Expression analysis of type I ARF small GTPases ARF1-3 during mouse brain development.

BACKGROUND: ARF (ADP-ribosylation factor) GTPases are major regulators of intracellular trafficking, and classified into 3 groups (Type I - III), among which the type I group members, ARF1 and 3, are responsible genes for neurodevelopmental disorders. METHODS: In this study, we analysed the expression of Type I ARFs ARF1-3 during mouse brain development using biochemical and morphological methods. RESULTS: Western blotting analyses revealed that ARF1-3 are weakly expressed in the mouse brain at embryonic day 13 and gradually increase until postnatal day 30. ARF1-3 appear to be abundantly expressed in various telencephalon regions. Biochemical fractionation studies detected ARF1-3 in the synaptosome fraction of cortical neurons containing both pre- and post-synapses, however ARF1-3 were not observed in post-synaptic compartments. In immunohistochemical analyses, ARF1-3 appeared to be distributed in the cytoplasm and dendrites of cortical and hippocampal neurons as well as in the cerebellar molecular layer including dendrites of Purkinje cells and granule cell axons. Immunofluorescence in primary cultured hippocampal neurons revealed that ARF1-3 are diffusely distributed in the cytoplasm and dendrites with partial colocalization with a pre-synaptic marker, synaptophysin. CONCLUSIONS: Overall, our results support the notion that ARF1-3 could participate in vesicle trafficking both in the dendritic shaft (excluding spines) and axon terminals (pre-synaptic compartments).

MED13L and its disease-associated variants influence the dendritic development of cerebral cortical neurons in the mammalian brain.

The mediator complex comprises multiple subcellular subunits that collectively function as a molecular interface between RNA polymerase II and gene-specific transcription factors. Recently, genetic variants to one subunit of the complex, known as MED13L (mediator complex subunit 13 like), have been implicated in syndromic intellectual disability and distinct facial features, frequently accompanied by congenital heart defects. We investigated the impact of five disease-associated MED13L variants on the subcellular localization and biochemical stability of MED13L protein in vitro and in vivo. In overexpression assays using cortical neurons from embryonic mouse cerebral cortices transduced by in utero electroporation-mediated gene transfer, we found that mouse orthologues of human MED13L-p.P866L and -p.T2162M missense variants accumulated in the nucleus, while the p.S2163L and p.S2177Y variants were diffusely distributed in the cytoplasm. In contrast, we found that the p.Q1922* truncation variant was barely detectable in transduced cells, a phenotype reminiscent of this variant that results in MED13L haploinsufficiency in humans. Next, we analyzed these variants for their effects on neuronal migration, dendritic growth, spine morphology, and axon elongation of cortical neurons in vivo. There, we found that overexpression of the p.P866L variant resulted in reduced number and length of dendrites of cortical layer II/III pyramidal neurons. Furthermore, we show that mMED13L-knockdown abrogated dendritic growth in vivo, and this effect was significantly rescued by co-electroporation of an RNAi-resistant mMED13L, but weakly by the p.T2162M variant, and not at all by the p.S2163L variant. However, overexpression of the p.S2163L variant inhibited mature dendritic spine formation in vivo. Expression of each of the 5 variants did not affect neuronal cell migration and callosal axon elongation in vivo. Taken together, our results demonstrate that MED13L expression is relevant to corticogenesis and influences the dendritic branching characteristics of cortical excitatory neurons. Our study also suggests that disease-associated MED13L variants may directly cause morphological and functional defects in cortical neurons in different ways.

Expression analyses of C-terminal-binding protein 1 (CtBP1) during mouse brain development.

INTRODUCTION: CtBP1 (C-terminal-binding protein 1) is a multi-functional protein with well-established roles as a transcriptional co-repressor in the nucleus and a regulator of membrane fission in the cytoplasm. Although CtBP1 gene abnormalities have been reported to cause neurodevelopmental disorders, the physiological role and expression profile of CtBP1 remains to be elucidated. METHODS: In this study, we used biochemical, immunohistochemical and immunofluorescence methods to analyze the expression of CtBP1 during mouse brain development. RESULTS: Western blotting analyses revealed that CtBP1 appeared to be expressed mainly in the central nervous system throughout the developmental process. In immunohistochemical analyses, region-specific nuclear as well as weak cytoplasmic distribution of CtBP1 was observed in telencephalon at embryonic day (E)15 and E17. It is of note that CtBP1 was barely detected in axons, but observed in the nucleus of oligodendrocytes in the white matter at E17. As to cerebellum at postnatal day 30, CtBP1 appeared to be expressed in the nucleus and cytoplasm of Purkinje cells, the nucleus of granule cells and cells in the molecular layer (ML), and the ML per se where granule cell axons and Purkinje cell dendrites are enriched. In addition, CtBP1 was detected in the cerebellar nuclei. CONCLUSION: The obtained results suggest involvement of CtBP1 in brain function.

Expression Analyses of Cep152, a Responsible Gene Product for Autosomal Recessive Primary Microcephaly, during Mouse Brain Development.

Centrosomal protein 152 (Cep152) regulates centriole duplication as a molecular scaffold during the cell cycle. Its gene abnormalities are responsible for autosomal recessive primary microcephaly 9 and Seckel syndrome. In this study, we prepared an antibody against mouse Cep152, anti-Cep152, and performed expression analyses focusing on mouse brain development. Western blotting analyses revealed that Cep152 with a molecular mass of ∼150 kDa was expressed strongly at embryonic day (E)13 and then gradually decreased during the brain development process. Instead, protein bands of ∼80 kDa and ∼60 kDa came to be recognized after postnatal day (P)15 and P30, respectively. In immunohistochemical analyses, Cep152 was enriched in the centrosome of neuronal progenitors in the ventricular zone at E14, whereas it was diffusely distributed mainly in the cytoplasm of cortical neurons at P18. In developing cerebellum at P7, Cep152 was localized at the centrosome in the external granular layer, where neurogenesis takes place. Notably, biochemical analysis revealed that Cep152 was also present in the postsynaptic density fraction. Subsequent immunofluorescent analyses showed co-localization of Cep152 with excitatory synaptic markers, PSD95 and synaptophysin, but not with an inhibitory synaptic marker gephyrin in differentiated primary cultured hippocampal neurons. The obtained results suggest that Cep152 takes part not only in neurogenesis during corticogenesis but also in the regulation of synaptic function in differentiated neurons.

Expression Analyses of Polo-Like Kinase 4, a Gene Product Responsible for Autosomal Recessive Microcephaly and Seckel Syndrome, during Mouse Brain Development.

Polo-like kinase 4 (Plk4) is a ser/thr kinase, which plays a central role in centriole duplication during the cell cycle. PLK4 gene abnormalities are responsible for autosomal recessive chorioretinopathy-microcephaly syndrome and Seckel syndrome. In this study, we performed expression analyses of Plk4 by focusing on mouse brain development. Western blotting analyses revealed that Plk4 with a molecular mass of ∼100 kDa was broadly expressed in adult mouse tissues with specific subcellular distribution. As to the central nervous system, Plk4 was expressed throughout the developmental process with drastic increase after P15, suggesting an essential role of Plk4 in differentiated neurons. In immunohistochemical analyses with mouse brain at embryonic day 14, Plk4 was detected dominantly at the cell-cell contact sites of neuronal progenitors in the ventricular zone. Plk4 was then diffusely distributed in the cell body of cortical neurons at P7, while it was enriched in the neuropil as well as soma of excitatory neurons in the cerebral cortex and hippocampus and Purkinje cells in the cerebellum at P30. Notably, biochemical fractionation analysis found an enrichment of Plk4 in the postsynaptic density fraction. Then, immunofluorescent analyses showed partial co-localization of Plk4 with excitatory synaptic markers, PSD95 and synaptophysin, in differentiated primary cultured hippocampal neurons. These results suggest that Plk4 takes part in the regulation of synaptic function in differentiated neurons.

Heterotrimeric G-protein, Gi1, is involved in the regulation of proliferation, neuronal migration, and dendrite morphology during cortical development in vivo.

Heterotrimeric G-proteins are composed of α, ß, and γ subunits, and function as signal transducers. Critical roles of the α-subunits of Gi/o family heterotrimeric G-proteins, Gαi2, and Gαo1, have so far been reported in brain development and neurodevelopmental disorders. In this study, we tried to clarify the role of Gαi1, α-subunit of another Gi/o family member Gi1, during corticogenesis, based on the recent identification of its gene abnormalities in neurodevelopmental disorders. In western blot analyses, Gαi1 was found to be expressed in mouse brain in a developmental stage-dependent manner. Morphological analyses revealed that Gαi1 was broadly distributed in cerebral cortex with relatively high expression in the ventricular zone (VZ) at embryonic day (E) 14. Meanwhile, Gαi1 was enriched in membrane area of yet unidentified early mitotic cells in the VZ and the marginal zone at E14. Acute knockdown of Gαi1 with in utero electroporation in cerebral cortex caused cell cycle elongation of the neural progenitor cells and promoted their cell cycle exit. Gαi1-deficient cortical neurons also exhibited delayed radial migration during corticogenesis, with abnormally elongated leading processes and hampered nucleokinesis. In addition, silencing of Gαi1 prevented basal dendrite development. The migration and dendritic phenotypes were at least partially rescued by an RNAi-resistant version of Gαi1. Collectively, these results strongly suggest a crucial role of Gi1 in cortical development, and disturbance of its function may cause deficits in synaptic network formation, leading to neurodevelopmental disorders.

Expression Analyses of Mediator Complex Subunit 13-Like: A Responsible Gene for Neurodevelopmental Disorders during Mouse Brain Development.

MED13L (mediator complex subunit 13-like) is a component of the mediator complex, which functions as a regulator for gene transcription. Since gene abnormalities in MED13L are responsible for neurodevelopmental disorders, MED13L is presumed to play an essential role in brain development. In this study, we prepared a specific antibody against MED13L, anti-MED13L, and analyzed its expression profile in mouse tissues with focusing on the central nervous system. In Western blotting, MED13L exhibited a tissue-dependent expression profile in the adult mouse and was expressed in a developmental stage-dependent manner in brain. In immunofluorescence analyses, MED13L was at least partially colocalized with pre- and post-synaptic markers, synaptophysin, and PSD95, in primary cultured hippocampal neurons. Immunohistochemical analyses revealed that MED13L was relatively highly expressed in ventricular zone surface of cerebral cortex, and was also located both in the cytoplasm and nucleus of neurons in the cortical plate at embryonic day 14. Then, MED13L showed diffuse cytoplasmic distribution throughout the cerebral cortex at the postnatal day (P) 30. In addition, MED13L appeared to be localized in cell type- and developmental stage-specific manners in the hippocampus and cerebellum. These results suggest that MED13L is involved in the development of the central nervous system and synaptic function.

Neuropathophysiological significance of the c.1449T>C/p.(Tyr64Cys) mutation in the CDC42 gene responsible for Takenouchi-Kosaki syndrome.

Takenouchi-Kosaki syndrome (TKS) is an autosomal dominant congenital syndrome, of which pathogenesis is not well understood. Recently, a heterozygous mutation c.1449T > C/p.(Tyr64Cys) in the CDC42 gene, encoding a Rho family small GTPase, has been demonstrated to contribute to the TKS clinical features, including developmental delay with intellectual disability (ID). However, specific molecular mechanisms underlying the neuronal pathophysiology of TKS remain largely unknown. In this study, biochemical analyses revealed that the mutation moderately activates Cdc42. In utero electroporation-based acute expression of Cdc42-Y64C in ventricular zone progenitor cells in embryonic mice cerebral cortex resulted in migration defects and cluster formation of excitatory neurons. Expression the mutant in primary cultured hippocampal neurons caused impaired axon elongation. These data suggest that the c.1449T > C/p.(Tyr64Cys) mutation causes altered CDC42 function and results in defects in neuronal morphology and migration during brain development, which is likely to be responsible for pathophysiology of psychomotor delay and ID in TKS.

Biochemical and morphological characterization of SEPT1 in mouse brain.

Septins are a highly conserved family of GTPases which are identified in diverse organisms ranging from yeast to humans. In mammals, nervous tissues abundantly contain septins and associations of septins with neurological disorders such as Alzheimer's disease and Parkinson's disease have been reported. However, roles of septins in the brain development have not been fully understood. In this study, we produced a specific antibody against mouse SEPT1 and carried out biochemical and morphological characterization of SEPT1. When the expression profile of SEPT1 during mouse brain development was analyzed by western blotting, we found that SEPT1 expression began to increase after birth and the increase continued until postnatal day 22. Subcellular fractionation of mouse brain and subsequent western blot analysis revealed the distribution of SEPT1 in synaptic fractions. Immunofluorescent analyses showed the localization of SEPT1 at synapses in primary cultured mouse hippocampal neurons. We also found the distribution of SEPT1 at synapses in mouse brain by immunohistochemistry. These results suggest that SEPT1 participates in various synaptic events such as the signaling, the neurotransmitter release, and the synapse formation/maintenance.

Expression Analyses of POGZ, A Responsible Gene for Neurodevelopmental Disorders, during Mouse Brain Development.

POGZ is a heterochromatin protein 1 α-binding protein and regulates gene expression. On the other hand, accumulating pieces of evidence indicate that the POGZ gene abnormalities are involved in various neurodevelopmental disorders. In this study, we prepared a specific antibody against POGZ, anti-POGZ, and carried out biochemical and morphological characterization with mouse brain tissues. Western blotting analyses revealed that POGZ is expressed strongly at embryonic day 13 and then gradually decreased throughout the brain development process. In immunohistochemical analyses, POGZ was found to be enriched in cerebrocortical and hippocampal neurons in the early developmental stage. The nuclear expression was also detected in Purkinje cells in cerebellum at postnatal day (P)7 and P15 but disappeared at P30. In primary cultured hippocampal neurons, while POGZ was distributed mainly in the nucleus, it was also visualized in axon and dendrites with partial localization at synapses in consistency with the results obtained in biochemical fractionation analyses. The obtained results suggest that POGZ takes part in the regulation of synaptic function as well as gene expression during brain development.

De novo PHACTR1 mutations in West syndrome and their pathophysiological effects.

Trio-based whole exome sequencing identified two de novo heterozygous missense mutations [c.1449T > C/p.(Leu500Pro) and c.1436A > T/p.(Asn479Ile)] in PHACTR1, encoding a molecule critical for the regulation of protein phosphatase 1 (PP1) and the actin cytoskeleton, in unrelated Japanese individuals with West syndrome (infantile spasms with intellectual disability). We then examined the role of Phactr1 in the development of mouse cerebral cortex and the pathophysiological significance of these two mutations and others [c.1561C > T/p.(Arg521Cys) and c.1553T > A/p.(Ile518Asn)], which had been reported in undiagnosed patients with intellectual disability. Immunoprecipitation analyses revealed that actin-binding activity of PHACTR1 was impaired by the p.Leu500Pro, p.Asn479Ile and p.Ile518Asn mutations while the p.Arg521Cys mutation exhibited impaired binding to PP1. Acute knockdown of mouse Phactr1 using in utero electroporation caused defects in cortical neuron migration during corticogenesis, which were rescued by an RNAi-resistant PHACTR1 but not by the four mutants. Experiments using knockdown combined with expression mutants, aimed to mimic the effects of the heterozygous mutations under conditions of haploinsufficiency, suggested a dominant negative effect of the mutant allele. As for dendritic development in vivo, only the p.Arg521Cys mutant was determined to have dominant negative effects, because the three other mutants appeared to be degraded with these experimental conditions. Electrophysiological analyses revealed abnormal synaptic properties in Phactr1-deficient excitatory cortical neurons. Our data show that the PHACTR1 mutations may cause morphological and functional defects in cortical neurons during brain development, which is likely to be related to the pathophysiology of West syndrome and other neurodevelopmental disorders.

Expression analyses of Dusp22 (Dual-specificity phosphatase 22) in mouse tissues.

Dusp22 (dual-specificity phosphatase 22) is considered to regulate various cellular processes through the regulation of protein dephosphorylation. In this study, we prepared a specific antibody against Dusp22, anti-Dusp22, and carried out expression analyses with mouse tissues and cultured cell lines. Western blotting analyses demonstrated a tissue-dependent expression profile of Dusp22 in the adult mouse, and strongly suggested the presence of isoforms with larger molecular masses. In fibroblast NIH3T3 cells, while both endogenous and Myc-tagged Dusp22 was diffusely distributed in the cytoplasm, Myc-Dusp22 was partially colocalized with actin cytoskeleton. From the obtained results, anti-Dusp22 was found to be a useful tool for biochemical and cell biological analyses of Dusp22.

Morphological characterization of Class III phosphoinositide 3-kinase during mouse brain development.

The mammalian Class III phosphoinositide 3-kinase (PIK3C3, also known as mammalian vacuolar protein sorting 34 homologue, Vps34) is a regulator of vesicular trafficking, autophagy, and nutrient sensing. In this study, we generated a specific antibody against PIK3C3, and carried out expression and morphological analyses of PIK3C3 during mouse brain development. In Western blotting, PIK3C3 was detected throughout the developmental process with higher expression in the early embryonic stage. In immunohistochemical analyses with embryonic day 16 mouse brain, PIK3C3 was detected strongly in the axon of cortical neurons. While PIK3C3 was distributed at the soma, nucleus, axon, and dendrites in primary cultured mouse hippocampal neurons at 3 days in vitro (div), it was also found in a punctate distribution with partial colocalization with synaptic marker, synaptophysin, at 21 div. The obtained results indicate that PIK3C3 is expressed and may have a physiological role in central nervous system during corticogenesis.

MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway.

We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.

Establishment of an in vivo electroporation method into postnatal newborn neurons in the dentate gyrus.

Electroporation-mediated gene transfer has been developed for the analysis of mammalian brain development in vivo. Indeed, in utero electroporation method is widely used for the investigation of the mouse embryonic cortical development while in vivo electroporation using neonatal mouse brain is employed for the analysis of the rostral migratory stream (RMS) and postnatal olfactory neurogenesis. In the present study, we established a stable gene-transfer method to dentate gyrus (DG) neurons by carefully determining the in vivo electroporation conditions, such as position and direction of electrode, voltage for electric pulses, and interval between electroporation and sample preparation. Consequently, GFP-positive cells in DG were observed to extend branched dendrites and long axons into the molecular layer and the hilus, respectively, 21 days after electrporation. They were morphologically identified as dentate granule neurons with many protrusions on dendrites, and some of them had wide head and thin neck that resembled matured mushroom spines. Expression of GFP in dentate neurons sustained for at least 9 months after electroporation under our experimental conditions. Taken together, the method developed here could be a powerful new tool for the analysis of the postnatal DG development.

Analyses of Conditional Knockout Mice for Pogz, a Gene Responsible for Neurodevelopmental Disorders in Excitatory and Inhibitory Neurons in the Brain.

POGZ (Pogo transposable element derived with ZNF domain) is known to function as a regulator of gene expression. While variations in the POGZ gene have been associated with intellectual disabilities and developmental delays in humans, the exact pathophysiological mechanisms remain unclear. To shed light on this, we created two lines of conditional knockout mice for Pogz, one specific to excitatory neurons (Emx1-Pogz mice) and the other to inhibitory neurons (Gad2-Pogz mice) in the brain. Emx1-Pogz mice showed a decrease in body weight, similar to total Pogz knockout mice. Although the two lines did not display significant morphological abnormalities in the telencephalon, impaired POGZ function affected the electrophysiological properties of both excitatory and inhibitory neurons differently. These findings suggest that these mouse lines could be useful tools for clarifying the precise pathophysiological mechanisms of neurodevelopmental disorders associated with POGZ gene abnormalities.

Biochemical and morphological characterization of A2BP1 in neuronal tissue.

A2BP1 is considered to regulate alternative splicing of important neuronal transcripts and has been implicated in a variety of neurological and developmental disorders. A2BP1 was found in neuronal cells and was analyzed biochemically and morphologically. In this study, we prepared a specific antibody against A2BP1, anti-A2BP1, and carried out protein expression and localization analyses of A2BP1 in rat and mouse tissues. By Western blotting, A2BP1 showed tissue-dependent expression profiles and was expressed in a developmental-stage-dependent manner in the brain. A2BP1 was detected at high levels in neocortex and cerebellum in the rat brain. Immunohistochemical analyses demonstrated that A2BP1 was highly expressed in differentiated neurons but not in mitotically active progenitor cells in the cerebral cortex during developmental stages. In cortical neurons, A2BP1 had accumulated mainly in the nucleus and diffusely distributed in the cell body and dendrites. In differentiated primary cultured rat hippocampal neurons, although A2BP1 was enriched in the nucleus and diffusely distributed in the cytoplasm, it was found in a punctate distribution adjacent to synapses. The results suggest that in neuronal tissues A2BP1 plays important roles, which are regulated in a spatiotemporal manner.

Possible role of a septin, SEPT1, in spreading in squamous cell carcinoma DJM-1 cells.

We performed biochemical, histochemical and cell biological characterization of septins by focusing on SEPT1 in human skin tissues and a squamous cell carcinoma (SCC) cell line DJM-1. In immunoblotting, SEPT1, together with other septins, was detected in normal human epidermis, SCC and DJM-1. In immunohistochemical analyses, SEPT1 was detected diffusely in the cytoplasm of human epidermal cells and eccrine gland epithelial cells, and the protein level was increased in some skin tumors. In DJM-1 cells, SEPT1 together with other members of SEPT2-subgroup, SEPT4 and SEPT5, was enriched in lamellipodia and the localization was dependent on the cortical actin structure. SEPT1 distribution at lamellipodia was also observed in melanoma B16 cells. SEPT9, SEPT11 and SEPT14, in contrast, were localized along with microtubules in DJM-1 cells. In immunoprecipitation assays, SEPT1 and SEPT5 were found to form a complex in DJM-1 cells, whereas SEPT9, SEPT11 and SEPT14 formed a distinct complex with other septins including SEPT7, SEPT8 and SEPT10, in which SEPT5 was not included. When SEPT1 was silenced in DJM-1 cells, cell spreading was inhibited. These results suggest that SEPT1 may participate in cell-cell and/or cell-substrate interaction in DJM-1 and exert its function in a coordinated manner with other septins.

Localization of multidomain adaptor proteins, p140Cap and vinexin, in the pancreatic islet of a spontaneous diabetes mellitus model, Otsuka Long-Evans Tokushima Fatty rats.

We have shown that two multidomain adaptor proteins, p140Cap and vinexin, interact with each other and are likely to be involved in neurotransmitter release. Because the basic molecular mechanism governing neurotransmitter and insulin secretion is conserved, these two proteins may also to play pivotal roles in insulin secretion. We therefore performed some characterization of p140Cap and vinexin in pancreas of a wild-type rat or a spontaneous type 2 diabetes mellitus (DM) model, the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. These two proteins were detected in Wistar rat pancreas by Western blotting. Immunohistochemistry revealed that p140Cap and vinexin are enriched in ß and α cells, respectively, in the rat pancreas. We then found that pancreatic islet structure was disorganized in the OLETF rat with hyperinsulinemia or with hyperglycemia, based on immunohistochemical analyses of vinexin. In ß cells of these model rats, p140Cap was distributed in a cytoplasmic granular pattern as in the control rats, although its expression was reduced to various extents from cell to cell. These results may suggest possible involvement of p140Cap in insulin secretion, and reduction of p140Cap might be related to abnormal insulin secretion in DM.

Biochemical and morphological characterization of MAGI-1 in neuronal tissue.

The membrane-associated guanylate kinase with inverted organization (MAGI) proteins consist of three members, MAGI-1, MAGI-2 (also known as S-SCAM), and MAGI-3. Although MAGI-2 has been analyzed and shown to interact with a variety of postsynaptic proteins, functional analyses and characterization of MAGI-1 in neuronal tissues have been rare. In this study, we prepared a specific antibody against MAGI-1, anti-MAGI-1, and carried out biochemical and morphological analyses of MAGI-1 in rat neuronal tissues. By Western blotting, a high level of MAGI-1 was detected in nervous tissues, especially in olfactory bulb. Biochemical fractionation clarified that MAGI-1 was relatively enriched in the synaptosomal vesicle and synaptic plasma membrane fractions, whereas MAGI-2 and MAGI-3 appeared to be in the synaptic plasma membrane and postsynaptic density fractions. Immunofluorescent analyses revealed diffuse distribution of MAGI-1 in the cell body and processes of primary cultured rat hippocampal neurons, whereas MAGI-2 and MAGI-3 were likely to be enriched at synapses. Immunohistochemical analyses demonstrated that MAGI-1 was expressed in Purkinje cells, in hypocampal neurons in CA1 region, in the glomerulus region of olfactory bulb, and at the dorsal root entry zone in embryonic rat spinal cord. These results suggest neuronal roles of MAGI-1 different from those of MAGI-2/3.