analyses of a BPAN patient with a loss-of-function mutation (p.Leu232Alafs*53) in WDR45 showed mild atrophy and Tau-positive neurofibrillary tangles in hippocampus, and mild Purkinje cell depletion in cerebellum 6 . It is also notable that cerebellar atrophy was observed in ~ 25% of BPAN patients 7 . As for the animal model, CNS-specific Wdr45-knockout (KO) mice have been shown to recapitulate some hallmarks of BPAN such as cognitive impairment, strongly suggesting that Wdr45-deficiency influences the function of hippocampus 10 . In addition, poor motor coordination was observed in the KO mice, suggesting impairment of cerebellar functions controlling coordination, precision and accurate timing of movements 10 . We thus carried out immunohistochemical analyses in hippocampus at E17, P0, P7 and P30 and in cerebellum at P30 ( Supplementary  Fig. S2). As in the case of cerebral cortex, Wdr45 appeared to be expressed in the nucleus and cytoplasm at E17 . COS7 cells were transfected with pSuper-Luc (control), pSuper-Wdr45#1(KD#1) or -Wdr45#2 (KD#2) together with pCAG-Myc-Wdr45 or -Myc-Wdr45R. After 48 h, cells were harvested and lysates were subjected to western blotting using anti-Wdr45. The blot was probed with anti-Sept11 as loading control. (B) Regional expression of Wdr45 in brain. Various regions were dissected from adult mouse and whole tissue extracts were prepared as in "Materials and methods" section. Each extract (20 µg of proteins) was subjected to western blotting using anti-Wdr45, anti-synaptophysin and anti-PSD95. (C) Whole brain lysates (30 µg protein per lane) at indicated developmental stages were subjected to western blotting with anti-Wdr45. GFAP and Sept11 were used as differentiation marker and loading control, respectively. (D) Subcellular fractions (30 µg protein per lane) prepared from adult forebrains by differential and density-gradient centrifugation were subjected to western blotting with anti-Wdr45. Anti-synaptophysin (SYP) and anti-PSD95 were used as presynaptic and postsynaptic markers, respectively. The relative levels of the ~ 40 kDa band were measured by densitometry and shown as fold-increase compared to the expression level of the homogenate. Hom homogenate, S1 soluble fraction, S2 crude cytosol fraction, P1 nuclear fraction, P2 crude membrane fraction, Syp synaptosomal fraction, PSDI PSD fraction-I, PSDII PSD fraction-II. Uncropped blots are shown in Supplementary Figs Fig. S2A,B). Then, the cytoplasmic distribution in the neurons of CA regions as well as dentate gyrus came to be detected at P7 and became clearer at P30 (Supplementary Fig. S2C,D). In cerebellum, almost all regions were positive for Wdr45 ( Supplementary  Fig. S2E). It is of note that Wdr45 appeared to be enriched in the nucleus of Purkinje cells at P30, in contrast to the cytoplasmic distribution in cortical neurons ( Supplementary Fig. S2E, Fig. 2A,e). Specificity of the anti-Wdr45 in immunohistochemistry was confirmed in Supplementary Fig. S3. These results are consistent with possible roles of Wdr45 in hippocampus and cerebellum.
Characterization of RNAi-and expression-vectors. Given that WDR45 gene abnormalities are responsible for the early onset of ID in BPAN, Wdr45 is most likely to participate in the formation of cor- The cytoplasmic (20 μg of protein) and nuclear (20 μg; proportionally assigned amount corresponding to the cytoplasmic proteins used) fractions from P7 and P30 cerebra were immunoblotted with anti-Wdr45. The fractions were also immunoblotted with anti-lamin and anti-HSP95 to confirm proper fractionation. (F) Cell lysates (20 mg) of cultured astrocytes (Ast) and cortical neurons (Neu) were immunoblotted with anti-Wdr45. The membrane was then re-probed with anti-synaptophysin (SYP) and anti-GFAP. Uncropped blots are shown in Supplementary Figs. S10 and S11. , as well as another mutant, c.439G > T (GVmut), with single amino acid substitution in the linker region between third and fourth β-propeller domains were hardly detected when expressed in primary cultured mouse cortical neurons, strongly suggesting that haploinsufficiency of WDR45 underlies the pathogenesis of BPAN (Fig. 4A). It is of note that Wdr45-Rstop and GVmut did not undergo degradation in COS7 cells, which might explain the CNS-dominant clinical feature of BPAN. We thus conducted acute knockdown experiments to recapitulate the pathophysiological conditions in BPAN. To this end, 2 RNAi vectors were designed against mouse Wdr45, pSuper-Wdr45#1 and #2, both of which effectively knocked down not only Myc-Wdr45 expressed in COS7 cells but also endogenous Wdr45 in primary cultured cortical neurons (Fig. 4B). Immunostaining analyses confirmed the knockdown of endogenous Wdr45 in cortical neurons in vitro (Fig. 4C). An RNAi-resistant version of Wdr45, Wdr45R, was shown to be resistant against pSuper-Wdr45#1 (Fig. 4D).

Role of Wdr45 in cortical neuron migration.
We asked whether Wdr45 is involved in the migration of newly generated cortical neurons by in utero electroporation-based acute knockdown of Wdr45. pSuper-H1. shLuc (control), pSuper-Wdr45#1 or #2 was co-electroporated with pCAG-Turbo RFP into VZ progenitor cells, and electroporated brains were fixed and analyzed at P2. When compared to the control experiments, Wdr45-  www.nature.com/scientificreports/ knockdown had little effects on the migration and the cells were considered to normally make it to the layer II-IV ( Supplementary Fig. S4).

Role of Wdr45 in axon extension and dendritic arbor development during brain development.
Since aberrant synaptic network formation is tightly associated with defective brain development and function, Wdr45 may take part in axon pathfinding and/or dendrite growth during brain development. We thus first examined the interhemispheric axon projection of Wdr45-deficient cortical neurons in vivo. To easily detect morphological changes, we analyzed pyramidal neurons in layer II/III which are generated at E14-16. To this end, Wdr45 was knocked down in the VZ cells at E14.5. Consequently, axon bundles from the hemisphere containing Wdr45-deficient pyramidal neurons normally reached the contralateral hemisphere at P2 (Fig. 5A) and this tendency was still observed at P30 (Fig. 5B). On the other hand, it is notable that axons of the Wdr45deficient cells did not penetrate efficiently into the layer structure of contralateral cortex at P60 when compared to control axons (Fig. 5C). Overall, Wdr45 appears to be important for callosal axon extension into the cortical structure after normal pathfinding from the ipsilateral to contralateral cortex. We then examined the knockdown effects on dendritic arbor development of cortical neurons in vivo. Electroporation of pSuper-Wdr45#1 or #2 into the VZ cells at E14.5 gave rise to highly suppressed dendritic www.nature.com/scientificreports/ arborization at P10 (Fig. 6A). Sholl analyses revealed a significant decrease in branch point number of both apical and basal dendrites in the deficient neurons at P10 (Fig. 6B). Rescue experiments were then performed to exclude off-target effects. When an RNAi-resistant version of Wdr45, Wdr45R, was co-electroporated into the VZ progenitor cells with pSuper-Wdr45#1, morphological defects were rescued, supporting the specificity of the effects caused by Wdr45 knockdown (Fig. 6A,B). We then measured the knockdown effects on the basal and apical dendrites, separately. As shown in Fig. 6C, Wdr45-knockdown by pSuper-Wdr45#1 or #2 suppressed basal dendrite development. In this analysis, while the phenotype tended to be rescued by Wdr45R, the effects were www.nature.com/scientificreports/ not statistically significant (Fig. 6C). Meanwhile, apically oriented dendritic length was abnormally shortened in pSuper-Wdr45#1-or #2-transfected cells at P10, and the phenotype by pSuper-Wdr45#1 was rescued at least partially (Fig. 6D). Collectively, we conclude that Wdr45-deficiency impairs neuronal connectivity through defective axon elongation as well as aberrant dendrite arborization.

Role of Wdr45 in dendritic spine morphology regulation in vivo.
Dendritic spines are the primary site of excitatory input on most neurons, and changes in their shape and size are correlated with the strength of excitatory synaptic connections. Spine morphology can be classified into 3 groups, although there is a continuum of shapes between these categories: mushroom-type spine is mature while stubby-type and thin filopodialike spines are immature. Mature spines form strong synaptic connections and have the longest lifetime, while filopodia-like spines show very mobile and flexible structures with a short lifetime 11 . Meanwhile, stubby spines typically do not have a neck and are likely to be formed during the disappearance of mature spines 12 . We here examined the physiological role of Wdr45 in the spine density and morphogenesis of layer II-III cortical neurons in vivo. To this end, pSuper-H1.shLuc (control), pSuper-Wdr45#1 or #2 was co-electroporated at E14.5 with pCAG-loxP-GFP plus pCAG-M-Cre for sparse GFP-labeling. Brains were then fixed and analyzed at P10. Total spine density was significantly decreased when Wdr45 was knocked down (Fig. 7A,B). The abnormal phenotype by pSuper-Wdr45#1 was rescued by co-expression of Wdr45R (Fig. 7A,B). In addition, knockdown of Wdr45 caused reduction of mature mushroom-type spine number (Fig. 7B). The phenotype by pSuper-Wdr45#1 was rescued by Wdr45R (Fig. 7B). We confirmed that Wdr45 was knocked down until P90 (Supplementary Fig. S5). We then examined the long-term effects of Wdr45-knockdown. While the reduced total spine density was still observed at P30, it was comparable to control neurons at P60 (Fig. 7C,D). In contrast, the number of mature mushroom-type spine remained lower at P60 (Fig. 7C,D). These results strongly suggest that Wdr45 plays a pivotal role in the synapse formation and/or maintenance.

Long term effects of Wdr45-knockdown on neuronal morphology in vivo.
Although individuals with BPAN develop sudden-onset progressive dystonia, parkinsonism and dementia in adolescence, pathophysiological mechanism(s) underlying these clinical manifestations is yet to be clarified. To gain some insight into this aspect, we examined the long-term effects of Wdr45-knockdown on neuronal morphology by focusing on dendritic arbor development at P30, P60 and P90. To this end, we measured the intersection number of dendritic arbor as well as the total length of apical and basal dendrites. As shown in Fig. 8A-E, dendritic arbor complexity of Wdr45-deficient neurons remained poor when compared to control cells at the 3 time points mentioned above. As for dendritic arborization, basal dendrites of deficient neurons were shorter than that of the control cells at all time points tested (Fig. 8F). Meanwhile, apical dendrite length was also decreased at P60 and P90, whereas it was statistically unchanged at P30, albeit showing a tendency to decrease (Fig. 8G). Since apical dendrite development was inhibited at P10 (Fig. 6D), the development might recover temporarily around P30 during brain development, although its pathophysiological meaning is unclear. We confirmed that Wdr45 was knocked down at P30 and P90, indicating the possibility that knockdown effects lasted until the adult stage (Supplementary Fig. S5). These results strongly suggest that Wdr45-deficiency prevents neuronal network formation even in the adult stage and causes sustained impairment of synaptic function.

Discussion
Autophagy, a basic process regulating intracellular degradation, is a key regulatory system for cell growth, differentiation, survival and physiological homeostasis 1,2,13,14 , and is thought to be impaired in various neurodegenerative disorders 13,15 . Given that autophagy is involved in synaptic remodeling and plasticity in neurons 16,17 , disruption of autophagy may cause disturbed neuronal functions related to BPAN symptoms, such as psychomotor retardation and ID in early childhood. In this context, it is of note that a large-scale genomic study of individuals with undiagnosed and severe developmental disorders revealed de novo mutations in WDR45 18 . We thus explored the role of WDR45 in the developing and adult brain. We first showed that Wdr45 is distributed in excitatory synapses of cortical and hippocampal neurons by biochemical and morphological analyses, implicating a specific role of Wdr45 at post synapses. Then, since WDR45 mutations responsible for BPAN are most likely to cause WDR45 instability 4 and subsequent degradation, we carried out acute gene knockdown of Wdr45 by in utero electroporation in order to mimic the pathophysiological conditions of BPAN in mouse brain. Wdr45deficiency was shown to cause defects in dendritic arborization as well as synaptogenesis in cortical excitatory neurons during brain development. We assume that the defects in synapse formation in cerebral cortex are responsible for a major BPAN symptom, ID. Regarding cortical axon development, interhemispheric extension was not affected at P2 and axons normally reached the contralateral hemisphere, whereas the pathfinding into the cortical layer structure and subsequent synapse network formation were hampered. These results also strongly suggest a pathophysiological role of WDR45 gene abnormalities in disrupted synaptic function related to ID, although the underlying molecular mechanisms are yet to be elucidated. WDR45 gene abnormalities are thought to be responsible for other clinical features of BPAN including sudden-onset of progressive dementia and parkinsonism in adolescence. While the molecular pathophysiological mechanism remains largely unknown, several reports have shown that some BPAN symptoms in adolescence-adult phase, such as learning impairment and memory disturbance, are due to defective autophagy 6,10 . On the other hand, constitutive Wdr45-KO mouse demonstrated apoptosis-mediated neuronal loss in several brain regions, such as prefrontal cortex and basal ganglion in aged mouse 19 , whereas the CNS-specific Wdr45-KO mouse exhibited a less characteristic autophagy phenotype; no significant changes were observed in the number of neuronal cells in cerebellum and hippocampus 10 . Instead, these conditional KO mice displayed scattered remarkable axonal swelling in various brain regions including cerebral cortex and cerebellum, as well as www.nature.com/scientificreports/ www.nature.com/scientificreports/ reactive astrogliosis in various brain regions including hippocampus 10 . From these results, Wdr45-deficiency appears to be involved in varying degree of neuronal damage in the mouse brain, where increased ER (endoplasmic reticulum) stress and impaired ER quality control may be underlying molecular mechanism 19 . While morphological alteration was not reported in cerebral cortex of the 2 types of Wdr45-KO mice, it is notable that other members of the WIPI protein family (Wipi1-3) may compensate for the deficiency of Wdr45 in contextdependent manners. However, acute knockdown of Wdr45 caused defects in cortical neuron morphology in this study. We consider that acute knockdown by the combination of in utero electroporation and RNAi may circumvent the compensatory effects of general gene-knockout approaches. The limitation of the electroporation method may be the difficulty in analyses of aged mice because the RNAi vector is only transiently expressed in Error bars indicate SD of the results from 10 to 15 neurons; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by t-test (vs. control). (F,G) Total dendritic length was calculated for basal (F) as well as apical dendrites (G). Error bars indicate SD of the results from 10 to 15 neurons; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by t-test (vs. control). www.nature.com/scientificreports/ cortical neurons. In this context, in utero electroporation-mediated GFP-labeling of developing cortical neurons of the KO mice followed by fine morphological examination may provide in vivo data for better understanding the pathophysiology of BPAN. Pathophysiological significance of autophagy in the abnormal morphological phenotypes observed in this study remains to be clarified. While disruption of ER function may be involved, further intensive investigation is needed to clarify the molecular basis of the functional defects of WDR45 as well as the relationship between hampered autophagy and BPAN etiology. In addition, given that some autophagy proteins participate in autophagyindependent processes 20 , results in this study might suggest dysregulation of yet unidentified Wdr45 function(s) involved in BPAN, although further studies are again required to address this possibility.
In conclusion, the present study suggests a role for Wdr45 in dendritic arborization, synaptogenesis and axon network formation of cortical excitatory neurons. We propose that WDR45-deficiency may be associated with BPAN neurodevelopmental symptoms by affecting the function of yet unidentified protein(s) involved in cytoarchitecture formation/maintenance such as cytoskeletal proteins and other linked cell signaling molecules. Further investigation will be essential to clarify the relationship between the pathophysiological mechanism of WDR45 gene abnormalities and the clinical features of BPAN.

Materials and methods
Ethics approval and consent to participate. We followed the fundamental guidelines for proper conduct of animal experiments in academic research institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. All the protocols for animal handling and treatment were reviewed and approved by the animal care and use committee of Institute for Developmental Research, Aichi Developmental Disability Center (approval number M-10). All experiments were conducted in compliance with ARRIVE guidelines. All methods, data presentation and statistical analyses were carried out in accordance with the 'TOP' guidelines. This study has not been pre-registered. Preparation of extracts from mouse tissues and western blotting. Sample preparation was performed essentially as described [23][24][25] . Briefly, tissues from ICR adult mice (Japan SLC, Shizuoka, Japan) were homogenized with the lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1% NaF, 5 mM EDTA, 1 mM Na 3 VO 4 and the protease inhibitor mix). Each suspension was sonicated on ice and solubilized with the lysis buffer containing 2% SDS. Protein concentration was estimated with BCA protein assay reagent kit (Pierce, Rockford, IL) with bovine serum albumin as a standard. SDS-PAGE (10% gel) and western blotting were performed as described 23-25 . Preparation of postsynaptic density (PSD) fractions. Sample preparation was performed essentially as described 24 . Briefly, 5 adult mouse brains were homogenized in 23 ml of 5 mM HEPES (pH 7.4) containing 1 mM MgCl 2 , 0.5 mM CaCl 2 , phosphatase inhibitors and the protease inhibitor mix with a Teflon homogenizer (H fraction). The resulting extract was centrifuged at low speed (1400×g for 10 min) to obtain S1 as a supernatant fraction. S2 fraction was then obtained by centrifugation of S1 at 13,800×g for 10 min. The resulting pellet (P2) was resuspended in 0.25 ml of 6 mM Tris (pH 8.0) containing 0.32 M sucrose, loaded onto a discontinuous sucrose gradient, and centrifuged at 82,500×g for 2 h. The synaptosome fraction was collected, extracted with 0.5% Triton X-100 and centrifuged at 32,800×g for 20 min. The resulting pellet (PSD-I; crude PSD fraction) was again extracted with 0.25 ml of 0.5% Triton X-100 and centrifuged again at 201,800×g for 1 h to obtain highly purified PSD fraction as pellet (PSD-II) which was dissolved in 0.25 ml of SDS-PAGE sample buffer.

Plasmids.
Immunohistochemical analyses. Mouse brains of either sex were fixed with 4% paraformaldehyde at various time points and cut into coronal section (6-µm thickness) at the level of dorsal hippocampus. After deparaffinization, sections were processed for immunohistochemistry as reported previously 22,23 . Images were captured using BZ-9000 microscope (Keyence Inc., Osaka, Japan). www.nature.com/scientificreports/ Cell culture, transfection and immunofluorescence. COS7 cell transfection experiments were performed with polyethyleneimine "MAX" reagent (Polysciences Inc., Warrington, PA). Immunofluorescence analyses were carried out as described [23][24][25] . Alexa Fluor 488-, 594-or 647-labeled IgG (Life Technologies) was used as a secondary antibody. Fluorescent images were captured with a Zeiss880 confocal microscopy. Primary cultured cortical neurons and astrocytes were isolated from E14.5 mouse embryos as described 23,26,27 . Briefly, dissected cortices were mechanically and enzymatically disrupted in the presence of trypsin and DNase I for 15 min at 37 ℃. Neurons were then cultured in Neurobasal medium with B27 supplement (Thermo scientific, Waltham, MA), 1 mM l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Astrocytes were then cultured in the DMEM medium.
In utero electroporation, and analyses of neuronal migration and axon elongation. In utero electroporation was carried out at E14.5 and analyses were conducted at P2 essentially as described 23 . E14.5 ICR mice were housed individually given ad libitum access to food and water. Both female and male pups were used. Pups were maintained in a 12:12 light-dark cycle together with their dams before being sacrificed at indicated time points, which were primary endpoints of this study. At least 5 electroporated brains from different dams were used for each experiment. After anesthetization of pups on ice for 5 to 10 m affording physical relief, brains were fixed overnight in 4% paraformaldehyde phosphate buffer solution. Sections (100 μm-and 150 μm-thickness for P2 and above P10 samples, respectively) were prepared by vibratome. Distribution of GFP-positive neurons was then analyzed as described 28,29 . Notably, neuronal progenitor cells present in the VZ at E14.5 are to become layer II/III pyramidal neurons in the adult stage. Thus, progenitor cells electroporated at E14.5 are to make it to the layer II/III under the normal conditions. Also, given that transfection efficiency into each cell depends on the size of the cell surface area which is physically exposed to the ventricular lumen (cerebrospinal fluid) where plasmids are injected, neurons incorporating low amount of a vector are supposed to undergo partial effects. For estimation of axon growth, GFP signal intensity of the callosal axons was measured in a 200 × 200 µm rectangle on both the ipsilateral and contralateral sides at the positions indicated in Fig. 5A. The ratio of the axonal RFP signals in the contralateral side to the corresponding ipsilateral side was calculated.
Quantitative analysis of dendritic arbor formation. To measure the length and branching point number of dendrites of post-migratory (mature) neurons, images of GFP-positive neurons in the layer II-III of cerebral cortex at P10, P30, P60 or P90 were acquired by a Zeiss880 confocal microscopy. Image J software was used for the quantitative analyses of dendritic length and Sholl test.
Quantitative analysis of spine morphology. Electroporated neurons were visualized by immunostaining of co-transfected GFP with anti-GFP. Electroporated cells were chosen randomly and images were obtained using a Zeiss880 confocal microscope. We took z-series stacks (0.2 μm-thickness) and used Zen software to generate image projections for quantitative analyses. Morphometric measurements were automatically performed using ImagePro PLUS software. To analyze spine density and morphology, more than 200 spines from 20 or more neurons were measured for each condition. Morphological assessments were conducted blindly. Statistical analysis. Data were analyzed using GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA). Results were expressed as means ± SD. When data were obtained from only 2 groups, Student's t-test was used for comparison. Values are expressed as means ± SEM. For other experiments, the rate of cell scores was initially analyzed using the one-way analysis of variance (ANOVA) with Tukey's or Dunnett's post hoc test for multiple comparisons. The level of statistical significance was considered to be p < 0.05.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.