Aizo Furukawa

Steroidogenic acute regulatory protein (StAR) transcripts constitutively expressed in the adult rat central nervous system : Colocalization of star, cytochrome P-450SCC (CYP XIA1), and 3β-hydroxysteroid dehydrogenase in the rat brain

Abstract: Steroidogenic acute regulatory protein (StAR) is a 30‐kDa protein involved in the transport of cholesterol to the inner mitochondrial membrane and thus plays a key role in steroid biosynthesis. To clarify the implications of this protein in neurosteroid biosynthesis, we examined the possible expression of a StAR transcript in the adult rat CNS and detected it. cDNA cloning and sequencing analysis revealed that two forms of StAR mRNAs are expressed in the brain in the same manner as in the adrenal gland, indicating that they are fully functional and not minor gene transcripts. An RNase protection assay quantitatively revealed that the amount of the rat StAR transcript in brain was two to three orders of magnitude lower than that in the adrenal gland. An in situ hybridization study, involving antisense riboprobes, revealed that StAR transcripts were abundant in the cerebral cortex, hippocampus, dentate gyrus, olfactory bulb, cerebellar granular layer, and Purkinje cells. Furthermore, other steroidogenic enzymes, side‐chain cleavage cytochrome P‐450SCC (CYP XIA1) and 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (EC 1.1.1.145), were found to be coexpressed in the hippocampus, dentate gyrus, cerebellar granular layer, and Purkinje cells. These findings strongly indicate that neurosteroids are synthesized in a region‐specific manner in the brain.

Enhanced carbonyl stress in a subpopulation of schizophrenia.

CONTEXTnVarious factors are involved in the pathogenesis of schizophrenia. Accumulation of advanced glycation end products, including pentosidine, results from carbonyl stress, a state featuring an increase in reactive carbonyl compounds (RCOs) and their attendant protein modifications. Vitamin B(6) is known to detoxify RCOs, including advanced glycation end products. Glyoxalase I (GLO1) is one of the enzymes required for the cellular detoxification of RCOs.nnnOBJECTIVESnTo examine whether plasma levels of pentosidine and serum vitamin B(6) are altered in patients with schizophrenia and to evaluate the functionality of GLO1 variations linked to concomitant carbonyl stress.nnnDESIGNnAn observational biochemical and genetic analysis study.nnnSETTINGnMultiple centers in Japan.nnnPARTICIPANTSnOne hundred six individuals (45 schizophrenic patients and 61 control subjects) were recruited for biochemical measurements. Deep resequencing of GLO1 derived from peripheral blood or postmortem brain tissue was performed in 1761 patients with schizophrenia and 1921 control subjects.nnnMAIN OUTCOME MEASURESnPentosidine and vitamin B(6) concentrations were determined by high-performance liquid chromatographic assay. Protein expression and enzymatic activity were quantified in red blood cells and lymphoblastoid cells using Western blot and spectrophotometric techniques.nnnRESULTSnWe found that a subpopulation of individuals with schizophrenia exhibit high plasma pentosidine and low serum pyridoxal (vitamin B(6)) levels. We also detected genetic and functional alterations in GLO1. Marked reductions in enzymatic activity were associated with pentosidine accumulation and vitamin B(6) depletion, except in some healthy subjects. Most patients with schizophrenia who carried the genetic defects exhibited high pentosidine and low vitamin B(6) levels in contrast with control subjects with the genetic defects, suggesting the existence of compensatory mechanisms.nnnCONCLUSIONSnOur findings suggest that GLO1 deficits and carbonyl stress are linked to the development of a certain subtype of schizophrenia. Elevated plasma pentosidine and concomitant low vitamin B(6) levels could be the most cogent and easily measurable biomarkers in schizophrenia and should be helpful for classifying heterogeneous types of schizophrenia on the basis of their biological causes.

Association between a novel polymorphism in the promoter region of the neuropeptide Y gene and schizophrenia in humans.

Hypoactivity of neuropeptide Y (NPY) is thought to be involved in the pathophysiology of schizophrenia, because post-mortem brain studies revealed a decrease of the NPY in schizophrenia, and antipsychotic treatments increase the NPY in animal brains and in cerebrospinal fluid of patients. We performed genetic analysis of the NPY gene in schizophrenia. Mutation screening of the gene detected nine single nucleotide polymorphisms, of which we typed a -485C>T variation from potential functional relevance. The -485T allele was overly represented in the disease group (P=0.0043). An in vitro promoter assay revealed that a C to T change at nt -485 significantly reduced transcriptional activity. These results suggest that the -485T allele in NPY may confer susceptibility to schizophrenia by decreasing the neuropeptide in brains.

Association analysis of the dopamine receptor D2 (DRD2) SNP rs1076560 in alcoholic patients

The dopamine system plays a well-established role in alcoholism. In this study, we examined the association between the single-nucleotide polymorphism (SNP) rs1076560 of the dopamine receptor D2 (DRD2) gene and susceptibility to alcoholism. SNP rs1076560 (C/A) is located in intron 6 of DRD2, where it is 1.4 kb downstream from alternative exon 6 and 83 bp upstream from exon 7. A total of 248 alcoholic patients and 322 healthy controls, all Japanese males, were genotyped for rs1076560 polymorphism by direct sequencing and allele-specific PCR. Data were analyzed using standard chi(2) statistics and a backwards logistic regression approach to adjust for the contribution of aldehyde dehydrogenase-2 (ALDH2) genotype status. The DRD2 risk allele A was more prevalent in the alcoholic patients (40.1%) than in the healthy controls (34.0%) (P=0.034, odds ratio=1.300, 95% confidence interval=1.020-1.657). These data identify SNP rs1076560 as a potentially important variable in the development of alcoholism.

Functional polymorphisms in the sigma1 receptor gene associated with alcoholism.

BACKGROUNDnSigma1 receptors are involved in the pathogenesis of drug abuse. Two polymorphisms (GC-241-240TT and Gln2Pro) in the sigma1 receptor gene (SIGMAR1) have been identified. To investigate the role of SIGMAR1 in conveying susceptibility to alcoholism, we performed a functional analysis of polymorphisms in the SIGMAR1 and a case-control study.nnnMETHODSnWe initially screened for polymorphisms in the 5-upstream region. The effects of the polymorphisms on transcriptional activity were determined using a gene reporter assay. The distribution of SIGMAR1 polymorphisms was analyzed in 307 alcoholic and 302 control subjects.nnnRESULTSnA novel T-485A polymorphism was identified. The transcriptional activity of the A-485 allele and the TT-241-240 allele was significantly reduced compared with that of the T-485 allele and the GC-241-240 allele. The frequencies of the A-485 allele (chi2=5.575, df=1, p=.0205) and the TT-241-240/Pro2 haplotype (chi2=21.464, df=1, p<.0001) were significantly higher in control subjects compared with alcoholic subjects. The T-485A and the GC-241-240TT may be functional polymorphisms, and the A-485 allele and TT-241-240/Pro2 haplotype are possible protective factors for the development of alcoholism.

Tissue‐specific alternative splicing of mouse brain type ryanodine receptor/calcium release channel mRNA

We detected alternative splicing of the mouse brain type ryanodine receptor (RyR3) mRNA. The splicing variant was located in the transmembrane segment. The non‐splicing type (RyR3‐II) included a stretch of 341 bp, and that of the 13th codon was stop codon TAA. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis shows that RyR3‐II mRNA was expressed in various peripheral tissues and brain at all developmental stages. However, interestingly, the splicing type (RyR3‐I) mRNA was detected only in the cerebrum. These findings suggest that the splicing variants RyR3‐I and RyR3‐II may generate functional differences of RyR3 in a tissue‐specific manner.

Synaptotagmin XI as a candidate gene for susceptibility to schizophrenia.

Synaptotagmin XI (Syt11) is a member of the synaptotagmin family, which is localized in cells either in synaptic vesicles or the cellular membrane, and is known to act as a calcium sensor. The Syt11 gene is located on chromosome locus 1q21‐q22, which was previously reported as a major susceptibility locus of familial schizophrenia. Here, we present evidence for an association between the number of 33‐bp repeats in the promoter region of the Syt11 gene and schizophrenia. We found that the transcriptional activity of the gene is affected by the number of 33‐bp repeats, which include an Sp1 binding site, suggesting that the excessive expression of Syt11 can be associated with schizophrenia. Another (single nucleotide) polymorphism in the Syt11 5′UTR region, where the potent transcription factor YY1 can bind, also affects the transcriptional activity of Syt11.

Molecular cloning and nucleotide sequences of cDNA clones of sheep and goat adrenocortical cytochromes P450scc (CYP11A1)

We isolated cDNA clones of the mRNAs for cytochromes P450scc (CYP11A1) from sheep and goat adrenocortices using the RT-PCR method. We determined the complete nucleotide sequences of the coding and 3-flanking regions of these cDNA clones. The results confirmed the amino terminal sequence of the sheep cytochrome P450scc reported previously [Miyatake et al., Biochim. Biophys. Acta 1215,176-182 (1994)]. On the basis of comparison of the deduced amino acid sequences of various animals and rainbow trout cytochromes P450scc, and other mitochondrial cytochrome P450 isozymes, we discussed the substrate-associated and adreno-ferredoxin-binding region of cytochrome P450scc.

Lack of association between sigma1 receptor gene variants and schizophrenia

Abstractu2003 Several pharmacological studies suggest the possible involvement of sigma1 receptors in the pathogenesis of schizophrenia. An association has been reported between schizophrenia and two variants (GC‐241–240TT and Gln2Pro) in the sigma1 receptor gene (SIGMAR1). We also previously reported that, along with T‐485u2003A, these two variants alter SIGMAR1 function. To investigate the role of SIGMAR1 in conveying susceptibility to schizophrenia, we performed a case‐control study. We initially screened for polymorphisms in the SIGMAR1 coding region using PCR‐single strand conformation polymorphism analysis. The distribution of SIGMAR1 polymorphisms was analyzed in 100 schizophrenic and 104 control subjects. A novel G620A variant was detected in exon4. G620A was predicted to alter the amino acid represented by codon 211 from arginine to glutamine. Our case‐control study showed no significant association between the T‐485u2003A, GC‐241–240TT, Gln2Pro, and G620A (Arg211Gln) variants and schizophrenia and clinical characteristics. These findings suggest that these SIGMAR1 variants may not affect susceptibility to schizophrenia.