Introduction: C-terminal-binding protein 1 (CtBP1) 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 the 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.

C-terminal-binding protein (CtBP) family is composed of CtBP1 and CtBP2 [1‒4], which are unique to higher eukaryotes and are highly conserved. CtBP1, also known as brefeldin A-ADP-ribosylation substrate, was discovered as a cellular protein that interacted with the C-terminus of adenovirus E1A proteins [1, 2]. Subsequent investigations clarified that CtBP1 functions as a transcriptional co-repressor in the nucleus [5, 6] and as a regulator of membrane fission in the cytoplasm [7, 8]. The CTBP1 gene locus codes for 2 proteins, CtBP1-S (where S stands for short; also named brefeldin A-ADP-ribosylation substrate 50) and CtBP1-L (L stands for long), which are generated by an alternative splicing and differ in their N-termini [9, 10]. Although precise physiological roles of these isoforms remain to be elucidated, both CtBP1 isoforms may share similar functions in the regulation of gene expression and membrane trafficking [7]. In the central nervous system (CNS), CtBP1 was also reported to be distributed in the presynaptic compartment and undergo activity-dependent shuttling from the presynaptic compartment to the nucleus in differentiated neurons [11]. This presynaptic localization of CtBP1 has been reported to relieve the nucleus of co-repressor activity, allowing for upregulation of activity-dependent genes such as ARC, BDNF, EGR1, EGR4, and FOS [11].

Recent genetic analyses revealed that CtBP1 is involved in neuronal development because its gene abnormalities have been shown to cause neurodevelopmental disorders (NDDs), including Wolf-Hirschhorn syndrome and Hypotonia, Ataxia, Developmental Delay, and Tooth Enamel Defect Syndrome (HADDTS) [12, 13]. However, while the spatial protein expression profile and synaptic localization of CtBP1 have been reported for the adult mouse brain [14, 15], the expression profile of CtBP1 in embryonic mouse brain, in which no functional synapses are formed, remains to be elucidated. In the present study, we carried out the expression analysis of CtBP1 by focusing on mouse brain development.

Plasmids

Mouse (m)CtBP1 cDNA was obtained by RT-PCR from a mouse brain RNA pool and constructed into pCAG-Myc vector (Addgene Inc., Cambridge, MA, USA). For RNAi experiments, a mCtBP1 target sequence (GAA​GAT​CTG​GAG​AAG​TTT​A, 250–268) was inserted into pSuper-puro vector (OligoEngine, Seattle, WA, USA), which was named as pSuper-mCtBP1#1. As a control RNAi vector, we used pSuper-Luc designed against luciferase, CGT​ACG​CGG​AAT​ACT​TCG​A (155–173) [16]. Numbers indicate the positions from translational start sites. All constructs were verified by DNA sequencing.

Antibodies

Mouse monoclonal anti-CtBP1 was from Santa Cruz Biotechnology (Cat# sc-398945, Dallas, TX, USA) [17]. Monoclonal anti-β-actin and anti-Myc (Cell Signaling Technology, Danvers, MA, Cat# 3700, RRID: AB_2242334 and Cat# 2276, RRID: AB_331783), anti-glial fibrillary acidic protein (GFAP) (Sigma-Aldrich Inc., Saint Louis, MO, Cat# G 3893, RRID: AB_477010) were also used. Rabbit polyclonal anti-calbindin-D was produced as described previously [18]. Rabbit polyclonal anti-Tbr1 (Abcam, Cambridge, UK, Cat# ab31940, RRID: AB_2200219), anti-NeuN (Abcam, Cambridge, UK, Cat# ab177487, RRID: AB_2532109), and anti-Olig2 (Proteintech, Rosemont, IL, USA, Cat# 13999-1-AP, RRID: AB_2157541) and goat polyclonal anti-Sox2 (Santa Cruz, Dallas, TX, USA, Cat# sc-17320, RRID: AB_2286684) were also used.

Preparation of Extracts from Mouse Tissues

Sample preparation was performed as described [19]. Briefly, tissues from ICR mice at P40 (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 Na3VO4, and 10 μg/mL each of aprotinin and leupeptin). Each suspension was sonicated on ice and centrifuged at 125,000 g for 20 min at 4°C. The supernatants were used as cytosolic extracts. The pellets were sonicated in phosphate-buffered saline (PBS), washed once with PBS, centrifuged, solubilized with the lysis buffer containing 2% SDS, and used as insoluble fractions. Protein concentration was estimated with BCA protein assay reagent kit (Thermo Scientific Inc., Waltham, MA, USA) with bovine serum albumin (BSA) as a standard. For Western blotting, cytoplasmic and insoluble fractions were prepared at the concentrations of 2 mg/mL and 1.5 mg/mL, respectively. Whole brain extracts were obtained as described previously [20]. Briefly, brains were dissected at indicated time points, weighed immediately, frozen in liquid nitrogen, and kept at −80°C until use. Brains were then homogenized with the lysis buffer containing 2% SDS. Brain samples were adjusted to 2 mg/mL of protein concentration.

SDS-PAGE (10% gel) and Western blotting were conducted as described [21]. Briefly, extracts were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 2% skim milk for 1 h, membranes were incubated overnight at 4°C with a primary antibody and then reacted with an HRP-conjugated secondary antibody for 1 h. Immunoreactive proteins were visualized using the ECL Plus Western blotting detection reagents (Perkin Elmer Inc., Waltham, MA) and the LAS 4000 mini imaging system (Fuji Film Inc., Tokyo, Japan).

Cell Culture and Transfection

COS7 (monkey kidney fibroblast), HEK293 (human embryonic kidney cell), B16 (human melanoma), C6 (rat glioma), and N2A (mouse neuroblastoma) cell lines were obtained from American Type Culture Collection (ATCC, Cat# CRL-1651, RRID: CVCL_0224), JCRB Cell Bank (Cat# JCRB9068, RRID: CVCL_0045), JCRB Cell Bank (Cat# JCRB0202, RRID: CVCL_F936), JCRB Cell Bank (Cat# JCRB9096, RRID: CVCL_0194), and ATCC (Cat# CCL-131, RRID:CVCL_0470), respectively, and cultured essentially as described [19]. Transient transfection into N2A cells, whose efficiency was ∼50%, was carried out using Lipofectamine 2000 (Life Technologies Inc., Japan, Tokyo).

Immunohistochemical Analyses

A whole mouse embryo at E15.5 and a brain at E17.5 were fixed with 4% paraformaldehyde. Then, brains were cut into serial sagittal (E15.5) or coronal (E17.5) sections (4-µm thickness). After deparaffinization, endogenous peroxidase activity in sections was blocked by incubation in 0.3% hydrogen peroxide for 20 min. Antigen retrieval was performed with Histo-VT One (Nacalai Tesque, Tokyo, Japan) at 70°C for 20 min. After blocking with PBS containing 0.5% Triton X-100 and 0.1% BSA at room temperature for 1 h, the slides were incubated with anti-CtBP1 (1:500) in 0.1% BSA/PBS overnight at 4°C. Staining signal detection was carried out with TaKaRa POD Conjugate Set Anti-Mouse kit (MK204) (Takara Bio Inc., Kyoto, Japan) together with TaKaRa DAB Substrate (MK210) (Takara Bio Inc., Tokyo, Japan) according to the manufacturer’s instructions. One embryonic slice was stained with hematoxylin and eosin. As to the staining of the cerebellum, after fixing with 4% paraformaldehyde at P30, sagittal sections (14 μm-thickness) were prepared with a cryostat (Leica CM1900, Leica Microsystems, Wetzlar, Germany). Frozen sections were incubated in the blocking solution followed by the primary antibody reaction as above. Signal detection was also performed as mentioned above. Where indicated, we stained slices using TaKaRa POD Conjugate Set Anti-Mouse Kit (MK200) in order to prevent the primary antibody from reacting with endogenous immunoglobulins, and suppress background staining. Images were captured using BZ-9000 microscope (Keyence Inc., Osaka, Japan).

Immunofluorescence Analyses

After fixation with 4% paraformaldehyde of telencephalon (E15 and E17) and cerebellum (P30), coronal sections (14 μm-thickness) were prepared with a cryostat. Frozen sections were incubated in PBS containing 0.5% Triton X-100 and 0.1% BSA at room temperature for 1 h. Then, a primary antibody reaction was done in PBS containing 0.05% Triton X-100 at 4°C overnight. Secondary antibody reaction together with nuclear staining was carried out in PBS containing 0.05% Triton X-100 at room temperature for 1 h. Stained sections were washed 3 times with PBS and mounted with the anti-fading mounting medium. Nuclei were visualized by DAPI. Alexa Fluor 488- and 568-labeled IgG were used as secondary antibodies. Fluorescence signals were detected with an LSM-880 confocal laser microscope (Carl Zeiss, Germany) at excitation/emission wavelengths of 401/421 nm (DAPI), 490/525 nm (Alexa Fluor® 488, green), 578/603 nm (Alexa Fluor® 568, red), and 651/672 nm (Alexa Fluor® 647, magenta).

Characterization of Anti-CtBP1 Antibody

The specificity of anti-CtBP1 was tested with N2A cell lysates expressing Myc-mCtBP1. While anti-CtBP1 recognized Myc-mCtBP1, the immunoreactivity was significantly reduced when Myc-mCtBP1 expression was silenced by pSuper-mCtBP1#1 (Fig. 1a). We next carried out Western blotting with whole cell lysates from various cell lines including COS7, HEK293, B16, C6V29, and N2A cells. Consequently, anti-CtBP1 detected a major protein band with ∼50 kDa in all the lines (Fig. 1b). Since CtBP1 is expressed in all cultured cell lines tested, it may be involved in essential cellular functions of these lines. Anti-CtBP1 detected a faint band with ∼65 kDa in human cell lines, HEK293 and B16 (Fig. 1b), suggestive of a human-specific posttranslationally modified form. When we looked into the effects of pSuper-mCtBP1#1 on endogenous CtBP1 in N2A cells, immunofluorescent signal was reduced by the knockdown vector (Fig. 1c). Together with the previously reported results [17, 22‒25], we concluded that this commercially available antibody specifically recognized CtBP1.

Fig. 1.

Characterization of anti-CtBP1. a N2A cells were transfected with pCAG-Myc-mCtBP1 (0.2 μg) together with pSuper vector or pSuper-mCtBP1#1 (1 μg each). After 48 h, cells were collected and the lysates (20 μg of protein) were separated by SDS-PAGE followed by Western blotting using anti-CtBP1 or anti-Myc. b Detection of CtBP1 in cultured cell lines. Whole lysates (20 μg of proteins) of COS7, HEK293, B16, C6V29, and N2A cells were subjected to Western blotting using anti-CtBP1. The blots were re-probed with anti-β-actin (a loading control). c Knockdown of endogenous CtBP1 in N2A cells. pCAG-EGFP (0.1 μg) was transfected with control pSuper-Luc or pSuper-mCtBP1#1 (0.5 μg). After 48 h, cells were fixed and immunostained for GFP (green) and CtBP1 (red). Control and deficient cells were marked with arrowheads, respectively. Scale bar: 10 μm. The fluorescent signals of endogenous CtBP1 were measured by ImageJ software. The ratio of CtBP1 signal of knockdown cell to that of control one was calculated (n = 30 cells each). ****p < 0.0001 by Student’s t test.

Fig. 1.

Characterization of anti-CtBP1. a N2A cells were transfected with pCAG-Myc-mCtBP1 (0.2 μg) together with pSuper vector or pSuper-mCtBP1#1 (1 μg each). After 48 h, cells were collected and the lysates (20 μg of protein) were separated by SDS-PAGE followed by Western blotting using anti-CtBP1 or anti-Myc. b Detection of CtBP1 in cultured cell lines. Whole lysates (20 μg of proteins) of COS7, HEK293, B16, C6V29, and N2A cells were subjected to Western blotting using anti-CtBP1. The blots were re-probed with anti-β-actin (a loading control). c Knockdown of endogenous CtBP1 in N2A cells. pCAG-EGFP (0.1 μg) was transfected with control pSuper-Luc or pSuper-mCtBP1#1 (0.5 μg). After 48 h, cells were fixed and immunostained for GFP (green) and CtBP1 (red). Control and deficient cells were marked with arrowheads, respectively. Scale bar: 10 μm. The fluorescent signals of endogenous CtBP1 were measured by ImageJ software. The ratio of CtBP1 signal of knockdown cell to that of control one was calculated (n = 30 cells each). ****p < 0.0001 by Student’s t test.

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Expression Profile of CtBP1 in Mouse Tissues

We performed Western blotting analyses to examine the tissue distribution of CtBP1 in a young adult mouse at P40. As shown in Figure 2a, CtBP1 with ∼50 kDa was detected strongly in the cytoplasm of the cerebrum, hippocampus, and cerebellum. CtBP1 was also detected in the insoluble membrane fractions of these neuronal tissues (Fig. 2a). These results indicate that CtBP1 may be involved in brain development. Meanwhile, a ∼55 kDa band was present mainly in the soluble fraction of the uterus (Fig. 2a). In this context, 2 splicing isoforms have been reported for CtBP1; CtBP1-L (440 aa) and CtBP1-S (429 aa) [9, 10]. It is possible that the main isoforms expressed in the brain and uterus may be CtBP1-S and CtBP1-L, respectively. Since the significantly diverse region between CtBP1-L and CtBP1-S was only the N-terminal stretch, further analyses with an antibody against the N-terminal are required to identify the ∼50 kDa and ∼55 kDa proteins.

Fig. 2.

Expression profiles of CtBP1 in mouse tissues and during brain development. a Cytosolic (10 μg of protein per lane) and insoluble membrane (15 μg) fractions from P40-mouse organs were subjected to Western blotting with anti-CtBP1. The blots were re-probed with anti-β-actin (bottom, narrow panels). Molecular size markers are shown at left. b Developmental changes of CtBP1 expression. Whole tissue extracts (20 μg protein) of whole brain (E13) or cerebral cortex (P0–P30) at indicated developmental stages were subjected to Western blotting with anti-CtBP1. The blot was re-probed with anti-GFAP (a differentiation marker) and anti-β-actin. For quantification, relative band intensity of CtBP1 to β-actin was calculated when the value at E13 was taken as 1.0. Error bars indicate SD (n = 3). c–e Immunohistochemical staining on sections of whole embryonic body at E15.5. Sections were stained with hematoxylin and eosin (c), anti-CtBP1 (d), and anti-CtBP1 after treatment with anti-mouse for mouse tissue kit (e). Arrowheads 1, heart; 2, liver; 3, small intestine.

Fig. 2.

Expression profiles of CtBP1 in mouse tissues and during brain development. a Cytosolic (10 μg of protein per lane) and insoluble membrane (15 μg) fractions from P40-mouse organs were subjected to Western blotting with anti-CtBP1. The blots were re-probed with anti-β-actin (bottom, narrow panels). Molecular size markers are shown at left. b Developmental changes of CtBP1 expression. Whole tissue extracts (20 μg protein) of whole brain (E13) or cerebral cortex (P0–P30) at indicated developmental stages were subjected to Western blotting with anti-CtBP1. The blot was re-probed with anti-GFAP (a differentiation marker) and anti-β-actin. For quantification, relative band intensity of CtBP1 to β-actin was calculated when the value at E13 was taken as 1.0. Error bars indicate SD (n = 3). c–e Immunohistochemical staining on sections of whole embryonic body at E15.5. Sections were stained with hematoxylin and eosin (c), anti-CtBP1 (d), and anti-CtBP1 after treatment with anti-mouse for mouse tissue kit (e). Arrowheads 1, heart; 2, liver; 3, small intestine.

Close modal

To gain some insight into the involvement of CtBP1 in neuronal development, we analyzed its expression in the whole extracts of mouse brains prepared from various developmental stages. CtBP1 with ∼50 kDa appeared to be expressed at similar levels throughout the developmental process from embryonic day (E)13 to P30 (Fig. 2b). The obtained results suggest a role of CtBP1 in ubiquitous brain function. This process and protein loading were confirmed by visualizing a glial cell differentiation marker, GFAP, and β-actin, respectively (Fig. 2b, middle and lower panels). Tissue-dependent CtBP1 expression profile was confirmed by immunohistochemical analyses using whole embryo slices (E15.5); CtBP1 was highly expressed in the CNS but little in non-neuronal organs such as the heart, liver, and small intestine (Fig. 2c, d). When endogenous mouse immunoglobulin present in mouse tissues was blocked by the TaKaRa-MK200 kit, the staining pattern was comparable to the control pattern (Fig. 2d, e).

Immunohistochemical and Immunofluorescent Analyses of CtBP1 in the Developing Telencephalon

To determine CtBP1 localization during brain development, we carried out immunohistochemical analyses of the mouse telencephalon at E15.5 (sagittal section) and E17.5 (coronal section), as well as immunofluorescent analyses to identify cell types. At E15.5, CtBP1 was detected in the nucleus of excitatory neurons in the cortical plate and progenitors in the ventricular zone (VZ) (Fig. 3a–c, 4a, b). The nuclear localization was also detected in VZ progenitor cells in the ganglionic eminences (Fig. 3a, b, d; 4c). On the other hand, cells in the subventricular zone and the intermediate zone appeared to express CtBP1 relatively weakly (Fig. 3a–c). Likewise, relatively low expression was observed in the subventricular zone of ganglionic eminences (Fig. 3a, b, and d). At E17.5, CtBP1 was notably visualized in the developing hippocampus; nuclear localization of CtBP1 was observed in future pyramidal neurons in the cornus ammonis 1 and cornus ammonis 3 (Fig. 3e, f, i, and j), whereas it was only weakly detected in the fimbria which contains axons from the hippocampus (Fig. 3f and k). In addition to excitatory neurons, CtBP1 was detected in medium spiny neurons (inhibitory neurons) in the striatum (Fig. 3e and g, 4d). Meanwhile, CtBP1 appeared to show weak expression in axon bundles of the striatum (Fig. 3g, inset) and was only detected in the nucleus of oligodendrocytes in the anterior commissure (Fig. 3e, h, 4e). These findings suggest that CtBP1 has limited distribution in axons.

Fig. 3.

Immunohistochemical analyses of CtBP1 in embryonic mouse telencephalon. Sagittal and coronal sections were prepared at E15.5 (a–d) and E17.5 (e–k), respectively, and stained with hematoxylin and eosin (a) or anti-CtBP1 (b–k). Boxed areas in (b) were magnified in (c) and (d). Boxed areas in (e) were magnified in (f–h). Boxed areas in (f) and (g) were magnified in (i–k) and the inset, respectively. Cortical layers were indicated in (c). MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars: 200 μm (b, e), 20 μm (c, d), 10 μm (f–h and i–k).

Fig. 3.

Immunohistochemical analyses of CtBP1 in embryonic mouse telencephalon. Sagittal and coronal sections were prepared at E15.5 (a–d) and E17.5 (e–k), respectively, and stained with hematoxylin and eosin (a) or anti-CtBP1 (b–k). Boxed areas in (b) were magnified in (c) and (d). Boxed areas in (e) were magnified in (f–h). Boxed areas in (f) and (g) were magnified in (i–k) and the inset, respectively. Cortical layers were indicated in (c). MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars: 200 μm (b, e), 20 μm (c, d), 10 μm (f–h and i–k).

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Fig. 4.

Staining of telencephalon and cerebellum with cell-type-specific markers. Cortical slices of telencephalon at E15.5 (a–c) or E17.5 (d, e), and cerebellum at P30 (f–h) were double-stained with anti-CtBP1 together with anti-Tbr1 for postmitotic neurons (a), anti-Sox2 for progenitors (b, c), anti-Ctip2 for medium spiny neurons (d), anti-Olig2 for oligodendrocytes (e, g), anti-calbindin D for PCs (f), or anti-NeuN for differentiated neurons (h). CP, cortical plate; VZ, ventricular zone; GE, ganglionic eminence; WM, white matter; PC, Purkinje cells. Scale bars: 10 μm.

Fig. 4.

Staining of telencephalon and cerebellum with cell-type-specific markers. Cortical slices of telencephalon at E15.5 (a–c) or E17.5 (d, e), and cerebellum at P30 (f–h) were double-stained with anti-CtBP1 together with anti-Tbr1 for postmitotic neurons (a), anti-Sox2 for progenitors (b, c), anti-Ctip2 for medium spiny neurons (d), anti-Olig2 for oligodendrocytes (e, g), anti-calbindin D for PCs (f), or anti-NeuN for differentiated neurons (h). CP, cortical plate; VZ, ventricular zone; GE, ganglionic eminence; WM, white matter; PC, Purkinje cells. Scale bars: 10 μm.

Close modal

We further compared the CtBP1 expression profile by focusing on specific brain regions across development. To this end, we selected and stained the cerebral cortex (Fig. 5) at E15, E17, P0, and P30. Consequently, CtBP1 appeared to be distributed mainly in the nucleus, while moderate-weak expression was observed in the cytoplasm at E15.5, E17.5, and P0 (Fig. 5a–c, e–g). It is notable that CtBP1 was distributed in the nucleus and dendrites but little in the cytoplasm of cortical neurons at P30 (Fig. 5d, h). For a negative control experiment, staining of telencephalic slices at P30 was carried out with or without incubation with anti-CtBP1 (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000534886).

Fig. 5.

Time-dependent expression of CtBP1 in cerebral cortex. Cerebral cortex sections of the somatosensory area at E15.5 (a, e), E17.5 (b, f), P0 (c, g), and P30 (d, h) were stained with anti-CtBP1. Boxed areas in (a–d) were magnified in (e–h). Scale bars: 200 μm (d), 100 μm (a–c), 20 μm (e–g), and 10 μm (h). CP, cortical plate; CP-U, upper cortical plate; CP-L, lower cortical plate; IZ, intermediate zone; SVZ/VZ, ventricular/subventricular zones; WM, white matter.

Fig. 5.

Time-dependent expression of CtBP1 in cerebral cortex. Cerebral cortex sections of the somatosensory area at E15.5 (a, e), E17.5 (b, f), P0 (c, g), and P30 (d, h) were stained with anti-CtBP1. Boxed areas in (a–d) were magnified in (e–h). Scale bars: 200 μm (d), 100 μm (a–c), 20 μm (e–g), and 10 μm (h). CP, cortical plate; CP-U, upper cortical plate; CP-L, lower cortical plate; IZ, intermediate zone; SVZ/VZ, ventricular/subventricular zones; WM, white matter.

Close modal

Immunohistochemical Analyses of CtBP1 in the Cerebellum

Despite emerging evidence suggesting a role of the cerebellum in cognitive and emotional functions, information regarding CtBP1 distribution in the cerebellum is currently unavailable [26, 27]. We therefore carried out expression analyses at P30. The cortex consists of 3 layers, including the molecular layer (ML), where granule cell axons and Purkinje cell (PC) dendrites are accumulated, the PC layer, and the granular layer (GL) which contains granule cells, Golgi cells, and axons of PC, from outermost to innermost (Fig. 6a, b). CtBP1 was detected in the nucleus of cells in the GL, although the expression levels are dependent on cells (Fig. 6b, c). On the other hand, PCs showed moderate expression of CtBP1 in the nucleus and cytoplasm (Fig. 4f, 6b, d). This clear cytoplasmic distribution was due to the broad cytoplasmic area of PCs. Staining signal was also detected in the nucleus of cells in the ML as well as its fiber components (Fig. 6b, e). These ML cells are possible to be interneurons such as stellate cells and basket cells. CtBP1-positive cells in the white matter are likely to be oligodendrocytes (Fig. 4g, 6b, f). CtBP1 was also enriched in the nucleus and visualized as occasional cytoplasmic dot-like patterns in excitatory neurons in the cerebellar nuclei (Fig. 4h, 6g–i, arrowheads). Meanwhile, CtBP1 was little detected in axon bundles like other brain areas (Fig. 3e–h, 6f–i). It is notable that staining patterns were comparable between the samples with or without the TaKaRa-MK200 kit treatment (Fig. 6b, j).

Fig. 6.

Immunohistochemical analyses of CtBP1 in mouse cerebellum. A P30 section was stained with anti-CtBP1. Boxed areas in (a) were magnified in (b) and (g). The boxed areas in (b) and (g) were magnified in (c–f) and (h), respectively. Arrowheads in (h) indicate excitatory neurons. The boxed area in (h) was magnified in (i). j A staining pattern of a slice with anti-mouse for mouse tissue kit was also shown. ML, molecular layer; PC, Purkinje cell layer; GL, granular layer; WM, white matter. Scale bars: 1 mm (a), 100 μm (g), 50 μm (b, j), 20 μm (h), and 10 μm (c–f, i).

Fig. 6.

Immunohistochemical analyses of CtBP1 in mouse cerebellum. A P30 section was stained with anti-CtBP1. Boxed areas in (a) were magnified in (b) and (g). The boxed areas in (b) and (g) were magnified in (c–f) and (h), respectively. Arrowheads in (h) indicate excitatory neurons. The boxed area in (h) was magnified in (i). j A staining pattern of a slice with anti-mouse for mouse tissue kit was also shown. ML, molecular layer; PC, Purkinje cell layer; GL, granular layer; WM, white matter. Scale bars: 1 mm (a), 100 μm (g), 50 μm (b, j), 20 μm (h), and 10 μm (c–f, i).

Close modal

CtBP1 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. Notably, recent clinical genetic analyses have identified CTBP1 as a responsible gene for NDDs [12, 28‒30]. While these results indicate an essential role of CtBP1 in brain development, its expression profile remains to be elucidated during brain development. In the present study, we conducted expression analyses of CtBP1 by focusing on mouse brain development.

Western blotting analyses revealed that CtBP1 was strongly detected in the CNS and uterus, although the apparent molecular masses of CtBP1 bands were different (Fig. 2a). These CtBP1 isoforms are possible to be CtBP1-S and CtBP1-L, respectively, and might exert not only similar transcriptional functions in the nucleus but also organ-specific functions. Further genetic analyses of CtBP1 as well as preparation of isoform-specific antibodies should be helpful to identify the 2 proteins and examine their organ-specific functions. As for the mRNA level, Northern blotting analyses revealed expression of CtBP1 in the heart, lung, kidney, and muscle [31]. A possible explanation for the discrepancy between the mRNA and protein expression may be that gene translation and mRNA transcription are differently regulated for CtBP1. Alternatively, CtBP1 protein degradation is highly accelerated in these non-neuronal tissues. It is notable that CtBP1 was detected in both soluble (cytoplasmic) and insoluble (membrane) fractions in the CNS. This may indicate that CtBP1 functions vary depending on subcellular localization. Although further analyses are crucial, cytoplasmic CtBP1 may be involved in gene expression, while membranous CtBP1 may interact with components of the presynapses and postsynaptic density.

Immunohistochemical analyses of the telencephalon revealed developmental stage-dependent expression pattern of CtBP1. It was distributed in the nucleus in cells in the cortical plate and VZs at E15.5 (Fig. 3a–d). CtBP1 also appeared to be expressed in various cell types such as median spiny (inhibitory) neurons, hippocampal neurons, and oligodendrocytes, with continuous enrichment in the nucleus at E17.5 (Fig. 3e–h). While the nuclear accumulation of CtBP1 is reasonable when considering its authentic role in the gene transcription, CtBP1 has been shown to serve as a regulator of membrane fission in the cytoplasm. However, cytoplasmic distribution of this molecule was not necessarily clear except for PCs because of the narrow area of neuronal cytoplasm.

As to the cerebellum, while strong nuclear enrichment of CtBP1 was observed in the GL cells, moderate staining was detected in the ML, which contains dendritic trees of PCs and the huge array of parallel fibers (axons) from granule cells (Fig. 4b). Given the CtBP1-immunoreactivity barely detected in axons in the cerebral cortex and hippocampus, the staining signal in the ML might reflect CtBP1 in PC dendrites. The entity of the CtBP1-positive cytoplasmic dot-like structure in excitatory neurons of cerebellar nuclei needs to be elucidated (Fig. 4h, arrowheads). While these results may suggest cell-type-specific CtBP1 functions in the cerebellum, further intensive analyses are essential to identify the cell types and organelle where CtBP1 is located.

CtBP1 was reported to be partially localized at presynapses in differentiated primary cultured hippocampal neurons [11]. Presynaptic and nuclear pools of CtBP1 are possible to be interconnected, and both synaptic retention and cytoplasm-nucleus shuttling of CtBP1 might be co-regulated by neuronal activity [11]. These results suggest that CtBP1 plays a role in synaptic network formation and/or maintenance in matured neurons, consistent with the assumption that NDDs are considered to affect the development of the CNS. In this context, CtBP1 was found to be expressed throughout the developing process from E13 to P30, indicating its function in adult neuronal cells (Fig. 2b). CtBP1 is also expressed in the hippocampus and cerebellum, suggesting that its gene abnormalities may cause impairment of memory, spatial learning, cognition, and motor control, which are frequently associated with NDDs. Since physiological significance of CtBP1 in immature neurons during brain development is enigmatic, further analyses are required to clarify the role of CtBP1 during brain development.

In the present study, we examined developmental stage-dependent expression of CtBP1. Western blotting analyses demonstrated that CtBP1 is dominantly expressed in the CNS in the adult mouse. Immunohistochemical analyses revealed that CtBP1 was expressed in various kinds of neuronal cells of developing telencephalon in spatiotemporal manners. These results suggest that CTBP1 may be involved in brain function, and its gene abnormalities may be associated with the pathophysiological mechanisms of NDDs.

For the animal experiments, we followed the fundamental guidelines for proper conduct of animal experiments and related activity in academic research institution under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. All 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: 2019-013).

The authors declare that they have no conflict of interest.

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (B) (Grant Number, JP22H03049), Grant-in-Aid for challenging exploratory research (JP20K21589), and Grant-in-Aid for Scientific Research (C) (JP19K07059).

Nanako Hamada performed antibody characterization and biochemical analyses and helped draft the manuscript. Tohru Matsuki and Atsuo Nakayama carried out immunohistochemical analyses, and helped draft the manuscript. Mariko Noda and Hidenori Tabata prepared mouse tissue extracts. Ikuko Iwamoto carried out immunofluorescence analyses. Takuma Nishijo prepared vectors for RNAi and expression experiments. Koh-ichi Nagata conceived and designed the experiment and wrote the manuscript. All authors read and approved the final manuscript.

Additional Information

Nanako Hamada and Tohru Matsuki contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

1.
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