小坂 仁

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-Iike and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor I (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen I [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

Rasmussen syndrome is an intractable epilepsy with a putative causal relation with cellular and humoral autoimmunity. Almost half of the patients have some preceding causative factors, with infections found in 38.2%, vaccinations in 5.9% and head trauma in 8.9% of Japanese patients. In a patient with seizure onset after influenza A infections, cross-reaction of the patient's lymphocytes with GluR epsilon 2 and influenza vaccine components was demonstrated by lymphocyte stimulation test. Database analyses revealed that influenza A virus hemagglutinin and GluR 1 2 molecules contain peptides with the patient's HLA class I binding motif ( HLA 2 A*0201). The relative risks of HLA class I genotypes for Rasmussen syndrome are 6.1 ( A*2402), 6.4 ( A*0201), 6.3 ( A*2601) and 11.4 ( B*4601). The relative risks of HLA class I-A and B haplotypes are infinity ( A*2601 + B*5401), 21.1 ( A*2402 + B*1501), 13.3 ( A*2402 + B*4801) and 5.1 ( A*2402 + B*5201). Some alleles and haplotypes of HLA class I may be the risk factors in Japanese patients. Cross-reactivity of cytotoxic T lymphocytes may contribute to the processes leading from infection to the involvement of CNS.

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked leukodystrophy caused by proteolipid protein 1 (PLP1) gene mutations. Previous studies indicated that proteolipid proteins (PLPs) with disease-associated mutations are misfolded and trapped in the endoplasmic reticulum (ER) during transportation to the cell surface, which eventually leads to oligodendrocyte cell death in PMD. Here we report a PMD patient with a very mild phenotype carrying a novel mutation (485G→T) in exon 4 of the PLP1 gene that causes a Trp162Leu substitution in the protein. We also investigated intracellular trafficking of this mutant PLP in COS-7 cells. Transiently transfected mutant PLPW162L fused to an enhanced green fluorescent protein (EGFP) or a short peptide tag was not carried to the plasma membrane. However, in contrast to previous studies, this mutant PLP was not retained in the ER, indicating an escape of the newly translated protein from the quality control machinery. We also found that the mutant PLP accumulated in the nuclear envelope (NE) in a time-dependent manner. This mutant PLP, with its distribution outside the ER and a very mild phenotype, supports the idea that accumulation of misfolded mutant protein in the ER causes the severe phenotype of PMD. © 2006 IBRO.

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice over-expressing EF1 alpha promoter-driven LICH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.

Mammalian neuronal cells abundantly express a de-ubiquitinating isozyme, ubiquitin carboxy-terminal hydrolase L1 (UCH L1). Loss of UCH L1 function causes dying-back type of axonal degeneration. However, the function of UCH L1 in neuronal cells remains elusive. Here we show that overexpression of UCH L1 potentiated ATP-induced currents due to the activation of P2X receptors that are widely distributed in the brain and involved in various biological activities including neurosecretion. ATP-induced inward currents were measured in mock-, wild-type or mutant (C90S)-UCH L1-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in both wild-type and C90S UCH L1-transfected cells, suggesting that hydrolase activity was not involved but increased level of mono-ubiquitin might play an important role. The increased currents were dependent on cAMP-dependent protein kinase (PKA) and Ca2+ and calmodulin-dependent protein kinase (CaMKII) but not protein kinase C. In addition, ATP-induced currents were likely to be modified via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) that is regulated by PKA and phosphatases. Our finding shows the first evidence that there is a relationship between UCH L1 and neurotransmitter receptor, suggesting that UCH L1 may play an important role in synaptic activity.

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

To seek a novel therapeutic approach to neuroblastoma (NBL), we used three NBL cell lines (SK-N-DZ, NH12, and SK-N-SH) to examine the underlining molecular mechanisms of cellular reactions and sensitivity to all-trans-retinoic acid (ATRA). SK-N-DZ cells expressed relatively high levels of retinoic acid receptor alpha (RAR-alpha) and underwent ATRA-induced cell death that was blocked by an RAR-alpha antagonist. By contrast, RAR-alpha expression gradually decreased in NH12 and SK-N-SH cells, which did not experience increased cell death in response to ATRA. We report here the ubiquitin-dependent down-regulation of RAR-alpha expression during ATRA treatment. Our data suggest that SK-N-DZ cells have a defect in RAR-alpha down-regulation, resulting in sustained high expression of RAR-alpha that confers high sensitivity to ATRA. Accordingly, treatment with a proteasome inhibitor dramatically increased ATRA-indueed cell death in NH12 and SK-N-SH cell lines. Our results reveal the crucial involvement of the RAR-alpha signaling pathway in NBL cell death and show that three NBL cell lines are differentially sensitive to ATRA. These data suggest a potential novel therapy for NBL involving retinoic acid treatment combined with the inhibition of RAR-alpha degradation.

The synuclein family includes three isoforms, termed alpha, beta and gamma. alpha-Synuclein accumulates in various pathological lesions resulting from neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. However, neither beta- nor gamma-synuclein has been detected in Lewy bodies, and thus it is unclear whether these isoforms contribute to neurological pathology. In the present study, we used immunohistochemistry to demonstrate accelerated accumulation of beta- and gamma-synucleins in axonal spheroids in gracile axonal dystrophy (gad) mice, which do not express ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). gamma-Synuclein immunoreactivity in the spheroids appeared in the gracile nucleus at 3 weeks of age and was maintained until 32 weeks. beta-Synuclein immunoreactivity appeared in spheroids around 12 weeks of age. In contrast, alpha-synuclein immunoreactivity was barely detectable in spheroids. Immunoreactivity for synaptophysin and ubiquitin were either faint or undetectable in spheroids. Given that UCH-L1 deficiency results in axonal degeneration and spheroid formation, our findings suggest that beta- and gamma-synuclein participate in the pathogenesis of axonal swelling in gad mice. (C) 2004 Elsevier B.V. All rights reserved.

Parkinson's disease (PD) is thought to be caused by environmental and genetic factors. Mutations in four genes, alpha-synuclein, parkin, DJ-1, and UCH-L1, have been identified in autosomal inherited forms of PD, The pathogenetic cause for the loss of neuronal cells in PD patients, however, remains to be determined. Due to the rarity of mutations in humans with PD, the analysis of animal models might help to further gain insights into the pathogenesis of familial PD. For UCH-L1, deficiency has been described in gad mice leading to axonal degeneration and formation of spheroid bodies in nerve terminals. Here, we investigated the gene expression pattern of the brain of 3-month-old Uch-l1-deficient gracile axonal dystrophy (gad) mice by microarray analysis. A total of 146 genes were differentially regulated by at least a 1.4-fold change with 103 being up-regulated and 43 being down-regulated compared with age and sex matched wildtype littermate mice. The gene products with altered expression are involved in protein degradation, cell cycle, vesicle transport, cellular structure, signal transduction, and transcription regulation. Most of the genes were modestly regulated, which is in agreement that severe alteration of these pathways might be lethal. Among the genes most significantly down-regulated is the brain-derived neurotrophic factor which might be one aspect of the pathogenesis in gad mice. Interestingly, several subunits of the transcription factor CCAAT/enhancer binding protein are up-regulated, which plays a central role in most altered pathways. (C) 2004 Elsevier B.V. All rights reserved.

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.

Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. in wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after fight stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival. (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed similar to70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo.

Neuronal nicotinic receptors (nAChR) are pentameric assemblies of subunits of a gene family where specified combinations of alpha and beta subunits form functional receptors. To extend our understanding of the role of spinal nAChR in the processing of sensory stimuli and regulation of autonomic and motor responses, we initiated investigations to localize nAChR subunit expression within discrete spinal regions and cell types. High-affinity epibatidine binding was present in the superficial dorsal and ventral horns, the mediolateral and central canal regions. RT-PCR identified transcripts for alpha3, alpha4, alpha5, beta2, and beta4 in both spinal cord parenchyma and dorsal root ganglia (DRG). Our affinity-purified antibodies against alpha3, alpha4, alpha5, beta2, and beta4 subunits identified specific protein bands of appropriate molecular mass (preadsorbed with the respective antigens) in specific tissues and cells that express nicotinic receptors, including the spinal cord and DRG neurons. Having established the absence of crossreactivity with related subunits, specific fluorescence labeling of nerve terminals and cell bodies was achieved and correlated with the distribution of defined marker proteins and nicotinic receptor binding sites determined autoradiographically. Our findings indicate that alpha3, alpha4, alpha5, beta2, and beta4 subunits are all expressed on primary afferents (IB4-positive terminals) in the spinal cord. The predominant presynaptic (synaptophysin colocalization) labeling is in the superficial layer of the dorsal horn. These receptor subunits, except for beta4, are also present in postsynaptic autonomic (anti-bNOS-positive) and somatic motor neurons (anti-VAChT-positive). The alpha3, alpha5, and beta2 subunits showed additional staining in glial (anti-GFAP-positive) cells. These studies reveal a dense and distinguishable distribution of nAChR subunits in the spinal cord and point toward future therapeutic targeting for specific spinal actions. (C) 2003 Wiley-Liss, Inc.

Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an 193M substitution results in a decrease in kat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in k(cat) (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease. (C) 2003 Elsevier Science (USA). All rights reserved.

Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin, We have isolated a novel mouse gene for ubiquitin carboxyl-terminal hydrolase isozyme L4. The gene named Uchl4 encodes a novel member of the family of ubiquitin carboxyl-terminal hydrolases (UCHs) whose predicted amino acid sequence shows 95% identity to mouse UCH-L3 and 94% identity to human UCH-L3, Genomic structure, chromosome localization, and expression pattern of Uchl3 and Uchl4 were characterized in the mouse. Both Uchl3 and Uchl4 were expressed in various tissues examined; however, expression level was quite lower in Uchl4. While Uchl3 consists of at least 9 exons spanning about 12 kb, Uchl4 was an intronless gene with a size of about 2 kb. By PCR-based analysis with T31 radiation hybrid mapping panel, Uchl3 and Uchl4 were mapped on mouse chromosome 9 and 14, respectively. (C) 2001 Academic Press.

We generated a yeast artificial chromosome (YAC)/bacterial artificial chromosome (BAC)-based physical and transcript map of a region containing the gracile axonal dystrophy (gad) locus on mouse chromosome 5. The YAC/BAC contig consists of 13 YAC and 49 BAC clones onto which 4 genes, 40 expressed sequence tags, and 7 new DNA polymorphisms were ordered. Using this physical map, we mapped Uchl1 encoding ubiquitin carboxyl-terminal hydrolase I, whose deletion has been determined to cause the gad mutation. We also mapped three other recently identified genes: Hip2, encoding Huntingtin interacting protein 2 Atp3a2, encoding a P-type ATPase and Pmx2b, encoding PHOX2b. (C) 2000 Academic Press.

To evaluate conduction abnormalities of the corticospinal tracts (CSTs) in Pelizaeus-Merzbacher disease (PMD), magnetic stimulation at three levels was carried out in 3 boys with PMD aged between 9 and 12 years. They were all diagnosed as having a duplicated proteolipid protein gene. The motor cortex and cervical spinal roots were stimulated with a round coil, whereas a double cone coil was used for brainstem stimulation. Surface electromyographic (EMG) recording was performed on the first dorsal interosseous muscles. Despite a normal EMG response to cervical stimulation, magnetic shock of the motor cortex elicited no EMG activity, even in the case with less motor symptoms. This discrepancy between the electrophysiological and clinical findings is likely due to slowing conduction, which reduces the temporal summation of multiple descending volleys magnetically elicited. A partial conduction block may also occur because of the lack of an EMG response to brain-stem stimulation. Thus, we speculated that the spastic paresis in PMD is associated with both slowing conduction and a partial conduction block in the CSTs.

Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the α-subunit and Asp-174 in the γ-subunit or the corresponding Asp-180 in the δ-subunit, are presumed to reside near the two agonist binding sites at the αγ and αδ subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. αAsp-152 affects agonist and antagonist affinity similarly, whereas γAsp-174 and δAsp-180 primarily affect agonist affinity. Combining charge neutralization on the a subunit with that on the γ and subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, αD152Y, reduced the affinities for agonists and antagonists, the γD174Y/SD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying αD152Q or γD174N/δD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the αD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of αAsp-152, γ/δAsp-174/180, and αAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.

We identified a novel mutation, L322P, in a patient with X-linked adrenoleukodystrophy (ALD) who underwent bone marrow transplantation (BMT). An identification of the ALD gene mutation enabled us to employ an approach not dependent on the use of radioisotopes for detecting mixed chimerism. This assay could show more than 99.0% of the patient's peripheral white blood cells were replaced by the donor's cells.

Earlier reports on T2-weighted magnetic resonance imaging (MRI) in the classical form of Pelizaeus-Merzbacher disease seemed to divide the patterns of the high-intensity lesions in the white matter into three subtypes: type I, diffusely hemispheric and corticospinal type II, diffusely hemispheric without brainstem lesions and type III, patchy in the hemispheres. The four boys presented in our study, between 10 and 17 years of age, with classical Pelizaeus-Merzbacher disease, who all had a duplicated proteolipid protein gene, invariably manifested type I despite their various clinical severities. Follow-up MRI after an interval of 5 years and proton magnetic resonance spectroscopy was performed in three of the patients. The white matter on the last MRI was unchanged in volume and the distribution of high-intense areas. Proton magnetic resonance spectroscopy revealed no abnormal peaks. These results were consistent with the lack of definite neurologic regression in the last 5 years and with the pathologic characteristics of well-preserved axons and the absence of sclerosis. Further study is required to precisely determine whether the patterns of MRI findings can be divided into subtypes corresponding to those of proteolipid protein gene abnormalities.

Proton MR spectroscopic findings in two patients with genetically defined Pelizaeus-Merzbacher disease revealed ratios of N- acetylaspartate/creatine and choline-containing compounds/creatine that were not significantly different from those found in a population of healthy subjects. These findings suggest that proton MR spectroscopy can aid in the diagnosis of Pelizaeus-Merzbacher disease.

We report the case of a 44-year-old woman with a partial 6-pyruvoyl tetrahydropterin synthase (6-PTS) deficiency, whose predominant clinical symptom was generalized dystonia with marked diurnal fluctuation. Dystonia was present in the eyelids, oromandibular region, trunk, and extremities (Meige syndrome plus double hemiplegia-like dystonia). A marked and sustained positive response to levodopa was observed. A molecular genetic study revealed a homozygous mutation (I114V) in the 6-PTS gene. This study indicates that genetic abnormality in the 6-PTS gene may be a hereditary dystonic disorder. We speculate that our patient has residual 6-PTS activity in the central nervous system, such as in the liver, and we suggest that residual, but insufficient production of tretrahydrobiopterin may play an important role in causing diurnal fluctuation of symptoms.

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked dysmyelinating disorder of the CNS resulting from abnormalities in the proteolipid protein (PLP) gene. Exonic mutations in the PLP gene are present in 10 to 25% of all cases. In investigating genotype-phenotype correlations, we screened five Japanese families with PMD for PLP gene mutations and compared their clinical manifestations. We identified two novel nucleotide substitutions in exon 5, at V208N and at P210L, in two families. In the remaining three families, there were no mutations detected. Although all patients satisfied the criteria for the classical form of PMD, two families not carrying the mutations showed milder clinical manifestations than those with the mutations. Since linkage analysis has shown homogeneity at the PLP locus in patients with PMD, our findings suggest that there may be genetic abnormalities other than exonic mutations that cause milder forms of PMD.

We report molecular analysis of thiamin-responsive pyruvate dehydrogenase complex (PDHC) deficiency in a patient with an X-linked form of Leigh syndrome. PDHC activity in cultured lymphoblastoid cells of this patient and his asymptomatic mother were normal in the presence of a high thiamin pyrophosphate (TPP) concentration (0.4 mmol/L). However, in the presence of a low concentration (1 x 10-4 mmol/L) of TPP, the activity was significantly decreased, indicating that PDHC deficiency in this patient was due to decreased affinity of PDHC for TPP. The patient's older brother also was diagnosed as PDHC deficiency with Leigh syndrome, suggesting that PDHC deficiency in these two brothers was not a de novo mutation. Sequencing of the X-linked PDHC E1α subunit revealed a C → G point mutation at nucleotide 787, resulting in a substitution of glycine for arginine 263. Restriction enzyme analysis of the E1α gene revealed that the mother was a heterozygote, indicating that thiamin-responsive PDHC deficiency associated with Leigh syndrome due to this mutation is transmitted by X-linked inheritance.

We analyzed follow-up magnetic resonance images (MRI) in eight children with clinical postinfectious encephalitis (PIE), and discussed their pathogeneses. Three categories of MRI findings were apparent: (1) multifocal lesions in the white matter with/without basal ganglia involvement consistent with acute disseminated encephalomyelitis (ADEM) (three patients) (2) single or multifocal lesions localized only in the gray matter (two patients) and (3) localized lesions in the brain stem, basal ganglia or cerebellum. Some lesions in the patients in Categories 1 and 2 migrated or were resolved quickly, sometimes within 10 days. Gadolinium caused linear or spotty enhancement in the patients in Category 2. These findings suggest that Categories 1 and 2 are a self-limiting allergic angiopathy without demyelination. In contrast, the lesions in the patients in Category 3 were fixed, and not resolved within 6 months (three patients). The pathogenesis of Category 3 is not known. All except one patient had no prednisolone (PSL) therapy, however all lesions were resolved completely or markedly reduced in size, which indicates PSL therapy is not always necessary in patients with PIE.

The coefficient of variation of R-R intervals (CVRR) was studied in 18 children having severe brain damage with a mean ± standard deviation (S.D.) age of 8.4 ± 5.9 years, who were divided into ten patients complicated with respiratory insufficiency (RI: group) and eight patients with severe athetotic cerebral palsy (SA group). CVRR was obtained in the resting supine position, and was compared with that in 22 neurologically normal controls. CVRR in the RI group (mean ± S.D., 2.19 ± 1.28%) was significantly lower than that in controls (5.56 ± 1.53%), while CVRR in the SA group (11.30 ± 3.91%) was significantly higher than that in controls (both P &lt 0.01, ANOVA). In particular, the four patients with brain death showed extremely low CVRR of 1.00-1.29%. Since CVRR was 4.09% in the patient aged 4 years with birth injury of the upper cervical spinal cord causing absence of spontaneous respiration, the extremely low CVRR in patients with brain death may be directly related to brainstem dysfunction. The cause of the high CVRR in the SA group was not determined. Thus, CVRR may be useful for quantitative evaluation of severe neurological disorder.

We studied the effect of digitalis on nerve conduction dysfunction in Pelizaeus-Merzbacher disease (PMD). The patients were three Japanese boys with PMD, aged 7-10 years. Digitalis was administered orally in a daily dose of 0.06 mg/kg for 2 consecutive months, and the obtained serum concentrations ranged from 0.33 to 0.55 ng/ml. The digitalis therapy induced slight improvement of severe dysarthria and cognitive dysfunction in the two older patients. Electrophysiological examinations revealed the following results: In brainstem auditory evoked potentials (BAEPs), while waves II (or III) to V were absent before treatment, on treatment all waves of BAEPs except a wave IV were restored in all patients. While visual evoked potentials (VEPs) in response to transient flash stimulation showed markedly prolonged latencies before treatment, digitalis produced a mild, although not statistically significant, shortening of the latency of N160. There were also no significant changes in inter-peak amplitudes of VEPs. Transcranial cortical magnetic stimulation continued to fail to elicit motor evoked potentials of the first dorsal interosseous muscles in all patients. Thus, although the serum concentrations were insufficient to elicit favorable therapeutic effects, digitalis therapy provided slight relief of clinical symptoms with evidence of improvement of conduction dysfunction. It is suggested that patients with PMD may respond to symptomatic treatment modulating nerve conduction.

We experienced five children having T1-shortening lesions in basal ganglia or thalami on magnetic resonance imaging (MRI), which were supposed to be caused by manganese (Mn) overdosis. Instead of the presence of above mentioned lesions, no neurological manifestations corresponding to them had developed in all patients. This observation suggests that a MRI is useful for detecting the side effects caused by overdosis of Mn in patients having parenteral nutrition.

Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10%-25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP gene duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be a important genetic abnormality in PMD and affect myelin formation.

We experienced a 15-year-old female, whose healthy parents were second cousins, who was suspected of having a dysmyelinating disease involving only the central nervous system (CNS). She was noticed to have congenital pendular nystagmus, and spastic gait disturbance developed at the age of 10 years. Mild athetosis of the upper limbs and ataxia were recognized at age 13 years, and dysarthria presented at age 15. MRI and electrophysiological findings showed the characteristics of Pelizaeus-Merzbacher disease (PMD), although the extensive nerve conduction slowing of the CNS was less severe than that in male patients with PMD. No promoter or exonic mutations of proteolipid protein (PLP) gene were detected. Although this patient might be heterozygous for a mutation of the extraexonic PLP gene sequences or of other unknown X-linked PLP associated genes, we speculate that this case had a dysmyelinating disease with an autosomal recessive trait characterized by the same phenotype as that of PMD.

Lymphoblastoid cells are useful materials for the diagnosis and basic studies of many human genetic disorder. To elucidate the etiology of Leigh syndrome, biochemical analyses and mitochondrial DNA analyses were performed on cultured lymphoblastoid cells from 20 patients with the clinical characteristics of this disorder. In 9 of 20 cases, we were able to define the following defects. Eight patients had biochemical defects, including 3 with pyruvate dehydrogenase complex (PDHC), 3 with cytochrome c oxidase (complex IV), and 2 with NADH cytochrome c reductase (complex I) deficiencies. Two of 3 patients with PDHC deficiency were diagnosed with thiamine-responsive PDHC deficiency. One patient had a point mutation (T→G) of mitochondrial DNA at nucleotide position 8993. These results indicate that the underlying defects in Leigh syndrome are heterogenous and cultured lymphoblastoid cells are very useful materials for diagnosis of the etiology of Leigh syndrome.

We report a rare polymorphism in the human proteolipid protein (PLP) gene. A synonymous mutation, 168 A→G, was detected in exon 2 of the PLP gene. Mutations in this gene have been reported in some cases of Pelizaeus-Merzbacher disease. © 1995 Springer-Verlag.

Two cases of a 13-year-old girl and a 14-year-old boy with postinfectious focal encephalitis due to influenza are reported. The clinical and magnetic resonance imaging (MRI) findings included: (1) partial motor seizures as the initial central nervous system manifestation, appearing more than 20 days after the influenzal infection, (2) no change in the level of consciousness although a boy demonstrated apraxia, and (3) high signal intensity lesions noticed with T2-weighted MRI located mainly in the cortex. The girl's lesion appeared to resolve within 10 days on MRI, while that of the boy (demonstrated in the thalamus on a third MRI) resolved within 1 week. However, a new lesion appeared in the cortex approximately 1 month later, that was visualized on a fourth MRI. Small gadolinium-enhanced lesions also were noticed during earlier stages in both patients. The pathogenesis of these MRI lesions is unknown, but the coexistence of small enhancing lesions, rapidly resolving lesions, and the elevated thrombin anti-thrombin III complexes, may indicate the presence of an angiopathy. Serial MRI examinations in patients with postinfectious encephalitis may lead to a better understanding of the pathogenesis of this disorder. © 1995.

An 12-old-year girl with Tolosa-Hunt syndrome(THS) complicated with pseudotumor cerebri is presented. She suffered from alternating, recurrent and painful ophthalmoplegia at the age of 8, and bilateral papilledema which did not affect visual acuity was also recognized. THS subsided readily on steroid therapy, and the remission lasted more than 3 years after discontinuing steroid. However, papilledema did not change despite remission of THS, and resulted in optic atrophy with a mildly enlarged scotoma and 10% decrease in visual acuity 6 months after the onset. MRI disclosed slightly contrasted masses in the bilateral enlarged cavernous sinuses and narrowing of the left carotid siphon. Another small mass with partial gadolinium enhancement was revealed adjacent to the left narrowing of the carotid siphon in the optochiasmatic cistern however, there was no lesion causing intracranial hypertension. The intracavernous MRI findings were considered characteristics of THS, and papilledema seemed to be due to pseudotumor cerebri by exclusion. Since subsequent MRI confirmed no progression of the above findings, the intracavernous and intracisternal masses were suspected to be non-specific inflammatory granulomas associated with THS. © 1995 Elsevier Science B.V. All rights reserved.

Brain lesions exhibited on MRI and CT scan in 2 patients with mitochondrial encephalomyelopathy representing Leigh syndrome were improved by administration of dichloroacetate (DCA). One patient had pyruvic acid dehydrogenase complex (PDHC) deficiency, the other had complex I deficiency. The efficacy of DCA was transient in the patient with the PDHC deficiency, lasting for about 2.5 months. The patient died at the age of 6, about 2 years after the initiation of DCA treatment. DCA administration was started in the patient with complex I deficiency when he was 15 months old and it is still effective at his present age of 24 months. His motor ability is developing, and he could walk without support at the age of 19 months. DCA administration should be tried in patients with mitochondrial diseases. © 1995.

We report a third mutation of the proteolipid protein gene in male Japanese patients withX-linked spastic paraplegia. Although the proteolipid protein gene encodes two myelin proteins, proteolipid protein and DM 20, our W144X mutation resides in the latter part of exon 3 (exon 3B), which is spliced out in DM 20. This mutation may reserve the function of DM 20. Findings in our patients support that this form of spastic paraplesiais allelic to Pelizaeus-Merzbacher disease and that the mild clinical phenotype of this disorder may be related to a mutation within exon 3B of thePLP gene. © 1995 by Academic Press, Inc.

We report a 12 year old girl with double cortex syndrome. She was moderately retarded and had bilateral tonic seizures of upper extremities and atonic seizures of lower extremities with consciousness disturbances. An interictal EEG showed two foci of spikes, left frontpolar and left posteriortemporal. She was hospitalized due to fluctuating disturbance of consciousness from a nearly normal to an almost unresponsive state. An electro encephalogram recorded during the unresponsive state disclosed a diffuse but posteriorly predominant 4-5 Hz hypersynchronous waves that began as a 10-11 Hz focal rhythmic waves at left parieto-occipital or frontal area 2-3 Hz spike and wave complex, following right frontal dominant 12-13 Hz wave, sometimes accompanied this high voltage wave. This pattern continued for a few minutes slid the consciousness returned to a nearly normal state as these waves disappeared. This complex partial status was composed of complex partial seizure for some minutes long and interictal state, in which the consciousness did not return to normal. These states were periodically repeated in a few minutes and were effectively controlled by intravenous diazepam. Although double cortex syndrome is a rare migratory disorder, it has often been reported together with intractable epilepsy. As far as we know, this is the first case of double cortex syndrome accompanied by complex partial status epilepticus.

A 13-year-old girl with nephrotic syndrome (NS) developed acute leucoencephalopathy during combination therapy with cyclosporin A (CyA) and prednisolone (PSL). The patient had a generalized motor seizure followed by coma at 19 days after CyA administration. Magnetic resonance scanning performed on the 1st hospital day revealed white matter lesions in the subcortices of the parietal and occipital lobes, brain stem and cerebellum. These lesions had completely resolved on the 10th hospital day. This episode might be caused by CyA because the clinical course and laboratory data revealed neither inflammation nor other causative factors. To our knowledge, this is the first report of acute leucoencephalopathy during combination therapy with CyA and PSL in a patient with NS. © 1994 IPNA.

We report clinical and neuropathological studies of 2 patients with glutaric aciduria type 1. A 10-month-old male with involuntary movements expired suddenly at home. The second, a 15-year-old female, died after three episodes of acute encephalopathy including a Reye syndrome-like episode and an episode of severe hypoglycemia. Hypocarnitinemia was also present. Selective involvement of type II muscle fibers was observed during the Reye syndrome-like episode. Magnetic resonance imaging of the 2 patients showed marked widening of the sylvian fissure, atrophy of the basal ganglia, and white matter lesions. Neuropathology of the 10-month-old patient showed: (1) temporal and frontal lobe hypoplasia, (2) degeneration of the putamen and the pallidum, (3) mild status spongiosus in the cerebral white matters, (4) heterotopic neurons in the cerebellum, and (5) hypoplasia of the cerebral white matter. This patient appeared to manifest a migration and/or maturation abnormality of the brain as well as previously observed basal ganglia and white matter degeneration. © 1994.

We report a 21-year-old man with agammaglobulinemia and chronic progressive encephalopathy. The patient was diagnosed as having X-linked agammaglobulinemia at 6 months of age, and gamma globulin supplementation was initiated. He exhibited normal development until he was 11 years old, when he showed a decline in school performance and a personality change. Computed tomography images at that time disclosed diffuse cerebral atrophy. Several generalized tonic-clonic convulsions, myoclonus and spasticity appeared at the age of 13 years. He lost his ability to walk and speak at the age of 17 years old. He is currently 21 years old and displays severe mental deterioration and spastic tetraplegia. Magnetic resonance imaging showed progressive diffuse cerebral atrophy with no change in intensity. The cerebellum and the brain stem were relatively well maintained. Viral isolations were negative and serum antibody titers for rubella, measles, and human immune deficiency virus were not elevated. Our patient's symptoms resemble those previously reported as chronic progressive encephalopathy without viral isolation. This condition may be a complication of agammaglobulinemia. It is possible that the encephalopathy of our patient has the same etiology as that described in the other reports. Further attempts to identify the etiology of the encephalopathy using molecular techniques are necessary.

A 7-year-old boy had symmetrical transient high signal lesions in the external capsules on T2-weighted image during the recovery phase of herpes simplex virus (HSV) encephalitis. Although this finding has never been reported in HSV or other viral encephalitis, a postinfectious allergic mechanism is suspected in this patient. With the development of magnetic resonance imaging and other neuroimaging studies, a more detailed analysis of central nervous system is now possible.