Thyroid Hormone Receptor α Regulates Autophagy, Mitochondrial Biogenesis, and Fatty Acid Use in Skeletal Muscle.

Skeletal muscle (SM) weakness occurs in hypothyroidism and resistance to thyroid hormone α (RTHα) syndrome. However, the cell signaling and molecular mechanism(s) underlying muscle weakness under these conditions is not well understood. We thus examined the role of thyroid hormone receptor α (TRα), the predominant TR isoform in SM, on autophagy, mitochondrial biogenesis, and metabolism to demonstrate the molecular mechanism(s) underlying muscle weakness in these two conditions. Two genetic mouse models were used in this study: TRα1PV/+ mice, which express the mutant Thra1PV gene ubiquitously, and SM-TRα1L400R/+ mice, which express TRα1L400R in a muscle-specific manner. Gastrocnemius muscle from TRα1PV/+, SM-TRα1L400R/+, and their control mice was harvested for analyses. We demonstrated that loss of TRα1 signaling in gastrocnemius muscle from both the genetic mouse models led to decreased autophagy as evidenced by accumulation of p62 and decreased expression of lysosomal markers (lysosomal-associated membrane protein [LAMP]-1 and LAMP-2) and lysosomal proteases (cathepsin B and cathepsin D). The expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial transcription factor A (TFAM), and estrogen-related receptor α (ERRα), key factors contributing to mitochondrial biogenesis as well as mitochondrial proteins, were decreased, suggesting that there was reduced mitochondrial biogenesis due to the expression of mutant TRα1. Transcriptomic and metabolomic analyses of SM suggested that lipid catabolism was impaired and was associated with decreased acylcarnitines and tricarboxylic acid cycle intermediates in the SM from the mouse line expressing SM-specific mutant TRα1. Our results provide new insight into TRα1-mediated cell signaling, molecular, and metabolic changes that occur in SM when TR action is impaired.

Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein.

Postsynaptic density (PSD)-93, a neuronal scaffolding protein, binds to and clusters N-methyl-D-aspartate receptor (NMDAR) subunits NR2A and NR2B at cellular membranes in vitro. However, the roles of PSD-93 in synaptic NR2A and NR2B targeting in the central nervous system and NMDAR-dependent physiologic and pathologic processes are still unclear. We report here that PSD-93 deficiency significantly decreased the amount of NR2A and NR2B in the synaptosomal membrane fractions derived from spinal cord dorsal horn and forebrain cortex but did not change their levels in the total soluble fraction from either region. However, PSD-93 deficiency did not markedly change the amounts of NR2A and NR2B in either synaptosomal or total soluble fractions from cerebellum. In mice deficient in PSD-93, morphine dose-dependent curve failed to shift significantly rightward as it did in wild type (WT) mice after acute and chronic morphine challenge. Unlike WT mice, PSD-93 knockout mice also showed marked losses of NMDAR-dependent morphine analgesic tolerance and associated abnormal sensitivity in response to mechanical, noxious thermal, and formalin-induced inflammatory stimuli after repeated morphine injection. In addition, PSD-93 knockout mice displayed dramatic loss of jumping activity, a typical NMDAR-mediated morphine withdrawal abstinence behavior. These findings indicate that impaired NMDAR-dependent neuronal plasticity following repeated morphine injection in PSD-93 knockout mice is attributed to PSD-93 deletion-induced alterations of synaptic NR2A and NR2B expression in dorsal horn and forebrain cortex neurons. The selective effect of PSD-93 deletion on synaptic NMDAR expression in these two major pain-related regions might provide the better strategies for the prevention and treatment of opioid tolerance and physical dependence.

Rodent BDNF genes, novel promoters, novel splice variants, and regulation by cocaine.

Results from studies using molecular and genetic methods in humans and rodents suggest that brain-derived neurotrophic factor (BDNF) is involved in the behavioral effects of abused drugs, making understanding of its genomic structure and regulation of substantial interest. Recently, we have reported that the human BDNF gene contains seven upstream exons that can each be spliced independently to the major BDNF coding exon to form diverse bipartite BDNF transcripts. We also identified a novel "BDNFOS" gene that is transcribed to produce alternatively spliced natural antisense transcripts (NATs); its fifth exon overlaps with the protein coding exon VIII of human BDNF. To better understand BDNF's genomic structure and differential regulation, we now describe the rodent BDNF gene and transcripts. This gene includes six bipartite transcripts that are generated by six independently transcribed exons, each of which is spliced to a major coding exon and a tripartite transcript that is composed of two upstream exons and one coding exon. In addition, we found no evidence for antisense, opposite strand BDNFOS gene transcripts in mice or rats. The BDNF rodent splice variants display specific patterns of differential expression in different brain regions and peripheral tissues. Acute cocaine administration increased striatal expression of a specific BDNF4 splice variant by up to 5-fold. Interestingly, however, neither experimenter- nor self-administered chronic cocaine administration enhanced striatal BDNF expression. These data suggest a role of specific BDNF promoter regions and regulatory sequences in stimulant-induced alterations in BDNF expression, and in the alterations that changed BDNF expression is likely to confer in the brain.

Decision support system for the response to infectious disease emergencies based on WebGIS and mobile services in China.

BACKGROUND: For years, emerging infectious diseases have appeared worldwide and threatened the health of people. The emergence and spread of an infectious-disease outbreak are usually unforeseen, and have the features of suddenness and uncertainty. Timely understanding of basic information in the field, and the collection and analysis of epidemiological information, is helpful in making rapid decisions and responding to an infectious-disease emergency. Therefore, it is necessary to have an unobstructed channel and convenient tool for the collection and analysis of epidemiologic information in the field. METHODOLOGY/PRINCIPAL FINDINGS: Baseline information for each county in mainland China was collected and a database was established by geo-coding information on a digital map of county boundaries throughout the country. Google Maps was used to display geographic information and to conduct calculations related to maps, and the 3G wireless network was used to transmit information collected in the field to the server. This study established a decision support system for the response to infectious-disease emergencies based on WebGIS and mobile services (DSSRIDE). The DSSRIDE provides functions including data collection, communication and analyses in real time, epidemiological detection, the provision of customized epidemiological questionnaires and guides for handling infectious disease emergencies, and the querying of professional knowledge in the field. These functions of the DSSRIDE could be helpful for epidemiological investigations in the field and the handling of infectious-disease emergencies. CONCLUSIONS/SIGNIFICANCE: The DSSRIDE provides a geographic information platform based on the Google Maps application programming interface to display information of infectious disease emergencies, and transfers information between workers in the field and decision makers through wireless transmission based on personal computers, mobile phones and personal digital assistants. After a 2-year practice and application in infectious disease emergencies, the DSSRIDE is becoming a useful platform and is a useful tool for investigations in the field carried out by response sections and individuals. The system is suitable for use in developing countries and low-income districts.

NCoR1 regulates thyroid hormone receptor isoform-dependent adipogenesis.

We previously showed that two thyroid hormone receptor (TR) isoforms--TRα1 and TRß1--differentially regulate thyroid hormone (triiodothyroxine, T(3))-stimulated adipogenesis in vivo. This study aims to understand the role of the nuclear receptor corepressor, NCoR1, in TR isoform-dependent adipogenesis. We found that T(3)-stimulated adipogenesis of 3T3-L1 cells was accompanied by progressive loss of NCoR1 protein levels. In 3T3-L1 cells stably expressing a mutated TRα1, PV (L1-α1PV cells), the T(3)-stimulated adipogenesis was more strongly inhibited than that in 3T3-L1 cells stably expressing an identical mutation in TRß1 (L1-ß1PV cells). The stronger inhibition of adipogenesis in L1-α1PV cells was associated with a higher NCoR1 protein level. These results indicate that the degree of loss of NCoR1 correlates with the extent of adipogenesis. siRNA knockdown of NCoR1 promoted adipogenesis of control 3T3-L1 cells and reversed the inhibited adipogenesis of L1-α1PV and L1-ß1PV cells, indicating that NCoR1 plays an essential role in TR isoform-dependent adipogenesis. An ubiquitin ligase, mSiah2, that targets NCoR1 for proteasome degradation was upregulated on day 1 before the onset of progressive loss of NCoR1. NCoR1 was found to associate with mSiah2 and with TR, TRα1PV, or TRß1PV, but a stronger interaction of NCoR1 with TRα1PV than with TRß1PV was detected. Furthermore, TRα1PV-NCoR1 complex was more avidly recruited than TRß1PV-NCoR1 to the promoter of the C/ebpα gene, leading to more inhibition in its expression. These results indicate that differential interaction of NCoR1 with TR isoforms accounted for the TR isoform-dependent regulation of adipogenesis and that aberrant interaction of NCoR1 with TR could underlie the pathogenesis of lipid disorders in hypothyroidism.

Adipogenesis is differentially impaired by thyroid hormone receptor mutant isoforms.

To understand the roles of thyroid hormone receptors (TRs) in adipogenesis, we adopted a loss-of-function approach. We generated 3T3-L1 cells stably expressing either TRalpha1 mutant (TRalpha1PV) or TRbeta1 mutant (TRbeta1PV). TRalpha1PV and TRbeta1PV are dominant negative mutations with a frameshift in the C-terminal amino acids. In control cells, the thyroid hormone, tri-iodothyronine (T(3)), induced a 2.5-fold increase in adipogenesis in 3T3-L1 cells, as demonstrated by increased lipid droplets. This increase was mediated by T(3)-induced expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha (C/EBPalpha), which are master regulators of adipogenesis at both the mRNA and protein levels. In 3T3-L1 cells stably expressing TRalpha1PV (L1-alpha1PV cells) or TRbeta1PV (L1-beta1PV cells), adipogenesis was reduced 94 or 54% respectively, indicative of differential inhibitory activity of mutant TR isoforms. Concordantly, the expression of PPARgamma and C/EBPalpha at the mRNA and protein levels was more repressed in L1-alpha1PV cells than in L1-beta1PV cells. In addition, the expression of PPARgamma downstream target genes involved in fatty acid synthesis - the lipoprotein lipase (Lpl) and aP2 involved in adipogenesis - was more inhibited by TRalpha1PV than by TRbeta1PV. Chromatin immunoprecipitation assays showed that TRalpha1PV was more avidly recruited than TRbeta1PV to the promoter to preferentially block the expression of the C/ebpalpha gene. Taken together, these data indicate that impaired adipogenesis by mutant TR is isoform dependent. The finding that induction of adipogenesis is differentially regulated by TR isoforms suggests that TR isoform-specific ligands could be designed for therapeutic intervention for lipid abnormalities.

Neurexin 3 polymorphisms are associated with alcohol dependence and altered expression of specific isoforms.

Neurexins are cell adhesion molecules that help to specify and stabilize synapses and provide receptors for neuroligins, neurexophilins, dystroglycans and alpha-latrotoxins. We previously reported significant allele frequency differences for single nucleotide polymorphisms (SNPs) in the neurexin 3 (NRXN3) gene in each of two comparisons between individuals who were dependent on illegal substances and controls. We now report work clarifying details of NRXN3's gene structure and variants and documenting association of NRXN3 SNPs with alcohol dependence. We localize this association signal with the vicinity of the NRXN3 splicing site 5 (SS#5). A splicing site SNP, rs8019381, that is located 23 bp from the SS#5 exon 23 donor site displays association with P = 0.0007 (odds ratio = 2.46). Including or excluding exon 23 at SS#5 produces soluble or transmembrane NRXN3 isoforms. We thus examined expression of these NRXN3 isoforms in postmortem human cerebral cortical brain samples from individuals with varying rs8019381 genotypes. Two of the splice variants that encode transmembrane NRXN3 isoforms were expressed at significantly lower levels in individuals with the addiction-associated rs8019381 'T' allele than in CC homozygotes. Taken together with recent reports of NRXN3 association with nicotine dependence and linkage with opiate dependence, these data support roles for NRXN3 haplotypes that alter expression of specific NRXN3 isoforms in genetic vulnerabilities to dependence on a variety of addictive substances.