Shohei Maekawa

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called “raft.” In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (akindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

Cholesterol-dependent Localization of NAP-22 on a Neuronal Membrane Microdomain (Raft)

A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-β-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca2+-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.

Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain

NAP-22 is a membrane-localized brain enriched acidic protein having a Ca(2+)-dependent calmodulin binding activity. Further fractionation of the NAP-22 containing membrane showed the localization of NAP-22 in a Triton insoluble fraction of low density. Besides NAP-22, this fraction was found to contain GAP-43 (neuromodulin), trimeric G proteins, and some GPI-anchored proteins such as Thy-1 and N-CAM-120. Presence of some protein tyrosine kinases, such as src and fyn, was also shown.

Immunohistochemical localization of a novel acidic calmodulin-binding protein, NAP-22, in the rat brain.

NAP-22 is a neuronal tissue-enriched acidic calmodulin-binding protein with a molecular mass of 22,000 and is recovered in the membrane fraction during biochemical fractionation. We observed the distribution pattern of this protein in the rat brain using an immunohistochemical method by light and electron microscopy. NAP-22 immunoreactivity was detected through the whole brain, and the most dense staining was observed in the forebrain including cerebral cortex, hippocampal formation, olfactory bulb, basal ganglia and thalamus. Immunoreactivity was distributed densely at the neuropil, whereas nerve cells and nerve fibres had little or no reaction. In the brain stem, immunonegative large nerve cell bodies were surrounded by immunopositive varicosities. In the cerebellar cortex, mossy fibre terminals and parallel fibres showed immunoreactivity, whereas Purkinje cells did not. Intracellular distribution was observed in the cerebral and cerebellar cortices. NAP-22 immunoreactivity was noted in the axon terminals, dendritic spines and thin nerve fibres. In these structures, reaction products were associated mainly with synaptic vesicles, pre- and postsynaptic membranes and microtubules. This study demonstrates that the immunoreactivity of NAP-22 is distributed widely in the brain, especially in the synapse, and suggests that this protein is involved in synaptic transmission both in the pre- and postsynaptic region.

Biochemical evidence for the presence of NAP-22, a novel acidic calmodulin binding protein, in the synaptic vesicles of rat brain

NAP-22 is a neuronal tissue-enriched acidic protein which is expressed predominantly in rat brain. In the present study, we quantitated the amount of NAP-22 in a highly purified synaptic vesicle fraction. NAP-22 comprised 1.3 +/- 0.15% of the total protein in the fraction, and NAP-22 was located on the external surface of the synaptic vesicle membrane. The results suggest NAP-22 may be involved in the synaptic vesicle cycling.

Localization of 2′,5′-oligoadenylate synthetase and the enhancement of its activity with recombinant interferon-α A/D in the mouse brain

Abstract Although the expression of 2′,5′-oligoadenylate synthetase (2–5AS) is induced by interferon (IFN), low constitutive levels can be detected in animals that have not been treated with IFN. In order to clarify which cells express 2-5AS in the mouse brain, the distribution of this enzyme in the brains of both normal healthy mice and mice treated with recombinant human IFN-α A/D was studied by Western blotting and immunohistochemistry, using a specific monoclonal antibody. On the Western blots, the antibody to 42-kD 2–5AS reacted slightly with extracts from the telencephalon, cerebellum, diencephalon, and medulla oblongata of normal mouse brain. 42-kD 2–5AS was predominantly found in the ependymal cells and epithelium of the choroid plexus, and to a lesser degree in neurons and glial cells. Injection of recombinant human IFN-α A/D into the left lateral ventricle enhanced the activity of the enzyme in the telencephalon, cerebellum, diencephalon, and medulla oblongata, but did not change the immunohistochemical localization of the enzyme. Direct injection of the IFN into the cortex of the telencephalon enhanced the activity of 2–5AS in all parts of the brain and immunoreactivity was observed in the neurons and glial cells surrounding the injection site. These data indicate that 42-kD 2–5AS activity in the mouse brain is enhanced by the injection of recombinant human IFN-α A/D either into the left lateral ventricle or cortex of the telencephalon. Expression of 42-kD 2–5AS in ependymal cells and epithelium of the choroid plexus may prevent viral infections in the brain.

Changes in the localization of NAP-22, a calmodulin binding membrane protein, during the development of neuronal polarity

NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca(2+)-dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.

Immunohistochemical demonstration of a neuronal calmodulin-binding protein, NAP-22, in the rat spinal cord

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a newly isolated calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. To reveal the NAP-22 distribution in vivo, we investigated the spinal cord of the 4-5-week old rats using light and electron microscopy. NAP-22 immunoreactivity was observed in the gray matter with dorsoventral gradient of reactivity. Distinct reactivity was demonstrated in the nerve terminals and dendritic spines. Some reactions were also observed in the thin nerve fibers. NAP-22 immunoreactivity was associated mainly with pre- and postsynaptic membranes, synaptic vesicles and outer mitochondrial membranes. In the nerve terminals, NAP-22 was colocalized with synaptic vesicle proteins such as synapsin I or synaptobrevin 2. About 80% of the nerve terminals having immunoreactivity for synapsin I or synaptobrevin 2 showed NAP-22 immunoreactivity. From these results, NAP-22 is confirmed to be distributed in the synaptic region of the spinal cord and is involved in the synaptic function relating to PKC.

Distribution of immunoreactive 2′,5′-oligoadenylate synthetase in mouse reproductive organs

Although 2′,5′-oligoadenylate synthetase (25AS) is an enzyme induced by inferferon (IFN) or viral infections and mediates one of the principal antiviral pathways turned on by IFN, low constitutive levels of the enzyme can be detected in various “normal” animals that have not been treated with IFN or virus. The distribution of this enzyme in the female and male reproductive organs of normal healthy mice was studied by Western blotting and by an immunohistochemical method, using a specific monoclonal antibody. On Western blotting, an antibody to 42-kD 2-5AS reacted with extracts from the ovary, oviduct, uterus, vagina, and placenta among the female reproductive organs, and testis, epididymis, and ductus deferens in the male. Immunohistochemically, the 2-5AS was localized on the following cells in the female reproductive organs: oocytes in the ovary; epithelium in the oviduct, uterus, and vagina; and trophoblasts in the placenta. Furthermore, the 2-5AS was localized on the epithelium and muscular layer in the ductus deferens and epithelium in the penis of the male mice, whereas the epithelium of the testis, epididymis, and seminal vesicle were stained faintly. It is well known that IFN is produced continuously in normal mice, so the 2-5AS in the tissues of normal mice is considered to be induced by such IFN produced under physiological conditions. Expression of the 2-5AS on the epithelium and trophoblasts in the reproductive organs may be responsible for the prevention of viral infections. However, the enzyme in oocytes may have some functions other than as an antiviral agent, since the enzyme was not detectable in embryos during early development.