A Novel Marker for Purkinje Cells, KIAA0864 Protein. An Analysis Based on a Monoclonal Antibody HFB-16 in Developing Human Cerebellum
Department of Pathology, St. Mary's Hospital, Kurume, Japan (YN,RF,KH); Department of Chemistry, Kurume University School of Medicine, Kurume, Japan (MY,EO); Tissue Engineering Research Center, National Institute of Advanced Industrial Science and Technology, Amagasaki, Hyogo, Japan (YK,EK,AY,HY); Institute for Clinical Research (YK,MY), and Department of Neurosurgery (MY), Osaka National Hospital, Osaka, Japan; Department of Reproductive Biology and Pathology, National Center for Child Health and Development, Tokyo, Japan (KM); Department of Physiology, Keio University School of Medicine, Tokyo, Japan (HJO,HO); and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan (HJO,HO)
Correspondence to: Yasuhiro Nakamura, MD, Department of Pathology, St. Mary's Hospital, 422, Tsubukuhon-machi, Kurume-shi, Japan 830-8543. E-mail: naka{at}st-mary-med.or.jp
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Summary |
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Key Words: Purkinje cell monoclonal antibody cerebellar development KIAA0864 protein immunohistochemistry expressional cloning
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Introduction |
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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.
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Materials and Methods |
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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 x 108 lymphocytes were obtained from the inguinal lymph nodes of 810 mice by rubbing the tissue between two frosted glass slides. The obtained lymphocytes were fused with 24 x 107 FO-1 cells by adding 50% polyethylene glycol (Sigma-Aldrich Japan; Tokyo, Japan). HAT (Sigma-Aldrich) medium selection was performed in 5 x 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 (520%), 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 (1923 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 34 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.
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Results |
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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 2024 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 3435 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 3A3C 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).
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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.
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 180200 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.
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Discussion |
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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.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Fujita S, Shimada M, Nakamura T (1966) 3H-thymidine autoradiographic studies on the cell proliferation and differentiation in the external and the internal granular layers of the mouse cerebellum. J Comp Neurol 128:191208[CrossRef][Medline]
Gebbink MF, Kranenburg O, Poland M, van Horck FP, Houssa B, Moolenaar WH (1997) Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology. J Cell Biol 137:16031613
Goodlett CR, Hamre KM, West JR (1990) Regional differences in the timing of dendritic outgrowth of Purkinje cells in the vermal cerebellum demonstrated by MAP2 immunocytochemistry. Dev Brain Res 53:131134[Medline]
Harding B, Copp AJ (1997) Malformations. In Graham DI, Lantos PL, eds. Greenfield's Neuropathology. London, Arnold, 479480
Isumi H, Mizuguchi M, Takashima S (1997) Differential development of the human cerebellar vermis: immunohistochemical and morphometrical evaluation. Brain Dev 19:254257[CrossRef][Medline]
Kato S, Ito M, Ohama E, Mikoshiba K, Maeda N, Yen S-H, Hirano A, et al. (1993) Immunohistochemical studies on cerebellar Purkinje cells of patients with Menke's kinky hair disease. Neuropathology 13:159166
Kato S, Hayashi H, Mikoshiba K, Hirano A, Yen S-H, Ohama E (1998) Purkinje cells in olivocerebellar atrophy and granular cell-type cerebellar degeneration: an immunohistochemical study. Acta Neuropathol 96:6774[CrossRef][Medline]
Larroche J-C, Encha-Razavi F, de Vries L (1997) Development of cerebellum. In Gilbert-Barness E, ed. Potter's Pathology of the Fetus and Infant. St Louis, Mosby, 10401043
Maeda N, Niinobe M, Mikoshiba K (1990) A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex. EMBO J 9:6167[Abstract]
Miyata M, Miyata H, Mikoshiba K, Ohama E (1999) Development of Purkinje cells in humans: an immunohistochemical study using a monoclonal antibody against the inositol 1,4,5-triphosphate type 1 receptor (IP3R1). Acta Neuropathol 98:226232[CrossRef][Medline]
Mulder J, Poland M, Gebbink MF, Calafat J, Moolenaar WH, Kranenburg O (2003) p116Rip is a novel F-actin-binding protein. J Biol Chem 278:2721627223
Nagase T, Ishikawa K, Suyama M, Kikuno R, Hirosawa M, Miyajima N, Tanaka A, et al. (1998) Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res 31:355364
Nakamura Y, Yamamoto M, Kumamaru E (1998) A variant very low density lipoprotein receptor lacking 84 base pairs of O-linked sugar domain in the human brain myelin. Brain Res 793: 4753[CrossRef][Medline]
NIH Guide for the Care and Use of Laboratory Animals (1985) NIH Publication No. 8523, revised
Norman RM (1966) Neuropathological findings in trisomies 1315 and 1718 with special reference to the cerebellum. Dev Med Child Neurol 8:170177[Medline]
Orlik O, Altaner C (1988) Modifications of hybridoma technology which improve the yield of monoclonal antibody producing cells. J Immunol Methods 115:5559[CrossRef][Medline]
Rakic P, Sidman RL (1970) Histogenesis of cortical layers in human cerebellum, particularly the lamina dessicans. J Comp Neurol 139:473500[CrossRef][Medline]
Rorke LB, Fogelson MH, Riggs H (1968) Cerebellar heterotopia in infancy. Dev Med Child Neurol 10:644650[Medline]
Takahashi-Iwanaga H, Kondo H, Yamakuni T, Takahashi Y (1986) An immunohistochemical study on the ontogeny of cells immunoreactive for spot 35 protein, a novel Purkinje cell-specific protein, in the rat cerebellum. Brain Res 394:225231[Medline]
Uzman LL (1960) The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J Comp Neurol 114:137159[CrossRef][Medline]
Wood TL, Kobayashi Y, Frantz G, Varghese S, Christakos S, Tobin AJ (1988) Molecular cloning of mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K): expression of calbindin-D28K RNAs in rodent brain and kidney. DNA 7:585593[Medline]
Zecevic N, Rakic P (1976) Differentiation of Purkinje cells and their relationship to other components of developing cerebellar cortex in man. J Comp Neurol 167:2747[CrossRef][Medline]