Akira Oka

Expression of the ataxia-telangiectasia gene (ATM) product in human cerebellar neurons during development

Ataxia-telangiectasia (A-T) is a hereditary disorder, exhibiting progressive cerebellar ataxia. We investigated the expression of the ATM protein in the human CNS. By western blotting, the ATM protein was detected in the cerebellar cortex, but not in the cerebral cortex, at the late gestational stage. Immunohistochemistry revealed that cerebellar neurons, particularly Purkinje cells, were markedly immunoreactive during late prenatal and early postnatal periods, followed by persistent and moderate reactivity in Purkinje cells. The ATM protein was distributed within the cytoplasm of Purkinje cells, but not within the nuclei. The ATM protein seems to play a role as a cytoplasmic protein in neurons of the cerebellar cortex.

Compound heterozygous mutations of cytochrome P450 oxidoreductase gene (POR) in two patients with Antley-Bixler syndrome.

Antley–Bixler syndrome (ABS) is characterized by skeletal defects including craniosynostosis and radiohumeral synostosis. Although mutations in the FGFR2 gene have been found in some patients called ABS, genetic heterogeneity of this syndrome has been proposed. We have previously reported three ABS patients with unique abnormalities in steroidogenesis (apparent decreased activity of 17α‐hydroxylase, 17,20‐lyase, and 21‐hydroxylase). Decreased activity of lanosterol 14α‐demethylase has also been described in an ABS patient. Since all these enzymes require cytochrome P450 oxidoreductase (encoded by POR) as an electron donor, we studied POR in two unrelated ABS patients with abnormal steroidogenesis. Direct sequencing of POR revealed that both patients had compound heterozygous mutations (1329insC and R454H in a male patient, 1698insC and R454H in a female patient). The two insertional mutations were assumed to generate truncated and unstable mRNAs. The R454H mutation was assumed to be deleterious because the R454 resides in the FAD‐binding domain and is highly conserved among diverse species. Our results demonstrate that mutations in POR cause the ABS phenotype with autosomal recessive inheritance and with characteristic abnormalities in steroidogenesis.

Developmental expression of monocyte chemoattractant protein-1 in the human cerebellum and brainstem

The developmental expression of monocyte chemoattractant protein-1 (MCP-1) in the cerebellum, medulla oblongata and pons was investigated in 26 normal human brains, ranging from 20 weeks of gestation (GW) to adulthood by means of an immunohistochemical method. Immunoreactivity to MCP-1 was observed in neurons of the cerebellum and brainstem, and was mainly distributed in the cytoplasm and dendrites. MCP-1-positive Purkinje cells appeared at 27 GW, reached a peak at 36 GW, and then decreased after 1 month of age, almost completely disappearing by 1-2 years of age. MCP-1-reactive neurons in the dentate nucleus and inferior olivary nucleus showed temporal increases similar to that of Purkinje cells. In the pons, however, MCP-1 reactivity was low in neurons of the pontine nuclei persisting from the fetal to the adult period, and was very low and short in the reticular formation and cranial nerve nuclei during the fetal and/or neonatal period. MCP-1 Western blotting of the cerebellar cortex confirmed the specificity of the immunohistochemistry. Our results suggest that MCP-1 may be related to the maturation of Purkinje cells, the dentate nucleus, the inferior olivary nucleus, and their network, promoting the growth of dendrites and synapses. Furthermore, MCP-1 may also be useful for the study of abnormal neuron development and ischemic damage.

The up-regulation of metabotropic glutamate receptor 5 (mGluR5) in Down’s syndrome brains

Abstract We investigated the expression of metabotropic glutamate receptor 5 (mGluR5), a subtype of group I mGluRs, in the cerebral cortex of cases with Down’s syndrome (DS), using immunohistochemistry and immunoblotting with this receptor. The up-regulation of mGluR5 was observed in DS by immunohistochemistry, and atrophic pyramidal neurons were immunolabelled in elderly DS cases. Western blotting confirmed the increased expression in DS brains. Since group I mGluR regulates the metabolism of amyloid precursor protein (APP) and accelerates its processing into non-amyloidogenic APP, the overexpression of mGluR5 may be related to the pathological state of APP metabolism in DS.

Fukutin protein is expressed in neurons of the normal developing human brain but is reduced in Fukuyama-type congenital muscular dystrophy brain.

Fukuyama‐type congenital muscular dystrophy (FCMD) results from a mutation in a gene on chromosome 9q31, fukutin, and is characterized pathologically by micropolygyria of the cerebral and cerebellar cortices. To elucidate the physiological function of fukutin as well as its pathological role in FCMD, we raised antisera against fukutin protein and observed its expression in developing human brains with or without FCMD. Western blotting using these antibodies demonstrated a 60‐kd band in the fetal but not in postnatal cerebral cortex of the controls. This band appeared negligible in the brains of FCMD fetuses. Immunohistochemistry revealed the localization of fukutin in Cajal‐Retzius cells, the subpial granular layer, the neuropil of the marginal zone, the cortical plate neurons, and the ventricular neuroepithelium of the fetal cerebrum. In the fetal cerebellum, fukutin immunoreactivity was localized to the external granule cell layer, molecular layer, Purkinje cells, and some internal granular cells. The immunoreactivity in these structures was reduced markedly in postnatal normal brains, as well as in an FCMD cerebrum at 23 gestational weeks. The spatial and temporal pattern of fukutin expression is compatible with its predicted role: the regulation of neuronal migration in the fetal cerebrum and cerebellum. Ann Neurol 2000;47:756–764

A Japanese girl with leukoencephalopathy with vanishing white matter

A Japanese girl with peculiar leukoencephalopathy was reported. Following normal development until 1 year of age, she showed progressive neurological deterioration with ataxia, epilepsy, pyramidal tract signs and choreic movement. Serial brain computed tomographies (CTs) revealed markedly low density and progressive volume loss in whole white matter. In extensive laboratory investigations, the level of glycine in the urine was elevated. She died at the age of 4 years, and the neuropathological findings were comprised of severe extensive changes in cerebral and cerebellar white matter, such as marked rarefaction or cystic degeneration with axonal loss. The pontine central tegmental tracts were also affected. Neuronal loss was seen in the cerebellar cortex. These features were compatible with leukoencephalopathy with vanishing white matter, which was recently established as a clinical entity. To our knowledge, this is the first report of a non-Caucasian patient with this new type of leukoencephalopathy.

Expression of DNA-dependent protein kinase catalytic subunit and Ku80 in developing human brains: implication of DNA-repair in neurogenesis.

We investigated expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku80 in developing human brain, both of which have been suggested to be involved in the repair of DNA double-strand break (DSB). Their expressions were well correlated, and the highest immunoreactivity was observed in post-mitotic immature neurons in the cerebral cortex as well as in progenitors in the periventricular germinal layer. The reactivity gradually decreased during development. Our results support the notion that DSB is generated during the ontogeny of the human central nervous system.

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.

The expression of late infantile neuronal ceroid lipofuscinosis (CLN2) gene product in human brains

We raised polyclonal antibodies against a gene product responsible for late infantile neuronal ceroid lipofuscinosis (CLN2). By Western blotting, all three antisera recognized the CLN2 protein at approximately 49 kDa in human brain homogenates. Immunohistochemistry using the antisera demonstrated the granular labelling in the cytoplasm of cerebral neurons and glial cells. The immunoreactivity on Western blots was absent from the brain of a patient with CLN2. Our results suggest the usefulness of these antibodies for the diagnosis of CLN2, which currently requires demonstration of characteristic ultrastructure by electron microscopy.

Enhanced GAP-43 gene expression in cortical dysplasia.

Growth-associated protein GAP-43, a phosphoprotein enriched at presynaptic nerve terminals, is thought to be involved in axonal outgrowth and plasticity in synaptic connections. To explore the synaptic remodeling under the epileptic conditions, we examined GAP-43 expression in brain specimens surgically resected as epileptogenic foci from 17 patients with cortical dysplasia. In situ hybridization with GAP-43 antisense riboprobe showed significantly increased signals in the dysplastic large neurons of cortical dysplasia. Specific distribution with increased immunoreactivity for GAP-43 was not shown in the dysplastic cortex. These results suggest that GAP-43 gene expression is over-expressed in the dysplastic large neurons, reflecting activated synaptic remodeling in the epileptic condition of cortical dysplasia, although the precise site of accelerated synaptic rearrangement remains unknown.