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To determine the epidemiological, clinical, and genetic characteristics of congenital hypomyelinating leukodystrophies, including Pelizaeus-Merzbacher disease (PMD), we conducted a nationwide epidemiological survey in Japan. A two-step survey targeting all medical institutions specializing in pediatric neurology and childhood disability (919 institutes) in Japan was performed. Detailed information was collected for 101 patients (86 males and 15 females) with congenital hypomyelinating leukodystrophies. The prevalence of congenital hypomyelinating disorders was 0.78 per 100,000 people (0-19 years old), and the incidence was 1.40 per 100,000 live births. Molecular testing was performed in 75 % of patients, and PLP1 gene abnormalities were observed in 62 %. The incidence of PMD with PLP1 mutations was estimated to be 1.45 per 100,000 male live births and that for congenital hypomyelinating disorders with unknown cause to be 0.41 per 100,000 live births. Patients with PLP1 mutations showed a higher proportion of nystagmus and hypotonia, both of which tend to disappear over time. Our results constitute the first nationwide survey of congenital hypomyelinating disorders, and provide the epidemiological, clinical, and genetic landscapes of these disorders.

We report an autopsy case of rare adult-onset spastic paraplegia type 2 (SPG2) with a novel missense mutation in exon 7 of the proteolipid protein 1 gene (PLP1). The patient was a 67-year-old man whose elder brother had died of a similar disease with onset in his 40s. Thirty-three years before death at the age of 35, he noticed difficulty in walking. He gradually became abasic over a period of 6 years. He also developed progressive dementia and eventually became bed-ridden by 28 years after onset. At autopsy, gross inspection revealed diffuse, moderate atrophy of the cerebrum with a dilated ventricular system and softening of the white matter throughout the central nervous system (CNS). Histopathologically, the CNS showed widespread myelin pallor in the white matter. By contrast, the gray matter and peripheral nerves were well preserved. Some white matter tracts, including the corticospinal tracts, were preferentially affected, and severe axonal degeneration was observed in these tracts. Genetic analysis revealed a novel mutation, p.Tyr263Cys, in exon 7 of PLP1. This case represents an adult-onset SPG2 patient with one of the oldest ages of onset reported to date. The late onset and long clinical course suggest that this novel mutation does not affect the maturation of oligodendrocytes, but is related to insufficient maintenance of myelin.

Basophilic inclusions (BIs), which are characterized by their staining properties of being weakly argyrophilic, reactive with Nissl staining, and immunohistochemically negative for tau and transactive response (TAR) DNA-binding protein 43 (TDP-43), have been identified in patients with juvenile-onset amyotrophic lateral sclerosis (ALS) and adult-onset atypical ALS with ophthalmoplegia, autonomic dysfunction, cerebellar ataxia, or a frontal lobe syndrome. Mutations in the fused in sarcoma gene (FUS) have been reported in cases of familial and sporadic ALS, and FUS immunoreactivity has been demonstrated in basophilic inclusion body disease (BIBD), neuronal intermediate filament inclusion disease (NIFID), and atypical frontotemporal lobar degeneration with ubiquitin-positive and tau-negative inclusions (aFTLD-U). In the present study, we immunohistochemically and ultrastructurally studied an autopsy case of sporadic adult-onset ALS with numerous BIs. The patient presented with the classical clinical course of ALS since 75 years of age and died at age 79. Postmortem examination revealed that both Betz cells in the motor cortex and motor neurons in the spinal cord were affected. The substantia nigra was spared. Notably, BIs were frequently observed in the motor neurons of the anterior horns, the inferior olivary nuclei, and the basal nuclei of Meynert. BIs were immunopositive for p62, LC3, and FUS, but immunonegative for tau, TDP-43, and neurofilament. Ultrastructurally, BIs consisted of filamentous or granular structures associated with degenerated organelles with no limiting membrane. There were no Bunina bodies, skein-like inclusions, or Lewy-like inclusions. All exons and exon/intron boundaries of the FUS gene were sequenced but no mutations were identified.

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA-type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1-4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long-term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T-maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.

Mutations in the fused in sarcoma gene (FUS) were recently found in patients with familial amyotrophic lateral sclerosis (ALS). The present study aimed to clarify unique features of familial ALS caused by FUS mutation in the Japanese population. We carried out clinical, neuropathological, and genetic studies on a large Japanese pedigree with familial ALS. In six successive generations of this family, 16 individuals of both sexes were affected by progressive muscle atrophy and weakness, indicating an autosomal dominant trait. Neurological examination of six patients revealed an age at onset of 48.2 ± 8.1 years in fourth generation patients, while it was 31 and 20 years in fifth and sixth generation patients, respectively. Motor paralysis progressed rapidly in these patients, culminating in respiratory failure within 1 year. The missense mutation c.1561 C>T (p.R521C) was found in exon 15 of FUS in the four patients examined. Neuropathological study of one autopsied case with the FUS mutation revealed multiple system degeneration in addition to upper and lower motor neuron involvement: the globus pallidus, thalamus, substantia nigra, cerebellum, inferior olivary nucleus, solitary nucleus, intermediolateral horn, Clarke’s column, Onuf’s nucleus, central tegmental tract, medial lemniscus, medial longitudinal fasciculus, superior cerebellar peduncle, posterior column, and spinocerebellar tract were all degenerated. Argyrophilic and basophilic neuronal or glial cytoplasmic inclusions immunoreactive for FUS, GRP78/BiP, p62, and ubiquitin were detected in affected lesions. The FUS R521C mutation in this Japanese family caused familial ALS with pathological features of multiple system degeneration and neuronal basophilic inclusions.

We studied a four-generation pedigree of a Japanese family with hereditary neuropathy to elucidate the genetic basis of this disease. Twelve members of the family were enrolled in this study. The clinical features were neurogenic muscle weakness with proximal dominancy in the lower extremities, sensory involvement, areflexia, fine postural tremors, painful muscle cramps, elevated creatine kinase levels, recurrent paroxysmal dry cough, and neurogenic bladder. We performed a genome-wide search using genetic loci spaced at about 13 Mb intervals. Although nine chromosomes (1, 3, 4, 5, 6, 10, 17, 19, and 22) had at least one region in which the logarithm of odds (LOD) score was over 1.0, no loci fulfilled the criteria for significant evidence of linkage. Moreover, we analyzed an extra 14 markers on 3p12-q13 (the locus of hereditary motor and sensory neuropathy, proximal dominant form) and an extra five markers on 3p22-p24 (the locus of hereditary sensory neuropathy with chronic cough) and observed LOD scores of

We have previously mapped autosomal dominant spinocerebellar ataxia (SCA) 16 to 3p26, overlapping with the locus of SCA15. Recently, partial deletions of ITPR1 and the neighbouring SUMF1 in the SCA15 and two additional families were reported. In the present study we determined the copy number of these genes by real time quantitative polymerase chain reaction (PCR) and found a heterozygous deletion of exons 1-48 of ITPR1, but not SUMF1 in SCA16. Breakpoint analysis revealed that the size of the deletion is 313,318 bp and the telomeric breakpoint is located in the middle of their intergenic region. Our data provide evidence that haploinsufficiency of ITPR1 alone causes SCA16 and SCA15.

Purpose We retrospectively evaluated the imaging spectrum of Pelizaeus–Merzbacher disease (PMD) in correlation with the clinical course and genetic abnormality. Methods We collected the magnetic resonance imaging (MRI) findings of 19 genetically proven PMD patients (all males, aged 0–29 years old) using our integrated web-based MRI data collection system from 14 hospitals. The patterns of hypomyelination were determined mainly by the signals of the cerebrum, corticospinal tract, and brainstem on T2-weighted images (T2WI). We assessed the degree of myelination age on T1-weighted images (T1WI) and T2WI independently, and we evaluated cerebellar and callosal atrophy. The clinical severity and genetic abnormalities (causal mutations of the proteolipid protein gene PLP1) were analyzed together with the imaging findings. Results The clinical stage tended to be more severe when the whole brainstem, or corticospinal tract in the internal capsule showed abnormally high intensity on T2WI. Diffuse T2-high signal of brainstem was observed only in the patients with PLP1 point mutation. Myelination age “before birth” on T1WI is a second manifestation correlated with the clinically severe phenotypes. On the other hand, eight patients whose myelination ages were > 4 months on T1WI were associated with mild clinical phenotypes. Four of them showed almost complete myelination on T1WI with a discrepancy in myelination age between T1WI and T2WI. A random and patchy pattern of myelination on T2WI was noted in one patient with PLP1 point mutation. Advanced myelination was observed in three of the seven followed-up patients. Four patients had atrophy of the cerebellum, and 17 patients had atrophy of the corpus callosum. Conclusion Our multicenter study has demonstrated a wide variety of imaging findings of PMD. Signal intensity of brainstem and corticospinal tract of internal capsule would be the points to presume clinical severity in PMD patients. The spectrum of MRI findings should be kept in mind to diagnose PMD and to differentiate from other demyelinating leukodystrophies.

Objective: To identify of the gene responsible for the onset of spinocerebellar ataxia type 16 (SCA16). Methods: We reanalyzed the linkage of the original Japanese pedigree using updated information, including three additional subjects. We then screened all exons located in the critical region. Results: We reassigned the locus of SCA16 to 3p26.2-pter (maximum logarithm-of-odds score = 5.177) and identified only one point mutation (4,256C→T) in the 3′ untranslated region of the contactin 4 gene (CNTN4) on chromosome 3p26.2-26.3, which cosegregated with the disease. This mutation was not detected in 520 control subjects; moreover, we revised the phenotype of SCA16 from pure to complicated SCA. Conclusion: The contactin 4 gene (CNTN4) is associated with cerebellar degeneration in spinocerebellar ataxia type 16. Additional studies are necessary to prove 4,256C→T to be a causative mutation.

alphaB-Crystallin, a member of the small heat shock protein (sHSP) family, is expressed in various tissues including lens, heart, and skeletal muscle. Previously we identified the gene of HSPB2, another member of the sHSP family, located 1-kb upstream of the alphaB-crystallin gene in a head-to-head manner. In the present study, we found a highly conserved region of 220 bp approximately 2.4-kb upstream of the alphaB-crystallin gene and examined its role in expression of the alphaB-crystallin gene. Transgenic mice containing 3 kb of the upstream sequence of the alphaB-crystallin gene showed lacZ reporter gene expression in the lens as well as the myotome and heart on embryonic day 12.5. Deletion analysis revealed that the -2656/-2267 region including the conserved region with four putative Sox binding elements (E1-E4) exhibits lens enhancer activity toward the alphaB-crystallin promoter. Gel shift assays showed that the Sox1 and Sox2 proteins preferentially bound to E2 and E4. Moreover, disruption of E2 and E4 abolished the reporter gene expression in the lens. These results indicate that the newly identified enhancer with Sox elements activates the alphaB-crystallin promoter in the lens, although they are separated by the entire HSPB2 gene.

An autopsied case of autosomal recessive hereditary spastic paraplegia with severe neurogenic muscular atrophy is described herein. This patient, a 16-year-old woman, presented with gait disturbance. She developed progressive spastic paralysis of the upper and lower limbs and mental deterioration. She became bedridden at approximately 40years of age. Dysarthria worsened at 45 years of age. She died of pneumonia at 50 years of age. Her younger sister has shown similar clinical symptoms and became bedridden at 37 years of age. Their parents were second cousins. Autopsy revealed a severely atrophic brain, weighing 720 g. The cerebral cortex was thin, and the white matter was extremely reduced in volume. Microscopically, neuronal loss and variable astrogliosis with diffuse spongy changes were evident at the cerebral cortex, thalamic nuclei, basal ganglia and hippocampus. The remaining neurons were atrophied with heavy deposition of lipofuscin. In the spinal cord, the pyramidal tracts as well as the dorsal spinocerebellar tracts were degenerated. In addition, marked loss of the anterior horn cells was seen. Severe neuronal loss of the nucleus gracilis was also detected. In contrast, only mild degeneration of the ventral spinocerebellar tracts and fasciulus cuneatus in the spinal cord were observed. In the frozen sections of skeletal muscle, severe neurogenic atrophy and fatty infiltration were evident. In addition, several rimmed vacuoles were observed in the atrophic fibers, and cytochrome coxidase-deficient fibers were present in part. Reduced nicotinamide adenine dinucleotide (NADH)-tetrazolium reductase reaction revealed abnormal accumulation of mitochondria around the center of the non-atrophic muscle fibers. It is suggested that an analysis of mitochondrial function of Japanese autosomal recessive hereditary spastic hemiplegia may provide additional information to clarify the pathogenesis.

Mutations in the fused in sarcoma (FUS, also known as translated in liposarcoma) gene have been recently discovered to be associated with familial amyotrophic lateral sclerosis (FALS) in African, European and American populations. In a Japanese family with FALS, we found the R521C FUS mutation, which has been reported to be found in various ethnic backgrounds. The family history revealed 23 patients with FALS among 46 family members, suggesting a 100% penetrance rate. They developed muscle weakness at an average age of 35.3 years, followed by dysarthria, dysphagia, spasticity and muscle atrophy. The average age of death was 37.2 years. Neuropathological examination of the index case revealed remarkable atrophy of the brainstem tegmentum characterized by cytoplasmic basophilic inclusion bodies in the neurons of the brainstem. We screened 40 FALS families in Japan and found 4 mutations (S513P, K510E, R514S, H517P) in exon 14 and 15 of FUS. Even in Asian races, FALS with FUS mutations may have the common characteristics of early onset, rapid progress and high penetrance rate, although in patients with the S513P mutation it was late-onset. Degeneration in multiple systems and cytoplasmic basophilic inclusion bodies were found in the autopsied cases.

We report the autopsy cases of two brothers which are pathologically compatible with Pelizaeus-Merzbacher disease (PMD). Both patients had a late onset (at the ages of 29 and 42 years) and chronic neurological symptoms including tremor, ataxia and dementia. The T2-weighted magnetic resonance imaging of the younger brother demonstrated increased signal areas with sparing of small areas in the cerebral white matter. The postmortem examinations, obtained at the ages of 45 and 61 years, showed similar neuropathological findings. Histologically, a cardinal finding was a lack of myelin in large parts of white matter with the preservation of islands of intact myelin, resulting in a "tigroid" appearance. Only small amounts of sudanophilic material were present. The axons were relatively well preserved, but oligodendrocytes were numerically reduced. Ultrastructurally, myelin sheaths in the white matter were markedly thin. Immunohistochemistry showed that proteolipid protein (PLP) was reduced in the affected white matter. However, genetic studies did not reveal exonic mutations or duplications of the PLP gene. We conclude that the two cases are a rare type of dysmyelinating disorder with PMD phenotype of adult onset and could be caused by previously unrecognized abnormalities of the PLP gene or other genes.

Dopamine D4 receptor (DRD4) has received attention in terms of pathogenesis of schizophrenia and association with human personalities. We isolated the human DRD4 gene containing the 5'-flanking region and determined its sequence. Analysis of the DRD4 transcripts by 5'-RACE (5'-rapid amplification of cDNA ends) revealed a region of the transcription initiation located between -501 and -436 relative to the first nucleotide of the initiation codon. There is a CpG island spanning from -900 to +500 but no TATA or CAAT boxes in the 5'-flanking region. Functional analysis of the 5'-flanking region of the DRD4 gene by a transient expression method revealed the presence of a negative modulator between -770 and -679. The region between -591 and -123 gave the highest transcriptional activity in IMR32 (neuroblastoma) and Y-79 (retinoblastoma) cells but not in HeLa cells, suggesting that this housekeeping gene-like promoter regulates the cell-type specific gene expression.

粉末X線回折とフーリエ変換赤外分光分析(FT-IR)を用いて研究した.粘度は保存期間とともに増加した.一方,糊化前にGu-HC1を添加しても粘度は保存期間とともに増加し,添加量が多いほど粘度は低かった.膨潤した粒子の相互作用により,それら粒子は水素結合により網目構造を形成する.デソプンの粘度の増加率の長期的な増加は可逆的な結晶化と,貯蔵中の粒子間内の分子間水素結合と関係する.そこでこれらの老化した糊液のX線回折を測定すると,B型の結晶構造の回折線が観測された.また糊化直後と老化後の糊液のFT-IRを測定すると,水素結合を示す3000-3800cm-1の波数でピークの相違を観測し,五つのピークがこの波数内で重なり合っていた.これらの結果から老化の際に,Gu-HC1がワキシコーンスターチの分子構造に与える変化を提案し,模式図として示した.

Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3′ region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3′ UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca2+ and Mg2+ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

The phenotypic effects of selectively altering the levels of alpha B-crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B-crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization.

Congenital cerebral hypomyelination includes a group of genetic disorders, such as Pelizaeus-Merzbacher disease (PMD), and is characterized by hypomyelination of the cerebral white matter. Until recently, no classification system was available for congenital hypomyelination disorders that are clinically and genetically excluded for PMD. However, the establishment of new disease entities with gene discoveries has generated a clinical need for a new classification and diagnostic criteria for this group of disorders. Here, we review the recent findings on congenital cerebral hypomyelination, which includes 11 diseases, with a novel disease classification and diagnostic criteria with flow charts.

We report herein a case of 2-year-old boy diagnosed with a mild form of Pelizaeus–Merzbacher disease due to deletion of the entire proteolipid protein 1 (PLP1) gene. The patient demonstrated spastic quadriplegia, mental retardation, and microcephaly. He exhibited brainstem auditory evoked potentials with prolonged interpeak latencies and magnetic resonance imaging characteristics suggestive of hypomyelination in most areas of the brain with the exception of the brainstem, cerebellar peduncles, corpus callosum, and the posterior limbs of the internal capsules. Proton magnetic resonance spectroscopy revealed a mildly reduced ratio of N-acetyl aspartate to creatine levels in the white matter, suggesting axonal involvement. Additionally, nerve conduction velocity of the lower extremities was mildly decreased. Genetic analysis showed a deletion of PLP1 in this patient. Further genome mapping followed by sequence analysis of the deletion breakpoints revealed that a genomic region, about 73 kb in length, including the entire PLP1 and RAB9B, was deleted. The size of the deletion was the smallest among those previously reported in this region. Except for the 1-base pair microhomology, there were no homologous sequences between the regions around the distal and proximal breakpoints, which suggests that the deletion occurred by nonhomologous end joining.

Spastic paraplegia type 4 (SPG4) is the most common autosomal dominant hereditary SPG caused by mutations in the SPAST gene. We studied the four-generation pedigree of a Japanese family with autosomal dominant hereditary SPG both clinically and genetically. Twelve available family members (ten affected; two unaffected) and two spouses were enrolled in the study. The clinical features were hyperreflexia in all four limbs, spasticity of the lower extremities, impaired vibration sense, mild cognitive impairment confirmed by the Wechsler Adult Intelligence Scale—Third Edition, and peripheral neuropathy confirmed by neurophysiological examinations. All four female patients experienced miscarriages. The cerebrospinal fluid tau levels were mildly increased in two of three patients examined. Linkage analyses revealed the highest logarithm of odds score of 2.64 at 2p23-p21 where the SPAST gene is located. Mutation scanning of the entire exonic regions of the SPAST gene by direct sequencing revealed no mutations. Exonic copy number analysis by real-time quantitative polymerase chain reaction revealed heterozygous deletion of exons 1 to 4 of the SPAST gene. Breakpoint analysis showed that the centromeric breakpoint was located within intron 4 of SPAST while the telomeric breakpoint was located within intron 3 of the neighboring DPY30 gene, causing a deletion of approximately 70 kb ranging from exons 1 to 3 of DPY30 to exons 1 to 4 of SPAST. To our knowledge, this is the first report of SPG4 associated with partial deletions of both the SPAST and DPY30 genes. The partial heterozygous deletion of DPY30 could modify the phenotypic expression of SPG4 patients with this pedigree.

Basophilic inclusions (BIs), which are characterized by their staining properties of being weakly argyrophilic, reactive with Nissl staining, and immunohistochemically negative for tau and transactive response (TAR) DNA-binding protein 43 (TDP-43), have been identified in patients with juvenile-onset amyotrophic lateral sclerosis (ALS) and adult-onset atypical ALS with ophthalmoplegia, autonomic dysfunction, cerebellar ataxia, or a frontal lobe syndrome. Mutations in the fused in sarcoma gene (FUS) have been reported in cases of familial and sporadic ALS, and FUS immunoreactivity has been demonstrated in basophilic inclusion body disease (BIBD), neuronal intermediate filament inclusion disease (NIFID), and atypical frontotemporal lobar degeneration with ubiquitin-positive and tau-negative inclusions (aFTLD-U). In the present study, we immunohistochemically and ultrastructurally studied an autopsy case of sporadic adult-onset ALS with numerous BIs. The patient presented with the classical clinical course of ALS since 75 years of age and died at age 79. Postmortem examination revealed that both Betz cells in the motor cortex and motor neurons in the spinal cord were affected. The substantia nigra was spared. Notably, BIs were frequently observed in the motor neurons of the anterior horns, the inferior olivary nuclei, and the basal nuclei of Meynert. BIs were immunopositive for p62, LC3, and FUS, but immunonegative for tau, TDP-43, and neurofilament. Ultrastructurally, BIs consisted of filamentous or granular structures associated with degenerated organelles with no limiting membrane. There were no Bunina bodies, skein-like inclusions, or Lewy-like inclusions. All exons and exon/intron boundaries of the FUS gene were sequenced but no mutations were identified.

Recently the authors have identified a major component of Rosenthal fibers as alpha B-crystallin, a major lens protein. In the current study the authors investigated the expression of alpha B-crystallin in four cultured glioma cell lines and in 115 human neuroectodermal tumors. alpha B-crystallin was expressed differentially by those glioma cell lines, but not by neuroblastoma cell lines. Northern blot analysis revealed two distinct messages for alpha B-crystallin in C-6, whereas only a single message in U-373MG and G26-24. In human surgical specimens positive immunostaining was frequently observed in the following brain tumors: pilocytic astrocytoma of the juvenile type, anaplastic astrocytoma, glioblastoma multiforme, and subependymal giant cell astrocytoma. The astrocytic elements of mixed oligoastrocytomas, glioblastomas with sarcomatous components, and gangliogliomas were likewise strongly stained. In contrast, little immunoreactivity was observed in ependymal and choroid plexus tumors. Thus, alpha B-crystallin is mainly expressed by astrocytic tumors among neuroectodermal neoplasms, without regard to the presence of Rosenthal fibers.

Pelizaeus-Merzbacher disease (PMD) is a rare dysmyelinating disorder due to mutations in the proteolipid protein (PLP) gene. PLP gene mutations are responsible for a broad spectrum of disease, from the most severe form, connatal PMD, to a less severe form, spastic paraplegia 2 (SPG2). We describe here a very mild case of PMD in a patient who presented with nystagmus in early infancy and was unable to walk until 1 year 7 months of age. Brain magnetic resonance imaging (MRI) at 1 year 7 months of age revealed white matter abnormalities typical of PMD. Genetic testing revealed a novel mutation of the PLP gene (Gly197Arg). The patient presented with only mildly ataxic gait and slurred speech at the age of 4 years. Gly197Arg is the first novel mutation located within exon 4 of the PLP gene and associated with mild PMD/SPG2 in a Japanese patient.

Two major classes of mRNAs for the alpha-crystallin B chain (or alpha(B)crystallin), about 0.9 and 1.2 kilobases in length, are expressed in rat brain. To examine the structures of these mRNAs, we isolated cDNA clones from rat brain and genomic DNA from rat liver. Characterization of these clones as well as Northern blot analysis indicated that the various mRNAs differed in the lengths of their 5' leader sequences. RNase protection assays revealed that the gene for alpha-crystallin B chain contains multiple start sites. The transcriptional start sites of the longer mRNAs are preceded by a putative CAAT box and that of the shorter mRNA by a putative TATA box. The shorter mRNA encodes the alpha-crystallin B chain protein, whereas the longer mRNA contained three extra small open reading frames upstream of the AUG start codon for the protein. The shorter mRNA is abundant in lens, heart, muscle, and kidney, while the longer mRNAs are constitutively expressed at low levels in a wide variety of tissues. The shorter mRNA was increased by treatment with phorbol 12-myristate 13-acetate in rat C6 glioma cells. Since there is only a single copy of the alpha-crystallin B chain gene, our results indicate that the two classes of mRNAs are generated by alternative transcriptional initiation from different promoters and their expressions are regulated differentially.

Recently, artificial microRNA (miRNA)-mediated RNA interference (RNAi) systems have been developed as useful tools to study gene functions. We report an artificial miRNA precursor motif (AMPM) containing several restriction sites in the loop and in the extended stem structures, and generated a vector-based expression system for AMPM under the control of an RNA polymerase II promoter. The AMPM located in the intron or the exon of the selection marker gene mediated silencing of the reporter luciferase gene expression in HeLa cells. Northern blotting and colony formation assays revealed that the AMPM was efficiently and appropriately processed into mature miRNA. The AMPA system also down-regulated endogenous p53 and laminA/C proteins in stable transfectants. Moreover, clustered AMPMs separated by an appropriate spacer, targeting different sites within a single mRNA mediated gene silencing, even if the sequences of the target and the AMPM were partially complementary. This indicates the potential therapeutic utility of clustered AMPMs for highly-mutable targets, such as human immunodeficiency virus type 1.

Pelizaeus-Merzbacher disease (PMD) is an X-linked neurological disorder characterized by dysmyelination in the central nervous system (CNS). Recently mutations of the myelin proteolipid protein (PLP) gene which encodes both PLP and its isoform, DM-20 generated by alternative splicing, have been demonstrated in PMD patients. We analyzed the seven exons of the PLP gene of a Japanese boy affected with PMD by direct sequencing and identified an insertion event in exon VII of the PLP gene. This mutation was also present in his carrier mother, but was absent in ninety-five X chromosomes of normal Japanese. The frame-shift mutation leads to the production of truncated PLP with altered carboxyl terminal amino acid sequences, resulting in considerable change of the structure of PLP and DM-20 necessary for functional purposes. This is the first report of a mutation in exon VII of the PLP gene associated with PMD.

We previously identified HSPB2, a new member of the small heat shock protein family, expressed in heart and skeletal muscles. In this study, we used a polyclonal anti-HSPB2 antibody and examined the subcellular localization of HSPB2 in differentiated C2C12 cells, KNS-81 cells, and NIH3T3 transfectants expressing human HSPB2. Double staining with anti-HSPB2 and various markers for cytoplasmic structures showed that HSPB2 was present in the cytosol as granules, some of which colocalized with mitochondria. This colocalization was not altered by a colchicine treatment, indicating that it is independent of microtubules. The subcellular fractionation of differentiated C2C12 cells revealed that HSPB2 was mainly detected in the postmitochondrial supernatant, but mild heat treatment enriched the amount of HSPB2 in the mitochondrial fraction. The expression of HSPB2 protected the cells from heat-induced cell death. In addition, Northern blot analysis revealed that expression of HSPB2 mRNA is higher in slow-twitch muscle than in fast-twitch muscle, which correlates with the amounts of mitochondria present in these two types of tissue. Taken together, these results suggest that HSPB2 may not localize in the matrix, but rather associates with the outer membrane components of the mitochondria and thus plays a role in the stress response.

alphaB-Crystallin, a member of the small heat shock protein (HSP) family, accumulates in reactive astrocytes in a variety of pathological conditions. We previously reported the upregulation of alphaB-crystallin in response to high extracellular potassium concentration. In the present study, we investigated the regulatory mechanism of alphaB-crystallin expression by KCl. When human glioma U-251MG cells were exposed to continuous KCl treatment, induction of alphaB-crystallin mRNA was observed after 8 h and persisted for a few days. Functional promoter analysis using deletion and mutation constructs revealed that the proximal heat shock element (HSE-P), which contributes to heat shock induction in HeLa cells, is essential for transcriptional activation of the alphaB-crystallin gene by KCl in U-251MG cells. Gel mobility shift and antibody supershift assays showed that KCl induces the HSE-binding activity of heat shock factor (HSF) 2, while heat shock induces that of HSF1. This is the first demonstration that HSF2 can be activated by KCl and is involved in the upregulation of alphaB-crystallin gene expression in glial cells.

Eleven 1,3,5-triazine derivatives were investigated by the use of a thermal-analysis techniques. These compounds showed a remarkable difference in pyrolytic behavior and decomposed thermally by a cleavage of the triazine ring and the liberation of water, isocyanic acid, and carbondioxide. The pyrolysis products were separated and identified by a gas chromatograph coupled to a mass spectrometer. All of the obtained information led to the proposal of a thermal degradation mechanism for the triazine derivatives, which is presented here. The main pyrolysis and fragmentation by electron impact mechanisms were classified into four categories, respectively.

alphaB-Crystallin is a member of the alpha-crystallin/small heat shock protein (hsp) family and under various neuropathologic conditions accumulates in reactive astrocytes and degenerating neurons. In the 5'-flanking region of the alphaB-crystallin gene on human chromosome 11q22-q23, where a constitutive DNase I hypersensitive site is located, we identified a gene transcribed in the opposite direction. Analysis of its mRNA structure by RT-PCR and 5'/3'RACE revealed that this gene is composed of two exons and encodes a new member of the alpha-crystallin/small hsp family. This gene was designated the HSPB2 gene by the HMGW Nomenclature Committee. The complete genomic structure of the rat homologue was also determined. Northern blot analysis revealed that the HSPB2 gene is expressed preferentially in skeletal muscle and heart but not in the lens, while the neighboring alphaB-crystallin gene is highly expressed in all three tissues. The two related genes are arranged in a head-to-head manner with an intergenic sequence of less than 1 kb, raising a possibility of shared regulatory elements for their expression.

AlphaB-crystallin and the small stress protein, heat shock protein of 27 kDa (HSP27), share structural similarities and are coordinately induced by classical stress stimuli. We have recently observed that hypertonic stress produced by high NaCl concentrations selectively induces alphaB-crystallin in glial cells. To examine divergence of the functional properties of these two related proteins, we have constructed stable alphaB-crystallin-expressing glial cell lines from the U-251 MG glioma cells, which are normally deficient in alphaB-crystallin expression but constitutively express HSP27. These transfected cells lines are more resistant to acute hypertonic stress than the parental line from which they were derived. Moreover, the parental line acclimates to stepwise increases in hypertonicity and upregulates endogenous alphaB-crystallin in the process but not HSP27. The overexpression of HSP27 and alphaB-crystallin in NIH/3T3 fibroblasts, a cell line that normally expresses little alphaB-crystallin and no HSP27, does not result in increased survival. This suggests that alphaB-crystallin interacts with cell-type specific mechanisms to aid in protection from hypertonic stress.

It has been shown by immunohistochemical studies that alpha B-crystallin accumulates in the reactive and neoplastic glial cells in a variety of pathologic situations. However, the molecular mechanism for the induction of alpha B-crystallin in diseased brains is still unknown. Since any destructive brain lesions cause an abnormal elevation in the potassium (K+) concentration of the extracellular space, which disturbs the regulatory mechanism of glial cell volume, we investigated the influence of elevated extracellular K+ on the expression of alpha B-crystallin in glial cells. The treatment of rat C6 glioma cells with augmented K+ in the culture media induced an accumulation of alpha B-crystallin mRNA in a dose-dependent manner and an accumulation of the alpha B-crystallin as well. Furthermore, an overexpression of alpha B-crystallin in the C6 transformant transfected with a rat alpha B-crystallin cDNA conferred a resistant phenotype against the insult of elevated extracellular K+ on the glioma cells. Thus, alpha B-crystallin may contribute to the survival of reactive glia in the presence of a high extracellular K+ concentration.

The alpha B-crystallin gene is abundantly expressed in the vertebrate lens and at lower levels in various non-lenticular tissues. Among the non-lenticular tissues, alpha B-crystallin is present at high levels in the heart and skeletal muscle. Using a specific antibody against alpha B-crystallin, the cellular localization of alpha B-crystallin was studied in biopsies of human skeletal muscles. Expression of alpha B-crystallin was observed in normal oxidative muscle fibers that show positive reactions for NADH-tetrazolium reductase and cytochrome c oxidase. In muscle diseases increased immunoreactivity for alpha B-crystallin was found in ragged-red fibers, which stained darkly with histochemistry for succinate dehydrogenase. Since alpha B-crystallin is related to small heat-shock proteins and can be induced by various stress conditions, the increased alpha B-crystallin immunoreactivity of ragged-red fibers could result from profound oxidative stress produced by the abnormal mitochondrial metabolism.

We investigated the proteolipid protein (PLP) gene of two boys in a Japanese family with Pelizaeus-Merzbacher disease (PMD), an X-linked neurologic disorder characterized by dysmyelination in the central nervous system (CNS). The patients showed similar clinical signs from birth and autopsy on the elder brother confirmed a connatal type of PMD. Direct sequencing of the PLP gene and PLP mRNAs from the brain of the PMD patient revealed a G to T transition in exon V of the PLP gene, which leads to a glycine to cysteine substitution at residue 220. Allele-specific oligonucleotide hybridization revealed that this mutation was also present in his brother, but was absent in 100 X chromosomes of normal Japanese individuals. Northern blot analysis showed that the mRNA levels of PLP and myelin basic protein, two major myelin proteins produced by oligodendrocytes, were much reduced in the PMD brain, hence, there was a specific loss of oligodendrocytes. It seems likely that the substitution is responsible for PMD (connatal type) in this particular family and causes oligodendrocytes death in the CNS.

alpha B-Crystallin is a major protein component of Rosenthal fibers, which massively accumulate in the brains of patients suffering from Alexander's disease. To examine whether or not accumulation of alpha B-crystallin is due to any abnormality of the gene structures, we determined the sequence of the alpha B-crystallin gene in two cases of pathologically confirmed Alexander's disease. Direct sequencing of the promoter and coding regions of the alpha B-crystallin gene in patients revealed them to have a normal sequence. Northern blotting showed a single alpha B-crystallin mRNA species expressed in the Alexander's disease brain.

Crystallins are well known to be water-soluble proteins in the lens that can pack together efficiently to form very large aggregates. α-Crystallin is a heterogeneous aggregate produced by the products of two genes, αA and αB. Of these two genes, αA-crystallin expression seems restricted to the lens [1], In contrast, αB-crystallin is expressed in various extra-ocular tissues such as heart, muscle, kidney, brain, nerve, and placenta [2,3]. The function of αB-crystallin in extra-ocular tissues is not yet known.

Expression of αB-crystallin in the developing rat kidney. The expression and cellular localization of αB-crystallin during rat renal development was studied by Northern blot analysis and by immunocytochemistry. Northern blotting of total RNA extracted from whole kidneys revealed that the messenger RNA for αB-crystallin rapidly increased after birth to reach adult levels by 20 days. At the same time, immunohistochemistry for αB-crystallin demonstrated that the prominent elongation of Henle's loop during the first 10 days of life was accompanied by increased αB-crystallin expression. Thus, the development of αB-crystallin expression is correlated temporally with the acquisition of tubule function in early post-natal life.

Background We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved. Results We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice. Conclusions These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice.