A Novel Marker for Purkinje Cells,KIAA0864 Protein. An Analysis Based on a Monoclonal Antibody HFB-16 in Developing Human Cerebellum

Abstract

In the search for immunohistochemical markers of the developing human brain, a monoclonal antibody, HFB-16, was raised against homogenates from the cerebrum of a 15-gestational-week-old (GW) human fetus and screened on paraffin-embedded human embryonic brain specimens. This antibody was particularly useful as a marker for Purkinje cells in the developing human cerebellum. Positive immunoreactivities with HFB-16 first appeared in the Purkinje cell layer at 17 GW. From 20 to 24 GW, positive immunoreactivities were found above the lamina dissecans. After 25 GW, dendrites of Purkinje cells were found with the HFB-16 antibody, and the nerve fibers of the Purkinje cells became positive after 35 GW. Neurons in the dentate nucleus and external and internal granular layers reacted negatively to this antibody. After 1 year, when the external granular layer faded out, the dendrites of the Purkinje cells reached the pial surface of the cerebellum, and nerve fibers began to develop in the white matter. This antibody was also useful for characterization of components in heterotopic neurons found in various anomaly syndromes such as trisomy 13. Expressional cloning indicated the antigen against HFB-16 to be human KIAA0864 protein, which is supposed to be an alternative splicing product of p116Rip, whose function has not yet been elucidated. The antigenicity of the KIAA0864 protein was confirmed using human cDNA of the KIAA0864 protein, a protein expression vector, and an HFB-16 antibody.

Keywords

Purkinje cell monoclonal antibody cerebellar development KIAA0864 protein immunohistochemistry expressional cloning

In developing mouse and human cerebella, the neuronal and glial cells migrate to their final location by two different pathways (Uzman 1960; Fujita et al. 1966; Rakic and Sidman 1970). In the human cerebellum, neuroblasts migrate radially outward from the germinal matrix layer in the wall of the fourth ventricle and settle in the deep cerebellar nuclei and the Purkinje cell layer between 9 and 13 gestational weeks (GW) (Rakic and Sidman 1970; Zecevic and Rakic 1976). The germinal matrix layer disappears at birth. Another pathway begins at 11 GW and 13 GW when the germinative cells migrate tangentially from the germinal matrix layer over the cerebellar surface, forming the external granular layer (EGL). Neuroblasts generated from the EGL begin to migrate inward around 16 GW and form the basket and stellate cells of the molecular layer (ML) and, finally, the internal granular layer (IGL). Between 20 and 32 GW, the lamina dissecans, composed of a dense meshwork of cellular processes and found only in the human cerebellum, separates a cellular layer from the IGL (Larroche et al. 1997). The EGL has its maximum cell number in the first few postnatal months and diminishes in size thereafter, until disappearance 1 year after birth.

Several markers for Purkinje cells such as microtubule-associated protein 2 (MAP2) (Goodlett et al. 1990), calbindin D28k (Wood et al. 1988), inositol 1,4,5-triphosphate type-1 receptor (IP3R1) (Maeda et al. 1990), and spot 35 protein (Takahashi-Iwanaga et al. 1986) have been reported. Among them, monoclonal antibodies against MAP2 and IP3R1 have been used to detect Purkinje cells with immunohistochemistry in formalin-fixed and paraffin-embedded brain samples from normal development (Isumi et al. 1997; Miyata et al. 1999) and diseased brains such as olivopontocerebellar atrophy (Kato et al. 1998), granule cell-type cerebellar degeneration (Kato et al. 1998), and Menke's kinky hair disease (Kato et al. 1993).

We describe here a new marker for Purkinje cells and the immunohistochemical development of Purkinje cells using a monoclonal antibody.

Materials and Methods

Materials

Brain samples for immunohistochemical study were obtained at autopsy from 33 cases ranging from 8 GW to 69 years (1 case each at 8, 9, 14, 15, 17, 18, and 19 GW, 10 cases at 20-29 GW, 7 cases at 30-40 GW, 3 cases at 3-6 mo of age, 1 at 1 year, 1 at 2 years, 1 at 5 years, and 3 at 30-69 years) and 4 cases with trisomy 13 (1 case at 35 GW, 1 case at 37 GW, and 2 cases at 38 GW). Most samples (36 out of 37 cases) were obtained at autopsy within 12 hr after death and placed in liquid nitrogen for Western blot preparations. Ten percent buffered formaldehyde-fixed and paraffin-embedded sections from the brain and other tissues were prepared for immunohistochemistry. Some tissues were fixed in 4% paraformaldehyde. In the cerebellum, transverse sections were obtained from the cerebellar hemisphere and/or vermis. In some cases, tangential and frontal sections were also available. This study was carried out in accordance with the principles of the Helsinki Declaration and was approved by the Ethical Committee of each institution.

Monoclonal Antibodies

For preparation of monoclonal antibodies, homogenates were obtained from the cerebrum of a 15-GW human fetus. Monoclonal antibodies were produced by fusion of FO-1 mouse myeloma cells with inguinal lymph node B-lymphocytes from female BALB/c mice immunized with three subcutaneous injections of 200 μg of homogenate with a Ribi adjuvant system (Orlik and Altaner 1988). About 1 × 10⁸ lymphocytes were obtained from the inguinal lymph nodes of 8-10 mice by rubbing the tissue between two frosted glass slides. The obtained lymphocytes were fused with 2-4 × 10⁷ FO-1 cells by adding 50% polyethylene glycol (Sigma-Aldrich Japan; Tokyo, Japan). HAT (Sigma-Aldrich) medium selection was performed in 5 × 96-well plates with feeder cells (mice thymocytes) and growth factors (Bryclone; Dainippon-pharm, Osaka, Japan). Hybridoma culture supernatants from 480 wells were screened by incubation with paraffin-embedded sections from developing human cerebrums and/or cerebellums at 15 and/or 35 GW. About one fifth of the supernatants were immunoreactive on the paraffin-embedded sections. One clone produced antibodies that reacted well with Purkinje cells. It was named HFB (human fetal brain)-16 after the well number. The hybridoma cells were subcloned three times by limiting dilution and injected into the peritoneal cavity of pristane-pretreated mice. The ascite fluid was obtained ~1 week later. The immunoglobulin subclass was determined using a kit (Amersham Pharmacia Biotech; Uppsala, Sweden) according to the instructions of the manufacturer. The IgG fractions were purified on a protein G-Sepharose column.

For the procedures using animals, we followed the NIH Guide for the Care and Use of Laboratory Animals (1985).

Light-microscopic Immunohistochemical Study

Paraffin-embedded specimens were used for light-microscopic immunohistochemistry. Light-microscopic immunohistochemical staining was performed according to previously reported methods colored with 3-amino-9-ethylcarbazole (AEC) as the chromophore (Nakamura et al. 1998). Pretreatment with proteinase or microwave exposure in 10 mM citrate buffer at pH 6.0 was applied in some cases. Dilution of the primary antibodies was 1:1000. For the controls, the primary antibodies were omitted or primary antibodies were absorbed with excess homogenate, or normal serum was used. Hematoxylin and eosin (HE) staining was also performed.

Western Blotting

Tissues stored in liquid nitrogen were pulverized, washed with PBS, homogenized with lysis buffer, and immunoprecipitated using a kit (Boehringer; Ingelheim, Ingelheim, Germany) according to the manufacturer's instructions, using HFB-16. Both non-immunoprecipitated and immunoprecipitated proteins were separated by electrophoresis on an SDS-polyacrylamide gel (5-20%), transferred to a nitrocellulose membrane, and blotted with HFB-16 antibody. Immunopositive bands were detected with an immunoperoxidase Western blotting kit (Amersham).

Expressional Cloning

cDNA libraries from whole fetal human brain male/female pools (19-23 GW) were purchased from Stratagene (catalog #937227; La Jolla, CA). Using appropriately prepared host bacteria, XL1-Blue MRF strains, titering of lambda phages was done according to the instructions of the manufacturer. Tubes containing titered phages and host bacteria were incubated for 15 min at 37C after which 5 ml LB top agarose was added. The plating culture was poured onto LB agar plates that were inverted and incubated overnight at 42C. Isopropyl thiogalactose (IPTG)-saturated filters were placed on the top of each plate and incubated at 37C for 3-4 hr. The filters were removed, rinsed, and placed in block solution for 1 hr, then in the HFB-16 1:1000 solution for 1 hr. They were then rinsed and placed in the secondary antibody (anti-mouse Ig; DAKO, Carpinteria, CA) with peroxidase conjugate 1:1000 solution for 1 hr, rinsed, and finally color developed with 3,3′-diaminobenzidine. Positive plaques with HFB-16 antibodies were selected and subcloned three times. Amplified cDNA by PCR with T1 and T3 primers was subcloned into pT7BlueTM T-vectors (Novagen; Darmstadt, Germany), transfected into Escherichia coli, and sequenced.

Protein Expression

Human full-length KIAA0864 cDNA was purchased from OriGene (Rockville, MD).

Subcloning was done with the plasmid vector pET16b, restricted enzymes Nde I/ Bam H1, cDNA of KIAA0864, and E. coli DH10B by TAKARA BIO dragon genomics center (Yokkaichi, Japan). Expressed protein induced by IPTG was screened with HFB-16 antibody by Dot blot and Western blot.

Results

Monoclonal Antibody HFB-16

Antibody HFB-16 belongs to the IgG1, κ subclass of immunoglobulins. HFB-16 revealed strong immunoreactivity in paraffin sections as well as fresh frozen sections. This antibody reacts with human, mouse, and rat materials (data not shown) both in buffered formaldehyde and 4% paraformaldehyde.

Light Microscopic Immunohistochemistry

Normal Developing Cerebellum. In the 8- to 15-GW cerebellum, there was negative immunoreactivity with HFB-16 (data not shown). In all 29 cases after 17 GW, optimal staining was obtained. In the 17- to 18-GW cerebellum, positive immunostaining appeared in three to five layers as short processes of Purkinje cells (Figures 1A and 1B). At 20-24 GW, a layer of cells, the so-called lamina dissecans, emerged below the Purkinje cell layer, separated from the internal granular layer. Positive immunoreactivity with HFB-16 was observed in the soma of the developing Purkinje cells and some short processes extending into the ML and in the lamina dissecans layer (Figures 1C and 1D). After 25 GW, HFB-16 recognized the soma as well as dendrites of the Purkinje that are prominent as a single layer. HFB-16 immunoreactive dendrites appeared to elongate into the ML and at 34-35 GW they were fully developed just under but never inside the EGL (Figure 2). Immunoreactivity with HFB-16 was also found along the nerve fibers in the IGL and in the white matter after 35 GW but not in the EGL or dentate nucleus neurons (Figure 2 and data not shown). After birth (up to 1 year), the distribution of HFB-16 immunoreactivity remained in fully developed dendrites, and the immunoreactivity along the nerve fibers appeared to be intensified. At about 1 year of age, the EGL disappeared. Strongly immunoreactive fully developed dendritic arborizations with spiny branches of the Purkinje cells reached the pial surface of the cerebellum as nerve fibers, also intensely stained with HFB-16, and were developed in the white matter (Figures 3A-3C and data not shown). In the adult cerebellum, HFB-16 marked immunoreactivity was found along the mature dendrites of the Purkinje cells in the ML and along nerve fibers in the white matter (Figure 3D).

Figure 1

Histology and immunohistochemical distribution of the KIAA0864 protein in a 17-GW (A,B) and a 23-GW (C,D) cerebellum. (A) External granular layer (EGL), thin molecular layer (ML), incomplete Purkinje layer (Pur), and migrated neuroblasts (Neu) are indicated. The internal granular layer was not yet detected. Hematoxylin and eosin (HE) staining. (B) Positive immunostaining with HFB-16 appeared in three to five layers with short processes of Purkinje cells. (C) The lamina dissecans layer (LD) and internal granular layer (IGL) became prominent. HE staining. (D) Positive staining with HFB-16 was observed in the soma of developing Purkinje cells' soma as well as in the short processes in the ML and the LD. Transverse section, without counterstaining. Bars = 20 μm.

Neuronal Heterotopia

Neuronal heterotopia was found multifocally in the white matter of all four cases with trisomy 13. It was composed of a mixture of large Purkinje cell-type neurons and small granular neurons of various proportions. HFB-16 immunoreactivity was confined to Purkinje cell-type neurons (Figure 4).

Cerebrum, Brain Stem, and Other Tissues

In the developing cerebrum, positive immunoreactivity with HFB-16 was found diffusely in areas including the radial fibers at 8-14 GW with the exception of the cortical plate. After 15 GW, HFB-16-positive areas remained in the ependymal and subependymal layers and then diffused out.

In the brain stem, nerve fibers were positive for HFB-16 from an early stage (8 GW).

Positivity for HFB-16 was specific for the central and peripheral nervous system, including the nasal epithelium, the retina, and the choroid plexus. Other main tissues were negative or only weakly positive for HFB-16.

No positive immunoreactivity was found by omitting primary antibodies or antibodies preabsorbed with excess homogenate or normal sera.

Figure 2

Histology and immunohistochemical distribution of KIAA0864 protein in a 35-GW cerebellum. (A) EGL, ML, a single prominent Purkinje cell layer, and IGL were clearly distinguished. HE staining. (B) Intense positive immunoreactivity with HFB-16 was found in the Purkinje cells' soma and dendrites that were elongated into the ML and just under but never inside the EGL (arrows). The axons of the Purkinje cells were also positive for the immunostaining (arrowheads). (C) Higher magnification of the immunopositive Purkinje cells. Transverse section, without counterstaining. Bars = 20 μm.

Western Blotting

The Western blot with HFB-16 antibody showed double bands at 200 kD and 140 kD in a non-immunoprecipitated extract of a 35-GW cerebellum (Figure 5, Lane 1), whereas a single band was shown at 200 kD in immunoprecipitated extracts of a 35-GW cerebellum and KIAA0864 bacterial fusion protein (Figure 5, Lanes 2 and 3). There were no positive bands in other tissue samples such as lung (Figure 5, Lane 4), kidney, heart, or liver. The expected size of the KIAA0864 protein was ~180-200 kD, and a positive band was found at the same level in KIAA0864 bacterial fusion protein; therefore, the band at 200 kD was considered to be specific for the HFB-16 corresponding antigen.

Expressional Cloning

Two positive plaques for HFB-16 were obtained by expression cloning using cDNA libraries from human fetal brains and 500-700 bp of each entrapped cDNA were sequenced. The cDNA sequence was searched for using the BLASTN 2.0.11 program and a homology search was performed. Both cDNAs for HFB-16 were identified (96% and 98%) as Homo sapiens KIAA0864. It may thus be concluded that antibodies HFB-16 recognize the KIAA0864 protein.

Discussion

KIAA0864 protein is a putative protein of a cDNA from 100 cDNA clones newly determined from a set of size-fractionated human brain cDNA libraries and their coding potentials of large proteins (180-200 kD) by using in vitro transcription/translation assays (Nagase et al. 1998). The human genome for the KIAA0864 protein is located on chromosome 17. A homolog genome is present on the mouse chromosome 11. From the mouse genomic composition, the KIAA0864 protein is thought to be an alternative splicing product of the p116Rip genome (Figure 6). The mouse genome for p116Rip is composed of exon 1-exon 23. The genome for KIAA0864 protein is composed of partial exon 14-exon 22 and exon beta. The sequence of cDNA obtained by expressional cloning was almost completely matched to the sequence of KIAA0864 protein and different from that of p116Rip. It suggests that HFB antibody does not recognize p116Rip, but does recognize the KIAA0864. p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA. It is indicated that p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth (Gebbink et al. 1997). Recently, p116Rip was reported as an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton (Mulder et al. 2003). Although the function of the KIAA0864 protein was originally categorized as a nucleic acid management protein (Gebbink et al. 1997), detailed functions of the KIAA0864 protein itself have not been elucidated. Contrary to the ubiquitous presence of p116Rip from this and other studies (Gebbink et al. 1997), KIAA0864 protein is relatively restricted in the nervous tissue.

Figure 3

Histology and immunohistochemical distribution of the KIAA0864 protein in a 1-year-old (A-C) and a 69-year-old (D) cerebellum. (A) The EGL had disappeared. HE staining. (B) Intense immunoreactivity with HFB-16 was found in the Purkinje cells' soma and their fully developed dendrites with spiny branches reaching the pial surface of the cerebellum (arrow) and nerve fibers developing in the white matter. (C) Higher magnification of the immunopositive Purkinje cells. (D) Intense immunoreactivity with HFB-16 was found in the Purkinje cells' soma and their fully developed dendrites and nerve fibers in IGL and in white matter (WM). Tangential section, without counterstaining. Bars = 20 μm.

KIAA0864 protein is the first protein reported to be expressed in growing Purkinje cells and their dendrites and nerve fibers as well as in neuronal components of the cerebrum and brain stem on formalin-fixed, paraffin-embedded human brain samples. Among several markers for Purkinje cell, MAP2 and IP3R1 have been applied for immunohistochemical study of Purkinje cells in normal and abnormal development on formalin-fixed, paraffin-embedded human brain samples (Isumi et al. 1997; Miyata et al. 1999). An immunohistochemical study using MAP2 antibodies showed that there were regional differences in the number of Purkinje cells during cerebellar development but did not describe details of Purkinje cell development (Isumi et al. 1997). Another study using IP3R1 immunohistochemistry described details of Purkinje cell development in normal and pathological conditions (Miyata et al. 1999). At 33-35 GW, positive HFB-16 immunoreactivity intensifies and is found in fully developed dendrites but never in the EGL. At about 1 year when the EGL has disappeared, immunoreactive dendrites of Purkinje cells reach the pial surface, and nerve fibers from Purkinje cells are widely distributed in the white matter.

Figure 4

Histology and immunohistochemical distribution of the KIAA0864 protein in neuronal heterotopia found in a 38-GW infant with trisomy 13. (A) Neural heterotopia was found in the subcortical white matter (arrowheads). (B) Neural heterotopia was composed of a mixture of large Purkinje cell-type neurons and small granular neurons. HE staining. (C) Positive immunoreactivity with HFB-16 was seen only in the large Purkinje cell-type neurons. Transverse section, without counterstaining. Bars = 20 μm.

Figure 5

Western blotting analysis with HFB-16 antibodies in the non-immunoprecipitated human fetal cerebellum (Lane 1) and immunoprecipitated human fetal cerebellum (Lane 2), lung (Lane 4), and KIAA0864 bacterial fusion protein extracts (Lane 3). A band at 200 kD was present both in non-immunoprecipitated and immunoprecipitated samples. An extra band at 140 kD was found only in the non-immunoprecipitated human fetal cerebellum. Cerebellum from human fetus of 35 GW.

Figure 6

Diagrams of human and mouse genome p116Rip genes and the mouse putative KIAA0864 protein genome gene.

Neuronal heterotopia in the cerebellum is seen in anomalous brains, particularly those with trisomy 13, as well as in normal brains (Norman 1966; Rorke et al. 1968). There are two forms of neuronal heterotopia in the cerebellum: clusters of large cells reminiscent of Purkinje or dentate neurons surrounded by a thin corona of a neuropil, and islands of heterotopic cortex, with individual layers being more or less well organized (Harding and Copp 1997). HFB-16 immunoreactivity was seen only in Purkinje neurons. Therefore, the antibody HFB-16 is a very useful tool for tracing Purkinje cells, dendrites, and nerve fibers on formalin-fixed, paraffin-embedded normal or malformed human cerebellar samples. This study suggests that the KIAA0864 protein may regulate neurite outgrowth, particularly in the Purkinje cells. Further studies using transgenic and/or knockout mice and rescue techniques are necessary.

Footnotes

Acknowledgments

This study was supported by Health Sciences Research Grants for Specific Diseases “Intractable Hydrocephalus” (1999-SD-17) from the Ministry of Health and Welfare, Japan.

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