Characterization of mutations in the gene doublecortin in patients with double cortex syndrome.

Mutations in the X-linked gene doublecortin, which encodes a protein with no dear structural homologues, are found in pedigrees in which affected females show "double cortex" syndrome (DC; also known as subcortical band heterotopia or laminar heterotopia) and affected males show X-linked lissencephaly. Mutations in doublecortin also cause sporadic DC in females. To determine the incidence of doublecortin mutations in DC, we investigated a cohort of eight pedigrees and 47 sporadic patients with DC for mutations in the doublecortin open reading frame as assessed by single-stranded conformational polymorphism analysis. Mutations were identified in each of the eight DC pedigrees (100%), and in 18 of the 47 sporadic DC patients (38%). Identified mutations were of two types, protein truncation mutations and single amino acid substitution mutations. However, pedigrees with DC displayed almost exclusively single amino acid substitution mutations, suggesting that patients with these mutations may have less of a reproductive disadvantage versus those patients with protein truncation mutations. Single amino acid substitution mutations were tightly clustered in two regions of the open reading frame, suggesting that these two regions are critical for the function of the Doublecortin protein.

Transcriptional regulation of mouse mu-opioid receptor gene.

Previously, the existence of dual promoters was reported in mouse mu-opioid receptor (mor) gene, with mor transcription in the mouse brain predominantly initiated by the proximal promoter. In this study, we further analyzed the proximal promoter region, base pairs -450 to -249, to identify cis-DNA regulatory elements and trans-acting protein factors that are important for mor promoter activity. The results revealed that a mor inverted GA (iGA) motif and a canonical Sp1 binding site are required for the promoter activity. Using electrophoretic mobility shift analysis, we identified nuclear proteins that specifically bind to the mor iGA motif and that are immunologically related to Sp1 and Sp3. Mutation of the mor iGA motif, resulting in a loss of Sp binding, led to a 50% decrease in activity. Mutation of the canonical Sp1 binding site yielded a lesser (approximately 25%) loss of activity. Mutation of both motifs together resulted in an approximately 70% decrease in activity. In cotransfection assays using Drosophila SL2 cells, Sp1 trans-activated the promoter in a manner dependent on the presence of mor iGA and canonical Sp1 binding motifs. Sp3 can also trans-activate the promoter, and furthermore, Sp1 and Sp3 can trans-activate the mor promoter additively. Our results suggest that combined or cooperative interaction of Sp transcription factors within the proximal promoter is necessary for activation of mor gene transcription.

Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein.

X-linked lissencephaly and "double cortex" are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. We identified a novel 10 kb brain-specific cDNA interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration.

Dual promoters of mouse mu-opioid receptor gene.

Two transcriptional initiation sites, distal and proximal, located at approximately 500 bp apart in mouse mu-opioid receptor gene were identified recently. Using deletional and transfection analyses, the distal or proximal promoter alone displayed similar activity in neuronal cells constitutively expressing mu-opioid receptors (SH-SY-5Y). The presence of both promoters did not result in an increase in activity. However, when distal promoter linked with 3'-downstream sequences without the proximal promoter region, the distal promoter activity was abolished. Transfection analysis using various cell lines further suggested that only the proximal promoter preferentially directed the neuronal subtype expression. To verify the physiological importance of each promoter, the ratio of mu-receptor transcripts initiated by either promoter was examined by quantitative RT-PCR using mouse adult brain mRNA. We found that receptor mRNA was predominantly initiated by the proximal promoter. These studies suggested that the proximal promoter was the major functional promoter.

Differential expression of opioid receptor genes in human lymphoid cell lines and peripheral blood lymphocytes.

The existence of receptors for opioid compounds on cells of the immune system has long been hypothesized, but has been very difficult to demonstrate unequivocally. We have used reverse-transcription polymerase chain reaction to obtain cDNA clones from the human MOLT-4 and CEM-3 T-leukemic cell lines which are nearly identical to portions of the delta and kappa opioid receptor cDNAs recently isolated from human brain and placenta, respectively. Northern analyses with riboprobes derived from the delta and kappa opioid receptor clones indicate these sequences are expressed at low levels in human peripheral blood lymphocytes and in several human lymphoid cell lines. Sequences corresponding to the mu opioid receptor cDNA were not detected in this study. The results suggest that delta and kappa opioid receptors may be responsible for mediating some direct effects of opioids in immune cells.

Expression of alternate forms of brain opioid 'orphan' receptor mRNA in activated human peripheral blood lymphocytes and lymphocytic cell lines.

We screened a PHA (phytohemagglutinin)-activated human lymphocyte cDNA library for clones with homology to the recently cloned brain opioid receptors. A cDNA clone, AT7-5EU, was isolated which encodes the opioid 'orphan' receptor, a molecule with very high homology to the opioid receptor gene family, but which has not been shown to bind opioids or any other known compounds. The protein coding region of AT7-5EU has complete homology with a reported opioid 'orphan' clone isolated from human brain, but the 5' untranslated regions of AT7-5EU and the human brain clones are divergent, suggesting mechanisms for tissue-specific expression of this receptor. Northern analysis of AT7-5EU mRNA demonstrates the expression of this message in human lymphocytic cell lines of both B-and T-cell lineages. Furthermore, analysis of mRNA from human peripheral blood lymphocytes demonstrates that activation of the lymphocytes with PHA results in at least a 10-fold induction of the AT7-5EU message. These results suggest that the opioid 'orphan' receptor may have an important immunological function in addition to its function in the nervous system.

Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors.

A rat brain cDNA library was screened for clones homologous to the recently cloned mouse delta-opioid receptor (DOR-1). Among the clones isolated was Hyp 8-1, a clone with a unique nucleotide sequence capable of encoding a putative protein which is 57-58% identical to the amino acid sequences of the cloned delta, mu and kappa opioid receptors, indicating a close relationship of Hyp 8-1 with the opioid receptor family. Several cDNAs representing possible splice variants of Hyp 8-1 were also isolated. Binding studies of COS-7 cells transfected with clone Hyp 8-1 failed to demonstrate specific binding with several 3H-opioid ligands. In situ hybridization studies indicate that the mRNA for Hyp 8-1 is distributed discretely throughout the rat brain, in an overall pattern which is different from that of several other G-protein-coupled seven transmembrane receptors. Thus, it is likely that the Hyp 8-1 cDNA encodes a novel peptide receptor.

Nerve glucose, sorbitol, fructose, and myo-inositol at various time after feeding in streptozotocin-induced diabetes in rats.

Because fasting nerve myo-inositol (myo-Ins) is not decreased in patients with diabetes treated conventionally, in whom neuropathy may develop or may have already developed, it seems unlikely that myo-Ins is the metabolic abnormality that initiates diabetic neuropathy, if nerve myo-Ins is not spuriously high during fasting. At 6 weeks after induction of diabetes with streptozotocin in Sprague-Dawley rats we found that endoneurial glucose, sorbitol, and fructose were substantially increased and myo-Ins was decreased, as reported by others. By use of multiple linear regression to assess the effect of disease condition, time after feeding (1.5, 4, and 20 hours), and duration of diabetes (1,2,6 and 12 weeks) on nerve sugars and alcohol sugars, for nerve glucose, sorbitol, and fructose only disease condition was statistically significant factor. For nerve myo-Ins, disease group (control or diabetic rats) (P = 0.08), duration of diabetes (P = 0.02), time after feeding (P less than 0.001), duration x time (P = 0.01), group x duration (P = 0.03), and group x time (P = 0.05) were significant variables. Because nerve myo-Ins was lower at 20 hours than at 1.5 or 4 hours after feeding for all durations of diabetes, one can infer that in human nerve the lowest nerve myo-Ins, not high values, occur in the fasting state. Therefore, one would not expect to find lower nerve myo-Ins levels in human diabetics at times other than the fasting condition.

Resistance to axonal degeneration after nerve compression in experimental diabetes.

To determine the effect of diabetes on the development of axonal degeneration after acute nerve compression, the mobilized peroneal nerves of rats with streptozotocin-induced diabetes and of control rats were compressed at 150 mmHg (1 mmHg = 133 Pa) for 30 min by using specially devised cuffs. At three intervals after compression--3 days, rats diabetic for 31 wk; 14 days, diabetic for 6 wk; and 24 days, diabetic for 31 wk--groups of nerves were studied to assess numbers and sizes of fibers above, at, and below the cuff and to assess frequency of fiber degeneration in teased fibers from nerve distal to the cuff. Teased fibers with pathologic abnormalities were more frequent in nerves from controls than in nerves from diabetic rats in all three groups but the difference was statistically significant only at 3 and 14 days after compression. The lack of significant difference at 24 days may be explained by higher rates of disappearance of degenerating products and of fiber regeneration at 24 than at 3 and 14 days. This study provides evidence that in addition to delaying the reported functional deficit of vibratory detection threshold and conduction block during nerve compression, diabetes also may partially prevent axonal injury. Low nerve myo-inositol concentration did not predispose diabetic nerve to acute compression injury. If these results also apply to human diabetes and if repeated acute compression is involved in the genesis of fiber degeneration in entrapment, then a higher frequency of entrapment neuropathy among diabetics might be due to mechanisms other than increased susceptibility of fibers to acute compression--e.g., possibly to greater constriction of nerve due to pathologic alterations of the carpal ligament.

Nerve glucose, fructose, sorbitol, myo-inositol, and fiber degeneration and regeneration in diabetic neuropathy.

We measured the alcohol sugars in sural nerves from 11 controls, 21 conventionally treated patients with diabetes and neuropathy, and 4 diabetics without neuropathy. The results were related to metabolic control and to clinical, neuropathological, and morphometric abnormalities in the nerves. The mean endoneurial glucose, fructose, and sorbitol values were higher in diabetic patients than in controls. Linear regression analysis revealed that nerve sorbitol content in the diabetics was inversely related to the number of myelinated fibers (P = 0.003). Mean nerve levels of myo-inositol were not decreased in the diabetic patients, with or without neuropathy, and were not associated with any of the neuropathological end points of diabetes. Our results indicate that myo-inositol deficiency is not part of the pathogenesis of human diabetic neuropathy, as had been hypothesized. Other accumulated alcohol sugars, however, were increased in diabetes and were associated with the severity of neuropathy. On repeat biopsy, six diabetics, treated for a year with the aldose reductase inhibitor sorbinil, had decreased endoneurial levels of sorbitol (P less than 0.01) and fructose (0.05 less than P less than 0.1), but unchanged levels of myo-inositol.