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The distribution of neuroligin4, an autism-related postsynaptic molecule, in the human brain
Molecular Brain volume 16, Article number: 20 (2023)
NLGN4X was identified as a single causative gene of rare familial nonsyndromic autism for the first time. It encodes the postsynaptic membrane protein Neuroligin4 (NLGN4), the functions and roles of which, however, are not fully understood due to the lack of a closely homologous gene in rodents. It has been confirmed only recently that human NLGN4 is abundantly expressed in the cerebral cortex and is localized mainly to excitatory synapses. However, the detailed histological distribution of NLGN4, which may have important implications regarding the relationships between NLGN4 and autistic phenotypes, has not been clarified. In this study, we raised specific monoclonal and polyclonal antibodies against NLGN4 and examined the distribution of NLGN4 in developing and developed human brains by immunohistochemistry. We found that, in the brain, NLGN4 is expressed almost exclusively in neurons, in which it has a widespread cytoplasmic pattern of distribution. Among various types of neurons with NLGN4 expression, we identified consistently high expression of NLGN4 in hypothalamic oxytocin (OXT)/vasopressin (AVP)-producing cells. Quantitative analyses revealed that the majority of OXT/AVP-producing neurons expressed NLGN4. NLGN4 signals in other large neurons, such as pyramidal cells in the cerebral cortex and hippocampus as well as neurons in the locus coeruleus and the raphe nucleus, were also remarkable, clearly contrasting with no or scarce signals in Purkinje cells. These data suggest that NLGN4 functions in systems involved in intellectual abilities, social abilities, and sleep and wakefulness, impairments of which are commonly seen in autism.
Autistic spectrum disorders (ASDs) are pervasive behavioral disorders characterized by impaired communication and social activities as well as repetitive and obsessive behaviors. Intensive studies in clinical genetics have revealed that genetic changes contributing to ASD pathogenesis are highly heterogeneous, and the number of genes disrupted de novo, hence responsible for sporadic ASDs with a severe phenotype, is approximately 400 . Among hundreds of genes considered to be involved in ASD pathogenesis, the NLGN4X gene has been identified for the first time as a single causative gene for nonsyndromic, familial ASDs, along with the related NLGN3 gene . To date, several NLGN4X variants associated with ASD and/or intellectual disability (ID) mainly in an X-linked recessive inheritance pattern have been reported, yet NLGN4X variants seem responsible for the pathogenesis in a limited population of ASD/ID [3,4,5,6,7,8,9,10].
NLGN4 is one of the highly conserved NLGN genes that encode neuroligin (NLGN) 1–5, all of which are regarded as postsynaptic cell-adhesion molecules. Knowledge about the functions of NLGNs has been derived mainly from studies on murine NLGNs, and they are considered essential for synaptic functions but not for primary synapse formation through interactions with presynaptic neurexins (NRXNs) [11, 12]. Although the specific functions of each NLGN are largely unknown, NLGN2 is considered to be involved mainly in inhibitory synaptic functions . NLGN1, NLGN3, and NLGN4, which are structurally more similar to each other than to NLGN2, are involved mainly, even if not exclusively, in excitatory synaptic functions . NLGN5, which is almost identical to NLGN4 and is encoded on the Y chromosome, is now designated NLGN4Y, and knowledge about its function is even more limited [14, 15]. The mouse homolog of NLGN4, which was initially not found in mouse genome assemblies, has been shown to be divergent from NLGN4 in other species and even between different mouse strains [10, 16, 17]. Although Nlgn4 KO mice exhibit a selective perturbation of social behavior and vocalization and are reported as genetic mouse models of ASD , accumulating lines of evidence have strongly suggested that human and mouse NLGN4 do not share sufficient structural homology and function for Nlgn4 KO mice to be a viable ASD model [10, 17].
The lack of a closely homologous gene in rodents has hampered research on NLGN4, and it has only recently been reported by Marro et al. that NLGN4 was detected at the highest level in the cerebral cortex . Unfortunately, however, detailed information about the distribution at the histological level was not available in the study, since the antibodies did not work for fixed tissue. In this study, we generated two kinds of antibodies against NLGN4 that are applicable for immunohistochemistry and examined NLGN4 distribution in developing and developed human brains using them. We revealed that NLGN4 is expressed almost exclusively in neurons in the brain and is especially enriched in several anatomical regions, including hypothalamic nuclei, and a large fraction of NLGN4 is located in the cytoplasm, despite its identity as a postsynaptic membrane protein. These results provide new insights into potential roles of NLGN4 in hypothalamic nuclei involved in social functions and in additional extrasynaptic functions.
We generated a rabbit polyclonal antibody against human NLGN4 (anti-NL4-p1) in a standard immunization protocol. Briefly, we immunized a rabbit (Chubu-Kagaku Co., Nagoya, Japan) with a synthetic peptide corresponding to amino acids 647–660 (PKHSKDPHKTGPED) of NLGN4 (NP-851849). We also generated a mouse monoclonal antibody against human Neuoroligin-4, named anti-NL4-m1, by immunizing three 8-week-old balb/c females with keyhole limpet hemocyanin–conjugated synthetic peptides corresponding to amino acids 111–122 (PQHLDERSLLHD). B cells were collected from lymph nodes and fused with SP2/0-AG14 myeloma cells with polyethylene glycol 4000. Clone selection was performed by ELISA and Western blot analyses. Both anti-NL4-p1 and anti-NL4-m1 antibodies purified by protein G affinity chromatography were used in the following experiments. The commercially available antibodies used in this study were as follows: AB911 against oxytocin (Chemicon International, Temecula, CA), AB1565 against vasopressin (Chemicon), A8592 against FLAG (Sigma‒Aldrich, St. Louis, MO), CLT9002 against α-tubulin (Cedarlane, Burlington, Ontario, Canada), AC-15 against β-actin (Abcam, Cambridge, MA, USA), GTX116674 against Neuroligin4 (GeneTex International Corporation, Hsinchu, Taiwan), F1804 against FLAG (Sigma‒Aldrich), Alexa Fluor 488–conjugated goat anti-mouse IgG (Molecular Probes, Carlsbad, CA, USA), and Alexa Fluor 568–conjugated goat anti-rabbit IgG (Molecular Probes).
Construction of expression vectors, transfection and Western blotting
The cDNA fragments encoding human NLGN1 (amino acid residues 46-823; GenBank Accession No. NM_014932), NLGN2 (amino acid residues 16-835; GenBank Accession No. AF376802), NLGN3 (amino acid residues 35-828; GenBank Accession No. BC051715), and NLGN4 (amino acid residues 39-816; GenBank Accession No. AF376803) were cloned into multicloning sites of the p3xFLAG-CMV-9 vector (Sigma) to yield p3xFLAG-NLs.
HEK293 cells were cultured in DMEM (Nacalai Tesque, Kyoto, Japan) with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin (Wako, Osaka, Japan). Transient transfection of expression vectors p3XFLAG-NLs into HEK293 cells was carried out using polyethylenimine MAX (Polysciences, Warrington, PA, USA) according to the manufacturer’s directions. Western blot analyses were performed as described previously .
Human induced pluripotent stem cell (iPSC) culture and generation of NLGN4X knockout iPSC clones
The human iPSC Line 610B1 was obtained from RIKEN BioResource Center (Tsukuba, Japan). The iPSCs were maintained in a pluripotent undifferentiated state on mitomycin C-treated mouse SNL feeder cells in ES medium [DMEM/F12 with 20% StemSure Serum Replacement, 0.5 mM StemSure monothioglycerol, 0.8% MEM nonessential amino acids solution, 2 mM l-alanyl-l-glutamine, 0.5% penicillin and streptomycin, and 10 ng/mL fibroblast growth factor (FGF) (FUJIFILM Wako Pure Chemical Co., Tokyo, Japan)]. To generate NLGN4X knockout (KO) iPSC clones, we applied CRISPR/Cas9-mediated genome editing with dual RNP complexes prepared by incubation with each CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and Alt-R® CRISPR‒Cas9 protein (IDT, Coralville, IA) with reference to previous reports [20, 21]. Two gRNAs, gRNA1 (CAGACCGGGGTGAACAACAA) complementary to exon 2 and gRNA2 (TGATTCCAAACACACTGACG) complementary to exon 6 of NLGN4X, were designed using Benchling (https://www.benchling.com) (Additional file 1: Fig. S1B). To assess NLGN4X knockout in iPSCs, genomic PCR was performed with the following primer sets: pEx2f (CTCGCCTCTGGGCTTTGTCTCCTTGGAGCC), pEx2r (TCATCAAAGTATCCAAATTGGCG), pEx6f (TTAAGTGTCACCATTGCCGTC), and pEx6r (GGAAAACACCAACGATAAGGG) (Additional file 1: Fig. S1B).
Human tissue samples
All human brain samples used for immunohistochemical studies were obtained by autopsy at the Central Hospital of Aichi Developmental Disability Center, Nagoya First Red Cross Hospital, and Nagoya Second Red Cross Hospital. Cases whose clinical records indicated diseases involving the central nervous system were excluded, and all brain specimens from the remaining cases were re-examined by pathologists (YH and AN) to confirm that they had no obvious brain damage or abnormalities. After exclusion, ten neonatal, two infantile, and eight adult brains remained as subjects for immunohistochemical studies. A summary of the cases is shown in Table 1A, and information on each case is listed in Table 1B.
Stored formalin-fixed and paraffin-embedded brain tissues were sectioned at 6 μm thickness, and the sections were used for immunohistochemistry and hematoxylin and eosin staining. Research use of autopsy samples was permitted with informed consent from the next of kin, and the study was carried out with the authorization of the Ethics Committee of Institute for Developmental Research, Aichi Developmental Disability Center, endorsed as #11-06.
Immunohistochemistry and immunocytochemistry
The staining procedure was performed as described previously . Briefly, antigen retrieval was performed by heat treatment in 10 mM citrate buffer (pH 6.0) in a microwave oven for 5 min (two cycles) at 600 W. After blocking with 4% normal goat serum in PBS/0.1% Triton-X, brain sections on glass slides were incubated with primary antibodies at 4 °C overnight. The dilutions of primary antibodies were as follows: 500 × (anti-NL4-p1), 5000x (anti-oxytocin), 5000x (anti-vasopressin), and 1000 × (anti-NL4-m1). For visualization of the antigens, a VECTASTAIN Elite ABC-HRP Kit (Vector Labs., Burlingame, CA, USA) was applied following the manufacturer’s protocol. In fluorescent immunocytochemistry, we applied Alexa Fluor 488–conjugated goat anti-mouse IgG at a 500 × dilution and Alexa Fluor 568–conjugated goat anti-rabbit IgG at a 500 × dilution. Fluorescent images were obtained by a confocal laser scanning microscope LSM880 (Carl Zeiss, Göttingen, Germany).
Generation and characterization of NLGN4-specific antibodies
We established NLGN4-specific polyclonal and monoclonal antibodies, named anti-NL4-p1 and anti-NL4-m1, respectively, targeting unique epitopes on NLGN4 to avoid cross reactions with the other NLGNs (Fig. 1A). As expected, both antibodies reacted with full-length human NLGN4 in Western blotting analyses but not with full-length human NLGN1, 2, or 3 when they were overexpressed in HEK293 cells (Fig. 1B; Additional file 1: Fig. S1A). We further confirmed the specificity in immune-fluorescent cytochemistry (Fig. 1C). Then, we assessed the sensitivities of the antibodies against endogenous NLGN4 by Western blotting. We, however, could not detect NLGN4 bands with anti-NL4-p1 or anti-NL4-m1 in several different human brain samples. We reasoned that sensitivities of the antibodies were not enough high to detect a low amount of NLGN4 in brain tissue lysates due to the low abundance of NLGN4-expressing cells. Therefore, we analyzed cell lysates prepared from human iPSCs, which are known to express NLGN4 . Both anti-NL4-p1 and anti-NL4-m1 visualized bands just above 100 kDa in accordance with the molecular size of NLGN4 reported previously , similar to GTX116674, a commercially available anti-NLGN4 antibody (Fig. 1D). These bands were not observed in lysates prepared from NLGN4X KO iPSCs (Fig. 1D; Additional file 1: Fig. S1B, C), confirming that the bands recognized by anti-NL4-p1 and anti-NL4-m1 were derived from endogenous NLGN4.
Distribution of NLGN4 in the brain
In initial studies, we confirmed that anti-NL4-p1 and anti-NL4-m1 provided very similar staining patterns with occasionally clearer images by anti-NL4-p1, and the signals were observed exclusively in neuronal lineage cells. In addition, both antibodies labeled the whole neuronal cell bodies but did not react in a cell-membrane-specific manner, yet NLGNs are regarded as postsynaptic membrane proteins. The pattern was, however, consistent with previous reports revealing subcellular distributions of other endogenous NLGNs in the tissue section [24,25,26]. We also noticed that the intensities of signals varied among subtypes of the neuron. Therefore, we will present detailed data divided into subsections, i.e., “The forebrain”, “The midbrain”, and “The hindbrain”, in that order. Representative images stained with anti-NL4-p1 at 1:500 dilution will be mainly shown unless otherwise mentioned. In addition, a summary of data in major anatomical structures is presented in Table 2.
In the adult cerebral cortex, NLGN4 signals were detected similarly in most neurons by anti-NL4-p1 and by anti-NL4-m1 and in both male and female samples (Fig. 2A–E). In particular, intense staining signals were observed in large pyramidal neurons in layer V (Fig. 2B, D, E). Neuronal NLGN4 expression was identified in all areas of the cerebral cortex we observed, although we did not thoroughly examine the cerebral cortical areas. In neonatal brains, most neurons that do not yet constitute the clear layer structure in the cortex showed NLGN4 signals (Fig. 2F, G). In a neonatal brain with further development, an ambiguous layer structure appeared, and intense NLGN4 signals were detected in some large pyramidal cells, as in the adult brain (Fig. 2H–J).
In the adult hippocampus, the pyramidal cell layer was immunolabeled in all available cases, while the granule cell layer was occasionally negative for NLGN4 (Fig. 3A). Most hippocampal pyramidal cells were clearly positive for NLGN4 regardless of the type of antibody and the gender of the donor (Fig. 3B–D). In all neonatal brains, both pyramidal cells and granular cells were well stained (Fig. 3E, F). In the infantile brain, pyramidal cells were positive for NLGN4 (Fig. 3G, H), but granular cells were scarcely stained in one case (not shown).
Neurons in additional telencephalic structures, such as the amygdala, the putamen, and the globus pallidus, were not labeled in adult brains, while immature neurons or precursors in the putamen and pallidus were labeled by anti-NLGN4 antibodies in some neonatal and infantile brains (not shown). Neurons in the caudate among the deep telencephalic structures were occasionally stained by the antibodies (not shown).
In the diencephalic region, the most remarkable immunolabeled signals were observed in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. Most, if not all, neurons were labeled by anti-NLGN4 antibodies in all available cases regardless of their developmental stages, kinds of antibodies, and sexes (Fig. 4A–H; Additional file 2: Fig. S2A–D). As shown in Fig. 4F, NLGN4 signals were remarkable in extended neurites in some cases (Fig. 4F).
Neurons in the thalamus of almost all cases were stained by the antibodies, yet NLGN4-positive neurons were sparsely distributed (Fig. 5A). At higher magnification, considerable numbers of neurons with very weak signals were identified (Fig. 5B).
Large neurons in the medial reticular formation were labeled intensely by anti-NLGN4 antibodies, which gave a sharp contrast with those lacking the signals in the red nucleus (Fig. 5C, D). Neurons in the oculomotor nucleus were labeled in all neonatal and infantile cases and approximately half of adult cases (Fig. 5E, F, Additional file 2: Fig. S2E, F). We noticed that signals in the accessory nucleus (Edinger-Westphal) looked more remarkable than those in the main nucleus (Fig. 5E, F). Neurons in the substantia nigra were weakly labeled in neonates and infants and in some adult cases (not shown).
In the cerebellum, Purkinje cells were scarcely labeled by the antibodies in adult brains (Fig. 6A). In contrast, the Purkinje cells in all neonatal and infantile brains were positive for NLGN4, but the signals were not very intense (Fig. 6B). Granule cells in the cerebellar cortex were negative or scarcely positive for NLGN4 in most adult cases and all neonatal cases. Some larger cells, putative Golgi cells, in the granular layer were occasionally labeled by the antibodies (Fig. 6A). Neurons in the dentate nucleus were also labeled in all adult brains and in most neonatal and infantile brains (not shown). The staining pattern of the dentate nucleus was similar to that of the olive nucleus shown below.
Among the nuclei and other neuronal structures in the pons, neurons in the locus coeruleus were intensely labeled by the antibodies in most of the infantile and adult brains (Fig. 6C–F; Additional file 2: Fig. S2G, H). In addition, neurons in the raphe nucleus of both infantile and adult brains were highly positive for NLGN4 (not shown). Neurons in cranial nerve nuclei of the pons were also positive for NLGN4, but the intensities were not as remarkable as in those described above (not shown).
In the medulla, neurons in the hypoglossal nucleus and the dorsal motor nucleus of the vagus were highly positive for NLGN4 (Fig. 6G). The olivary nucleus showed a unique staining pattern; large neurons were positive for NLGN4 at various intensities, from scarcely detectable to high levels (Fig. 6H). In contrast, neurons in the medial accessory nucleus were consistently positive (Fig. 6G).
The majority of OXT/AVP-producing neurons in the hypothalamus express NLGN4
We were interested in intensive NLGN4 signals in the PVN and SON, since these nuclei are the centers producing social neuropeptides, oxytocin (OXT) and arginine vasopressin (AVP) and impaired OXT/AVP system can be involved in the pathophysiology of ASD. We therefore examined whether NLGN4-positive cells in the PVN and SON produce these neuropeptides using mirror sections. Because of the limitation of archival autopsy samples, we could perform this additional analysis only on the PVN of adult case A2. As shown in Fig. 7A–D, most of AVP-positive cells appeared NLGN4-positive. In contrast, less OXT-positive cells expressed NLGN4 in this case (not shown). Then we quantified the percentages of NLGN4-positive cells among AVP-positive cells and OXT-positive cells. Quantification analyses revealed that approximately three-quarters of AVP-positive neurons expressed NLGN4, while less than half of OXT-positive neurons expressed NLGN4 in case A2 (Fig. 7I). To obtain further information about the proportion of NLGN4-expressing cells among AVP/OXT-producing neurons, we additionally examined PVNs and SONs in cases that had been excluded from the study owing to the presence of neurodevelopmental or neurological disorders. Three PVNs and SONs without histological abnormalities were available from two neonatal cases, Ni (diagnosed as 4p-syndrome (Fig. 7E–H)) and Nii (unspecified multiple anomalies), and one adult case, Ai (diagnosed as Parkinson’s disease). Much less OXT-producing neurons (Fig. 7G) compared with NLGN4-positive neurons (Fig. 7E) were considered due to the fact that the great majority (90% or more than 95%) of the neurosecretory neurons were AVP-producing neurons, while OXT-producing neurons were very minor population in SON [27, 28]. Yet most of the scattered OXT-producing neurons appeared NLGN4-positive (Fig. 7F, H). In these three cases, 71.6–84.6% of AVP-positive neurons in PVN and 78.0–86.7% of those in SON were NLGN4 positive and 63.6–69.8% of OXT-positive neurons in PVN and 86.5–100% of those in SON were NLGN4 positive (Fig. 7I). Thus, we concluded that the majority of OXT-producing neurons as well as AVP-producing neurons in both the PVN and SON were considered to express NLGN4 (Fig. 7I).
We established two kinds of antibodies against unique amino acid residues of NLGN4 and confirmed that they were highly specific for NLGN4 among the extremely conserved NLGN protein family. Utilizing these antibodies, we examined the distribution of NLGN4-positive cells in the human brain. This study revealed a couple of unique and intriguing features of NLGN4, which may be relevant to the neuronal functions impaired in ASD.
Among various subtypes of neurons that express NLGN4, neurons functioning in intellectual abilities, i.e., the cerebral cortical and hippocampal neurons; in social abilities, i.e., neurons in the PVN and SON; in sensory processing, i.e., the thalamic neurons; and in the regulation of wakefulness and consciousness, i.e., neurons in the locus coeruleus and the raphe nucleus, were found to express NLGN4 constantly at high levels (Table 2). In contrast, neurons functioning in motor processing, i.e., neurons in the putamen, globus pallidus, substantia nigra, red nucleus, and cerebellar cortex, expressed NLGN4 not at all or very weakly, especially in the mature brain. Such preferential distribution firmly supports that NLGN4 is involved, especially in intellectual, social, sensory, and consciousness functions in the brain, while it is less involved in motor function. This seemingly explains why the disruption of the NLGN4X gene results in autism accompanying intellectual disability [2, 3]. It is intriguing that neurons expressing NLGN4 intensely were identified exceptionally in the motor nuclei of the cranial nerves, i.e., the oculomotor nucleus and the hypoglossal nucleus, since the former is important for normal visual attention and the latter is involved in speaking ability, both of which are affected in autism.
Intensive expression of NLGN4 in OXT/AVP-producing neurons is especially noteworthy, since it can be tightly relevant to impaired social function, which is one of the major symptoms of ASD. OXT has emerged as a neuromodulator of diverse social behaviors, and extensive studies have been conducted on its possible pathophysiological involvement and therapeutic effects in ASD, yet many controversial issues have remained [29,30,31,32]. At least its therapeutic effects in ASD were questioned, since the most recent, large-scale, placebo-controlled trial of intranasal oxytocin therapy in ASD patients showed no significant effects on social or cognitive function of them . AVP and OXT confer mostly opposite effects on anxiety and depression-related behavior; however, they have effects in the same direction on social behavior . Therefore, AVP is considered another potential biomarker as well as a therapeutic reagent for ASD; promising data in support of this hypothesis have been accumulated recently [35,36,37]. Our quantitative examination revealed that the majority of AVP-producing cells and OXT-producing cells in both the PVN and SON expressed NLGN4, with the exception of OXT-producing cells in the PVN of case A2. We could not completely rule out the possibility that the expression of AVP, OXT, and/or NLGN4 was affected somehow by the disease conditions in additional cases; however, significant changes seemed improbable. Thus, it is suggested that NLGN4 functions in the majority of social neuropeptide-producing neurons.
Intriguingly, our data demonstrated that the subcellular distribution of NLGN4 in neurons appeared as a cytoplasmic pattern but not as a surface membrane protein pattern. This is consistent with previous studies that reported cytoplasmic localization of other NLGNs [24,25,26], and the widespread cytoplasmic distribution seems to be a common feature of NLGNs despite their primary emergence as synaptic cell-adhesion molecules interacting with their binding partner NRXNs [11, 12]. We tried to obtain further information about the subcellular distribution of NLGN4 by immunofluorescent multiple staining, but archival brain samples did not provide sufficient resolution to distinguish subcellular organelles. Recently, Nguyen et al. reported that autism-associated variants of NLGN4X have deficits in trafficking to the cell surface, phenocopying NLGN4Y . They demonstrated that NLGN4Y did not efficiently traffic to the cell surface, which hindered its ability to induce synapses. They also suggested that NLGN4Y was distinct from NLGN4X regardless of their extreme structural similarities and that it could act as a negative regulator of other NLGNs heterodimerizing with them. Since the antibodies we generated and applied in this study were designed to target peptide sequences that were almost identical in NLGN4X and NLGN4Y, they might react with both NLGN4X and NLGN4Y. Therefore, it is possible that the cytoplasmic NLGN4 signals were derived from NLGN4Y that failed to traffic to the cell surface in male cases; however, this should not occur in female cases. We observed a widespread cytoplasmic distribution of NLGN4 signals equally in male and female cases, as presented in the results, and insist that a considerable fraction of NLGN4X localizes in the cytoplasm, independent of NLGN4Y expression. Intriguingly, OXT/AVP-producing neurons secrete OXT/AVP from their axon terminals into plasma and, in independently regulated manners, also release OXT/AVP from dendrites locally in the brain, which may induce many behavioral effects [38,39,40]. It is tempting to speculate that the transmembrane protein NLGN4, which is distributed throughout the cytoplasm of OXT/AVP-producing neurons, might localize to small vesicles involved in dendritic secretion of neuropeptides. Further studies on the nonsynaptic functions of NLGN4 are necessary and important to understand the entire roles of this transmembrane protein in the brain.
Availability of data and materials
Materials including antibodies and NLGN4 KO iPSC clones are available on request for noncommercial use.
Induced pluripotent stem cell
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We thank Drs. M Ito, M Fujino (Nagoya First Red Cross Hospital), T Tsuzuki, and E Maeda (Nagoya Second Red Cross Hospital) for providing autopsy samples.
This study was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant numbers 18590397, 16K10202 to AN, and 17K08778 to AI.
Ethics approval and consent to participate
Research use of autopsy samples was permitted with informed consent from the next of kin, and the study was carried out with the authorization of the Ethics Committee of Institute for Developmental Research, Aichi Developmental Disability Center, endorsed as #11-06.
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The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1: Figure S1.
Specificity of anti-NL4-m1 antibodies and generation of NLGN4X knockout human iPSC clones (hiPSC NL4-KO). (A) Anti-NL4-m1 antibodies specifically react with NLGN4 but not with the other NLGNs in Western blotting. Lysates of HEK293 cells expressing FLAG-tagged NLGN1 (FLAG-NL1), NLGN2 (FLAG-NL2), NLGN3 (FLAG-NL3), or NLGN4 (FLAG-NL4) were electrophoresed and immunoblotted with anti-FLAG and anti-NL4-m1 antibodies. Anti-NL4-m1 antibodies reacted with FLAG-NL4 and did not cross-react with FLAG-NL1, 2, and 3. (B) Schematic structures of the partial human NLGN4X gene (upper schematic diagram) and an expected edited gene in the hiPSC NL4-KO clone (KO: lower schematic diagram). The hiPSC NL4-KO clone was generated by engineering the NLGN4X gene of human iPSC clone 610B1 using the CRISPR/Cas9 system. Target sites of two guide RNAs (gRNA1 and gRNA2) are indicated by white arrows. Positions of primer sets (pEx2f and r, pEx6f and r) flanking the target sites to confirm expected gene editing and sizes of respective PCR amplicons are also indicated in the schema. (C) Agarose gel electrophoresis images of PCR products from human iPSC clone 610B1 (hiPSC WT) genomic DNA or hiPSC NL4-KO genomic DNA. Expected PCR products were amplified from hiPSC WT genomic DNA with primer sets pEx2f/pEx2r and pEx6f/pEx6r but not from hiPSC NL4-KO genomic DNA. In contrast, PCR products were amplified only from hiPSC NL4-KO genomic DNA with the primer set pEx2f/pEx6r. These data are compatible with the finding that the hiPSC NL4-KO clone has the NLGN4X gene with targeted deletion spanning from the middle of exon 2 to the middle of exon 6. As an internal control, the X-linked gene PUDP was used.
Additional file 2: Figure S2
. Additional images stained with anti-NL4-p1 and anti-NL4-m1. (A, B) Low magnification views of a neonatal paraventricular nucleus (case ID: N1) stained with either anti-NL4-p1 (A) or anti-NL4-m1 (B). Similar staining patterns are confirmed, yielding a higher signal-to noise ratio by anti-NL4-p1. (C, D) Low magnification views of a neonatal supraoptic nucleus (case ID: N1) stained with either anti-NL4-p1 (C) or anti-NL4-m1 (D). Similar staining patterns were confirmed. (E, F) Low magnification views of adult oculomotor nuclei (case ID: A7) stained with either anti-NL4-p1 (E) or anti-NL4-m1 (F). Intense signals are observed in neurons by anti-NL4-p1, while weak signals are identified in neurons by anti-NL4-m1. (G, H) Low magnification views of an adult locus coeruleus (case ID: A4) stained with either anti-NL4-p1 (G) or anti-NL4-m1 (H). Similar staining patterns are confirmed, yielding a higher signal-to noise ratio by anti-NL4-p1. Scale bars = 250 µm (A-H).
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Toya, A., Fukada, M., Aoki, E. et al. The distribution of neuroligin4, an autism-related postsynaptic molecule, in the human brain. Mol Brain 16, 20 (2023). https://doi.org/10.1186/s13041-023-00999-y
- Periventricular nucleus
- Supraoptic nucleus