小坂 仁

BACKGROUND: Mutations of voltage-gated sodium channel αII gene, SCN2A, have been described in a wide spectrum of epilepsies. While inherited SCN2A mutations have been identified in multiple mild epilepsy cases, a de novo SCN2A-R102X mutation, which we previously reported in a patient with sporadic intractable childhood localization-related epilepsy, remains unique. To validate the involvement of de novo SCN2A mutations in the etiology of intractable epilepsies, we sought to identify additional instances. METHODS: We performed mutational analyses on SCN2A in 116 patients with severe myoclonic epilepsy in infancy, infantile spasms, and other types of intractable childhood partial and generalized epilepsies and did whole-cell patch-clamp recordings on Nav1.2 channels containing identified mutations. RESULTS: We discovered 2 additional de novo SCN2A mutations. One mutation, SCN2A-E1211K, was identified in a patient with sporadic infantile spasms. SCN2A-E1211K produced channels with altered electrophysiologic properties compatible with both augmented (a ∼18-mV hyperpolarizing shift in the voltage dependence of activation) and reduced (a ∼22-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation and a slowed recovery from inactivation) channel activities. The other de novo mutation, SCN2A-I1473M, was identified in a patient with sporadic neonatal epileptic encephalopathy. SCN2A-I1473M caused a ∼14-mV hyperpolarizing shift in the voltage dependence of activation. CONCLUSIONS: The identified de novo mutations SCN2A-E1211K,-I1473M, and-R102X indicate that SCN2A is an etiologic candidate underlying a variety of intractable childhood epilepsies. The phenotypic variations among patients might be due to the different electrophysiologic properties of mutant channels. Copyright © 2009 by AAN Enterprises, Inc.

11歳男児。患者は四肢の不随意運動、歩行障害を主訴とした。既往として9歳時から足底に異常感があり、10歳頃からは転倒することが多く、易疲労感があった。今回、所見ではジストニア歩行で、体の種々の部位にミオクローヌスが認められた。だが、血液検査、腹部エコー、CT検査では肝病変を示唆する所見はみられなかった。入院時の臨床経過、MRI等から若年型Huntington病を疑い、検査では血清銅ならびにセルロプラスミンの低値、尿中の銅排泄亢進が認められ、診断的治療の目的で塩酸トリエンチン・亜鉛薬が開始された。その結果、治療開始後の尿中銅排泄は著明な増加を示し、Wilson病に矛盾しない所見であった。確定診断のため肝生検を行ったところ、肝細胞内に銅蓄積が認められ、Wilson病と診断、その経過、画像所見により「神経型」と考えられた。クロナゼパムの内服によりミオクローヌスは改善し、手指の巧緻性は改善傾向にあったが、ジストニアは増悪し、退院時は車椅子移動であった。

We reported two cases of Pelizaeus-Merzbacher disease. Both cases visited our hospital manifesting horizontal nystagmoid movements present front birth, and delayed motor development. Magnetic resonance imaging of the brain showed diffuse dysmyelination of the cerebral white matter, and auditory brainstem response showed waves I and II but absence of all Subsequent components. Conditioned orientation reflex (COR) audiometry showed poor reactions in an infantile case whose development was severely retarded, and who spoke no meaningful words. Auditory steady-state response (ASSR) was it helpful tool for identifying her auditory ability; thereafter, her communication skills improved naturally. The other case was mildly developmentally retarded, and the results of COR audiometry and ASSR were considered the same level. The discrepancy between results Of these hearing tests may arise under the influence of developmental level of the case. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy(1). Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3-q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species(2-4). Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.

Numerous numbers of pre-, peri- and postnatal damages cause West syndrome in early infancy, however, etiology in many cases are not still elucidated despite intensive biochemical and neuroradiologic investigations. We described four patients having early onset epileptic encephalopathy with severe hypomyelination and reduction in cerebral white matter. The clinical symptoms of these patients are impaired Visual attention, acquired microcephaly, spastic tetraplegia, profound psychomotor delay and infantile spasms since early infancy. All patients had striking hypomyelination of cerebrum, reduced volume of white matter and cortical atrophy on MRI. Serial MRI investigations in three patients showed absence of myelination of the white matter. On EEG, one patient revealed suppression-burst and other three had hypsarrhythmia. Despite having intractable seizures, no patient showed deterioration of neurological development. The group of these findings is mimicking to clinical manifestations of 3-phosphoglycerate dehydrogenase deficiency, and has some overlap with progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) like syndrome, however it is not compatible with these two conditions. The findings observed in our patients can be regarded as a new clinical condition associated with early onset

We investigated the roles of mutations in voltage-gated sodium channel alpha 1 subunit gene (SCN1A) in epilepsies and psychiatric disorders. The SCN1A gene was screened for mutations in three unrelated Japanese families with generalized epilepsy with febrile seizure plus (GEFS+), febrile seizure with myoclonic seizures, or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). In the family with GEFS+, one individual was affected with panic disorder and seizures, and another individual was diagnosed with Asperger syndrome and seizures. The novel mutation V13661 was found in all probands and patients with psychiatric disorders of the three families. These results suggest that SCN1A mutations may confer susceptibility to psychiatric disorders in addition to variable epileptic seizures. Unidentified modifiers may play critical roles in determining the ultimate phenotype of patients with sodium channel mutations. (c) 2007 Elsevier B.V. All rights reserved.

The 193M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the 193M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing 193M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing 193M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in 193M Tg mice. The midbrains of 193M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(193M) leads to the degeneration of dopaminergic neurons. (c) 2006 Elsevier Ltd. All rights reserved.

Loss-of-function mutations of the parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP). Parkin was shown to function as a RING-type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin-potentiated adenosine triphosphate (ATP)-induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP-induced inward currents were measured in mock-, wild-type or mutant (T415N)-parkin-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in wild-type parkin-transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP-dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca (2+) and calmodulin-dependent protein kinase (CaMKII). ATP-induced currents were also regulated by phosphatases and cyclin-dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32), though the phosphorylation at Thr-34 and Thr-75 were unchanged or rather attenuated. We also tried to investigate the effect of a-synuclein, a substrate of parkin and also forming Lysine 63-linked multiubiquitin chains. Expression of a-synuclein did not affect the amplitude of ATP-induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity.

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.