Narcolepsy is a disabling sleep disorder affecting humans and animals. It is characterized by daytime sleepiness, cataplexy, and striking transitions from wakefulness into rapid eye movement (REM) sleep. In this study, we used positional cloning to identify an autosomal recessive mutation responsible for this sleep disorder in a well-established canine model. We have determined that canine narcolepsy is caused by disruption of the hypocretin (orexin) receptor 2 gene (Hcrtr2). This result identifies hypocretins as major sleep-modulating neurotransmitters and opens novel potential therapeutic approaches for narcoleptic patients.

In myotonic dystrophy (DM), expression of RNA containing expanded CUG or CCUG repeats leads to misregulated alternative splicing of pre-mRNA. The repeat-bearing transcripts accumulate in nuclear foci, together with proteins in the muscleblind family, MBNL1 and MBNL2. In transgenic mice that express expanded CUG repeats, we show that the splicing defect selectively targets a group of exons that share a common temporal pattern of developmental regulation. These exons undergo a synchronized splicing switch between post-natal day 2 and 20 in wild-type mice. During this post-natal interval, MBNL1 protein translocates from a predominantly cytoplasmic to nuclear distribution. In the absence of MBNL1, these physiological splicing transitions do not occur. The splicing defect induced by expanded CUG repeats in mature muscle fibers is closely reproduced by deficiency of MBNL1 but not by deficiency of MBNL2. A parallel situation exists in human DM type 1 and type 2. MBNL1 is depleted from the muscle nucleoplasm because of sequestration in nuclear foci, and the associated splicing defects are remarkably similar to those observed in MBNL1 knockout mice. These results indicate that MBNL1 participates in the post-natal remodeling of skeletal muscle by controlling a key set of developmentally regulated splicing switches. Sequestration of MBNL1, and failure to maintain these splicing transitions, has a pivotal role in the pathogenesis of muscle disease in DM.

Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. Here we use a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with Muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.

Human narcolepsy-cataplexy, a sleep disorder associated with a centrally mediated hypocretin (orexin) deficiency, is tightly associated with HLA-DQB1*0602. Few studies have investigated the influence that additional HLA class II alleles have on susceptibility to this disease. In this work, 1,087 control subjects and 420 narcoleptic subjects with cataplexy, from three ethnic groups, were HLA typed, and the effects of HLA-DRB1, -DQA1, and -DQB1 were analyzed. As reported elsewhere, almost all narcoleptic subjects were positive for both HLA-DQA1*0102 and -DQB1*0602. A strong predisposing effect was observed in DQB1*0602 homozygotes, across all ethnic groups. Relative risks for narcolepsy were next calculated for heterozygous DQB1*0602/other HLA class II allelic combinations. Nine HLA class II alleles carried in trans with DQB1*0602 were found to influence disease predisposition. Significantly higher relative risks were observed for heterozygote combinations including DQB1*0301, DQA1*06, DRB1*04, DRB1*08, DRB1*11, and DRB1*12. Three alleles-DQB1*0601, DQB1*0501, and DQA1*01 (non-DQA1*0102)-were found to be protective. The genetic contribution of HLA-DQ to narcolepsy susceptibility was also estimated by use of lambda statistics. Results indicate that complex HLA-DR and -DQ interactions contribute to the genetic predisposition to human narcolepsy but that additional susceptibility loci are also most likely involved. Together with the recent hypocretin discoveries, these findings are consistent with an immunologically mediated destruction of hypocretin-containing cells in human narcolepsy-cataplexy.

Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUG(exp)) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUG(exp) RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUG(exp) RNA, when compared with Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUG(exp) RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUG(exp) RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding pre-mRNAs. These results support the idea that trans-dominant effects of CUG(exp) RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.

The s allele variant of the serotonin transporter gene (5-HTT) has recently been observed to moderate the relationship of stress to depression and anxiety. To date no study has considered interactive effects of 5-HTT genotype, stress and hypothalamic-pituitary-adrenal (HPA) function on cognition in healthy, older adults, which may reflect developmental, functional or neurodegenerative effects of the serotonin transporter polymorphism. We investigated whether 5-HTT genotype interacts with cumulative life stress and HPA-axis measures of waking and diurnal cortisol slope to impact cognition in 154 non-depressed, older adults. Structural images of hippocampal volume were acquired on a subsample of 56 participants. The 5-HTT s allele was associated with both significantly lower delayed recall and higher waking cortisol levels. Presence of the s allele interacted with higher waking cortisol to negatively impact memory. We also observed a significant interaction of higher waking cortisol and the s allele on lower hippocampal volume. Smaller hippocampi and higher cortisol were associated with lower delayed recall only in s allele carriers. No impact or interactions of cumulative life stress with 5-HTT or cortisol were observed. This is the first investigation to identify an association of the 5-HTT s allele with poorer memory function in older adults. The interactive effects of the s allele and waking cortisol levels on reduced hippocampal volume and lower memory suggest that the negative effect of the serotonin polymorphism on memory is mediated by the HPA axis. Further, given the significant association of the s allele with higher waking cortisol in our investigation, future studies may be needed to evaluate the impact of the serotonin transporter polymorphism on any neuropsychiatric or behavioral outcome which is influenced by HPA axis function in older adults.

No abstract

The first committed step in the formation of taxol has been shown to involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer, taxa-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-2H2,20-2H3] and [20-2H3]geranylgeranyl diphosphates were employed as substrates with the partially purified taxadiene synthase from Pacific yew (Taxus brevifolia) stems to examine the possibility of a preliminary cyclization to taxa-4(20),11(12)-diene followed by isomerization to the more stable endocyclic double bond isomer. GLC-MS analysis of the derived taxa-4(5),11(12)-diene, via selected ion monitoring of the parent ion and the P-15 and C-ring fragment ions, compared to those of unlabeled standard, showed the olefin product to possess a deuterium enrichment essentially identical to that of the acyclic precursor, thus ruling out the putative isomerization step. With [4-2H2]geranylgeranyl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5),11(12)-diene to contain only one deuterium atom, consistent with direct formation from a taxenyl cation by deprotonation at C5. (+/-)-Casbene, (+/-)-verticillene, and (+/-)-taxa-4(20),11(12)-diene were tested as possible olefinic intermediates in taxa-4(5),11(12)-diene formation by a series of inhibition, trapping, and direct conversion experiments; no evidence was obtained that these exogenous olefins could serve as intermediates of the cyclization reaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-3H]geranylgeranyl diphosphate in 2H2O indicated little incorporation of deuterium from the medium and suggested a rapid internal proton transfer in a tightly bound olefinic intermediate. Analysis of the enzymatic product generated from [10-2H1]geranylgeranyl diphosphate confirmed the intramolecular hydrogen transfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5),11(12)-diene. From these results, a stereochemical mechanism is proposed for the taxadiene synthase reaction involving the initial cyclization of geranylgeranyl diphosphate to a transient verticillyl cation intermediate, with transfer of the C11 alpha-proton to C7 to initiate transannular B/C-ring closure to the taxenyl cation, followed by deprotonation at C5 to yield the taxa-4(5),11(12)-diene product directly.