Inhibition of Hypothalamic FTO Activates STAT3 Signal through ERK1/2 Associated with Reductions in Food Intake and Body Weight.

INTRODUCTION: Fat mass and obesity-associated (FTO) gene is strongly associated with obesity which brings a major health threat. Altered expression of its encoded protein FTO in the hypothalamus has been identified to contribute to central control of appetite and body weight. However, its molecular mechanisms remain elusive. METHODS: Mouse hypothalamic POMC cell line N43/5 was treated with FTO inhibitor rhein, FTO shRNA, or extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 to inhibit FTO or ERK1/2. Rhein and U0126 were injected into lateral ventricle of the mice by intracerebroventricular cannulation. Western blotting and immunofluorescent assays were performed to monitor protein level. RESULTS: This study identified that inhibition of FTO in N43/5 cells led to phosphorylation of signal transducer and activator of transcription 3 (STAT3) at S727 site and induced p-STAT3-S727 nuclear translocation. We further showed that FTO inhibition promoted phosphorylation of ERK1/2; specific inhibition of ERK1/2 signaling by U0126 could abolish the effect of FTO inhibition on STAT3-S727 phosphorylation and nuclear translocation. Furthermore, we found that inhibition of hypothalamic FTO promoted STAT3-S727 phosphorylation in the hypothalamic arcuate nucleus, and the mice showed reductions in food intake and body weight. In addition, inhibition of hypothalamic ERK1/2 could abolish the effects of FTO inhibition on STAT3-S727 phosphorylation, reductions of food intake and body weight. CONCLUSION: Our in vitro and in vivo data suggest that the inhibition of hypothalamic FTO could activate STAT3 through ERK1/2, which is potentially associated with reductions in food intake and body weight.

Characterization of a silent azaphilone biosynthesis gene cluster in Aspergillus terreus NIH 2624.

Filamentous fungal secondary metabolites are an important source of bioactive components. Genome sequencing ofAspergillus terreusrevealed many silent secondary metabolite biosynthetic gene clusters presumed to be involved in producing secondary metabolites. Activation of silent gene clusters through overexpressing a pathway-specific regulator is an effective avenue for discovering novel fungal secondary metabolites. Replacement of the native promoter of the pathway-specific activator with the inducible Tet-on system to activate thetazpathway led to the discovery of a series of azaphilone secondary metabolites, among which azaterrilone A (1) was purified and identified for the first time. Genetic deletion of core PKS genes and transcriptional analysis further characterized thetazgene cluster to consist of 16 genes with the NR-PKS and the HR-PKS collaborating in a convergent mode. Based on the putative gene functions and the characterized compounds structural information, a biosynthetic pathway of azaterrilone A (1) was proposed.

Expression Pattern of ALOXE3 in Mouse Brain Suggests Its Relationship with Seizure Susceptibility.

Arachidonic acid (AA), a polyunsaturated fatty acid, is involved in the modulation of neuronal excitability in the brain. Arachidonate lipoxygenase 3 (ALOXE3), a critical enzyme in the AA metabolic pathway, catalyzes the derivate of AA into hepoxilins. However, the expression pattern of ALOXE3 and its role in the brain has not been described until now. Here we showed that the levels of Aloxe3 mRNA and protein kept increasing since birth and reached the highest level at postnatal day 30 in the mouse hippocampus and temporal cortex. Histomorphological analyses indicated that ALOXE3 was enriched in adult hippocampus, somatosensory cortex and striatum. The distribution was restricted to the neurites of function-specific subregions, such as mossy fibre connecting hilus and CA3 neurons, termini of Schaffer collateral projections, and the layers III and IV of somatosensory cortex. The spatiotemporal expression pattern of ALOXE3 suggests its potential role in the modulation of neural excitability and seizure susceptibility. In fact, decreased expression of ALOXE3 and elevated concentration of AA in the hippocampus was found after status epilepticus (SE) induced by pilocarpine. Local overexpression of ALOXE3 via adeno-associated virus gene transfer restored the elevated AA level induced by SE, alleviated seizure severities by increasing the latencies to myclonic switch, clonic convulsions and tonic hindlimb extensions, and decreased the mortality rate in the pilocarpine-induced SE model. These results suggest that the expression of ALOXE3 is a crucial regulator of AA metabolism in brain, and potentially acts as a regulator of neural excitability, thereby controlling brain development and seizure susceptibility.

High fat suppresses SOD1 activity by reducing copper chaperone for SOD1 associated with neurodegeneration and memory decline.

High fat consumption leads to reactive oxygen species (ROS) which is associated with age-progressive neurological disorders. Cu/Zn superoxide dismutase (SOD1) is a critical enzyme against ROS. However, the relationship between SOD1 and the high-fat-induced ROS and neurodegeneration is poorly known. Here we showed that, upon treatment with a saturated fatty acid palmitic acid (PA), the SOD1 activity was decreased in mouse neuronal HT-22 cell line accompanied by elevation of ROS, but not in mouse microglial BV-2 cell line. We further showed that PA decreased the levels of copper chaperone for SOD1 (CCS) in HT-22 cells, which promoted the nuclear import of SOD1 and decreased its activity. We demonstrated that the reduction of CCS is involved in the PA-induced decrease of SOD1 activity and elevation of ROS. In addition, compared with the adult mice fed with a standard diet, the high-fat-diet adult mice presented an increase of plasma free fatty acids, reduction of hippocampal SOD1 activity and CCS, mitochondrial degeneration and long-term memory decline. Taken together, our findings suggest that the high-fat-induced lower CCS level is essential for SOD1 suppression which may be associated with neurodegeneration and cognitive decline.

FMRP-absence-induced up-regulation of hypothalamic MAP1B expression decreases AgRP level linking with reduces in food intake and body weight.

Fragile X mental retardation protein (FMRP), strongly associated with fragile X syndrome, plays important roles by regulating gene expression via interacting with other RNA binding proteins in the brain. However, the role of FMRP in hypothalamus, a central part responsible for metabolic control, is poorly known. Our study shows that FMRP is primarily located in the hypothalamic arcuate nucleus (ARC). Using proteomic analysis, we identified 56 up-regulated and 22 down-regulated proteins in the hypothalamus of Map1b KO mice, with microtubule-associated protein 1 B (MAP1B) being the most outstanding increased protein (more than 10 folds). Immunofluorescent assays showed that MAP1B significantly increased in the Map1b-KO ARC, in which the number of agouti-related peptide (AgRP)-staining neurons significantly reduced, but not altered for pro-opiomelanocortin (POMC) neurons. We further showed an age-dependent reduces in food intake and body weight of the KO mice, along with the decreases of MAP1B and AgRP at the same time points. In hypothalamic GT1-7 cells, the AgRP expression decreased upon knockdown of FMRP or overexpression of MAP1B, and increased in response to overexpression of FMRP or knockdown of MAP1B. Co-knockdown or co-overexpression of FMRP and MAP1B led to a reverse expression of AgRP compared to overexpression of knockdown of FMRP alone, demonstrating that MAP1B is essential for the regulatory effect of FMRP on AgRP expression. Taken together, these data suggest that FMRP-deficiency-induced increase of hypothalamic MAP1B and decrease of AgRP might be associated with reduces in food intake and body weight.

A Species-Correlated Transitional Residue D132 on Human FMRP Plays a Role in Nuclear Localization via an RNA-Dependent Interaction With PABP1.

Fragile X mental retardation protein (FMRP), a key determinant of normal brain development and neuronal plasticity, plays critical roles in nucleocytoplasmic shuttling of mRNAs. However, the factors involved in FMRP nuclear localization remain to be determined. Using cross-species sequence comparison, we show that an aspartate in position 132 (D132), located within the conserved nuclear localization signal (NLS) of FMRP, appears in human and other mammals, while glutamate 132 (E132) appears in rodents and birds. Human FMRP-D132E alters the secondary structure of the protein and reduces its nuclear localization, while the reciprocal substitution in mouse FMRP-E132D promotes its nuclear localization. Human FMRP could interact with poly(A)-binding protein 1 (PABP1) which is impeded by the D132E mutation. Reversely, mouse FMRP could not interact with PABP1, but the E132D mutation leads to the FMRP-PABP1 interaction. We further show that overexpression of human FMRP-D132E mutant promotes the formation of cytoplasmic aggregates of PABP1 in human cells, but not of mouse FMRP-E132D in mouse cells. PABP1 knockdown reduces the nuclear localization of human FMRP, but not mouse FMRP. Furthermore, RNase A treatment decreases the PABP1 levels in the anti-V5-immunoprecipitates using the V5-hFMRP-transfected cells, suggesting an interaction between human FMRP and PABP1 in an RNA-dependent fashion. Thus, our data suggest that the FMRP protein with the human-used D132 accommodates a novel protein-RNA-protein interaction which may implicate a connection between FMRP residue transition and neural evolution.

Genome-based deletion analysis in Aspergillus terreus reveals the acetylaranotin bis-thiomethyltransferase gene.

Acetylaranotin is an epipolythiodiketopiperazine (ETP) secondary metabolite with a broad range of bioactivities. We demonstrated that ATEG_01465.1 located outside of acetylaranotin gene cluster is responsible for catalyzing the S-methylation of its biosynthetic pathway. Combining the previous characterization of acetylaranotin biosynthetic gene cluster together with the identification of its S-methyltransferase provides a means to obtain second-generation acetylaranotin derivatives previously inaccessible. By permutations of targeted deletions of ATEG_01465.1, acetyltransferase (AtaH), and benzoate hydroxylase (AtaY), three novel acetylaranotin derivatives were produced by Aspergillus terreus.

MDH2 is an RNA binding protein involved in downregulation of sodium channel Scn1a expression under seizure condition.

Voltage-gated sodium channel α-subunit type I (NaV1.1, encoded by SCN1A gene) plays a critical role in the excitability of brain. Downregulation of SCN1A expression is associated with epilepsy, a common neurological disorder characterized by recurrent seizures. Here we reveal a novel role of malate dehydrogenase 2 (MDH2) in the posttranscriptional regulation of SCN1A expression under seizure condition. We identified that MDH2 was an RNA binding protein that could bind two of the four conserved regions in the 3' UTRs of SCN1A. We further showed that knockdown of MDH2 or inactivation of MDH2 activity in HEK-293 cells increased the reporter gene expression through the 3' UTR of SCN1A, and MDH2 overexpression decreased gene expression by affecting mRNA stability. In the hippocampus of seizure mice, the upregulation of MDH2 expression contributed to the decrease of the NaV1.1 levels at posttranscriptional level. In addition, we showed that the H2O2 levels increased in the hippocampus of the seizure mice, and H2O2 could promote the binding of MDH2 to the binding sites of Scn1a gene, whereas ß-mercaptoethanol decreased the binding capability, indicating an important effect of the seizure-induced oxidation on the MDH2-mediated downregulation of Scn1a expression. Taken together, these data suggest that MDH2, functioning as an RNA-binding protein, is involved in the posttranscriptional downregulation of SCN1A expression under seizure condition.

A novel role of fragile X mental retardation protein in pre-mRNA alternative splicing through RNA-binding protein 14.

Fragile X mental retardation protein (FMRP), an important RNA-binding protein responsible for fragile X syndrome, is involved in posttranscriptional control of gene expression that links with brain development and synaptic functions. Here, we reveal a novel role of FMRP in pre-mRNA alternative splicing, a general event of posttranscriptional regulation. Using co-immunoprecipitation and immunofluorescence assays, we identified that FMRP interacts with an alternative-splicing-associated protein RNA-binding protein 14 (RBM14) in a RNA-dependent fashion, and the two proteins partially colocalize in the nuclei of hippocampal neurons. We show that the relative skipping/inclusion ratio of the micro-exon L in the Protrudin gene and exon 10 in the Tau gene decreased in the hippocampus of Fmr1 knockout (KO) mice. Knockdown of either FMRP or RBM14 alters the relative skipping/inclusion ratio of Protrudin and Tau in cultured Neuro-2a cells, similar to that in the Fmr1 KO mice. Furthermore, overexpression of FMRP leads to an opposite pattern of the splicing, which can be offset by RBM14 knockdown. RNA immunoprecipitation assays indicate that FMRP promotes RBM14's binding to the mRNA targets. In addition, overexpression of the long form of Protrudin or the short form of Tau promotes protrusion growth of the retinoic acid-treated, neuronal-differentiated Neuro-2a cells. Together, these data suggest a novel function of FMRP in the regulation of pre-mRNA alternative splicing through RBM14 that may be associated with normal brain function and FMRP-related neurological disorders.

Discovery of McrA, a master regulator of Aspergillus secondary metabolism.

Fungal secondary metabolites (SMs) are extremely important in medicine and agriculture, but regulation of their biosynthesis is incompletely understood. We have developed a genetic screen in Aspergillus nidulans for negative regulators of fungal SM gene clusters and we have used this screen to isolate mutations that upregulate transcription of the non-ribosomal peptide synthetase gene required for nidulanin A biosynthesis. Several of these mutations are allelic and we have identified the mutant gene by genome sequencing. The gene, which we designate mcrA, is conserved but uncharacterized, and it encodes a putative transcription factor. Metabolite profiles of mcrA deletant, mcrA overexpressing, and parental strains reveal that mcrA regulates at least ten SM gene clusters. Deletion of mcrA stimulates SM production even in strains carrying a deletion of the SM regulator laeA, and deletion of mcrA homologs in Aspergillus terreus and Penicillum canescens alters the secondary metabolite profile of these organisms. Deleting mcrA in a genetic dereplication strain has allowed us to discover two novel compounds as well as an antibiotic not known to be produced by A. nidulans. Deletion of mcrA upregulates transcription of hundreds of genes including many that are involved in secondary metabolism, while downregulating a smaller number of genes.

[A method for combining Fluoro-Jade B staining and immunofluorescent staining].

OBJECTIVE: To explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction. METHOD: Permanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order. RESULTS: FJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells. CONCLUSION: FJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus.

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis.

Postnatal high-protein diet improves learning and memory in premature rats via activation of mTOR signaling.

PURPOSE: The present study investigated whether a high-protein diet affects spatial learning and memory in premature rats via modulation of mammalian target of rapamycin (mTOR) signaling. METHODS: Pre- and full-term Sprague-Dawley pups were fed a normal (18% protein) or high-protein (30% protein) diet (HPD) for 6 or 8 weeks after weaning. Spatial learning and memory were tested in the Morris water maze at week 6 and 8. The activation of mTOR signaling pathway components was evaluated by western blotting. RESULTS: Spatial memory performance of premature rats consuming a normal and HPD was lower than that of full-term rats on the same diet at 6 weeks, and was associated with lower levels of ribosomal protein S6 kinase p70 subtype (p70S6K) and initiation factor 4E-binding protein 1 (4EBP1) phosphorylation in the hippocampus. Spatial memory was improved in 8-week-old premature rats on an HPD as compared to those on a normal diet. Premature rats on an HPD had p70S6K and 4EBP1 phosphorylation levels in the hippocampus that were comparable to those of full-term rats on an HPD. CONCLUSION: Long-term consumption of a protein-rich diet can restore the impairment in learning and memory in pre-term rats via upregulation of mTOR/p70S6K signaling.

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity.

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.

Molecular genetic characterization of terreic acid pathway in Aspergillus terreus.

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, combined with bioinformatic analyses, allowed for the proposition of a biosynthetic pathway for terreic acid.

A conserved region in the 3' untranslated region of the human LIMK1 gene is critical for proper expression of LIMK1 at the post-transcriptional level.

LIM kinase 1 (LIMK1), a cytosolic serine/threonine kinase, regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function. Abnormal expression of LIMK1 is associated with several neurological disorders. In this study, we performed a conservation analysis using Vector NTI (8.0) software. The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene, respectively. We found that a region ranging from nt +884 to +966 in the human LIMK1 3' untranslated region (UTR) was highly conserved in the mouse Limk1 3' UTR and formed a structure containing several loops and stems. Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted, pGL4-hLIMK1-3U-M, in SH-SY5Y and HEK-293 cells was only ~60% of that of the wild-type construct pGL4-hLIMK1-3U, indicating that the conserved region is critical for the reporter gene expression. Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to ~50% of that in cells transfected with pGL4-hLIMK1-3U, suggesting an important role of the conserved region in maintaining Luc2 mRNA stability. Our study suggests that the conserved region in the LIMK1 3' UTR is involved in regulating LIMK1 expression at the post-transcriptional level, which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3'-UTR mutant and neurological disorders.

[Expression of iNOS in the testis of Fmr1 knockout mice in different developmental stages].

OBJECTIVE: To analyze the expression of inducible nitric oxide synthase (iNOS) in the testis tissues of Fmr1 (fragile X mental retardation 1) knockout and wild-type male mice in different developmental stages, and provide background information for researches on fragile X syndrome. METHODS: This study included 4, 6, 8 and 10 weeks old Fmr1 knockout and wild-type male mice, 6 in each age group. We identified the genotype of the mice by PCR, and detected and compared the expression of iNOS in the testis tissues of the Fmr1 knockout and wild-type mice by immunohistochemistry. RESULTS: The iNOS expression was weakly positive in the Leydig cells of the 4-week-old mice, moderately positive in the 6-week-old ones, and strongly positive in 8- and 10-week-old ones, significantly weaker in the Fmr1 knockout than in the wild-type ones. CONCLUSION: The expression of iNOS significantly decreases in the testis of Fmr1 knockout mice, suggesting that iNOS may be involved in the pathogenesis of fragile X syndrome.

Early continuous inhibition of group 1 mGlu signaling partially rescues dendritic spine abnormalities in the Fmr1 knockout mouse model for fragile X syndrome.

RATIONALE: Abnormal dendritic spine morphology is a significant neuroanatomical defect in fragile X mental retardation. It has been suggested that overactive group 1 metabotropic glutamate receptor (mGlu) signaling is associated with the spine dysmorphology occurring in fragile X syndrome (FXS). Thus, group 1 mGlu became a new therapeutic target for the treatment of FXS. OBJECTIVE: The purpose of this study was to identify the effect of inhibition of mGlu signaling in FXS. METHODS: We observed the changes in dendritic spines after pharmacological modulation of mGlu signaling in an Fmr1 knockout (KO) mouse model. RESULTS: The activation of group 1 mGlu resulted in elongation of dendritic spines in the cultured neurons derived from Fmr1 KO mice and wild-type (WT) mice. Antagonism of group 1 mGlu reduced the average spine length of Fmr1 KO neurons. Furthermore, systemic administration of the selective group 1 mGlu5 antagonist 2-methyl-6-phenylethynyl pyridine (MPEP) reduced the average spine length and density in the cortical neurons of Fmr1 KO mice at developmental age. For the adult mice, MPEP administration was less effective for the restoration of spine length. The percentage of immature spines showed a similar reduction in parallel to the changes of spine length. Temporary MPEP intervention with single-dose treatment did not show any effect. CONCLUSION: These results show that MPEP administration could partially rescue the morphological deficits of dendritic spines in Fmr1 KO mice at developmental age.

Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: associated with loss of function of Na(v) 1.1.

PURPOSE: Generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy in infancy (SMEI) are associated with sodium channel α-subunit type-1 gene (SCN1A) mutations. Febrile seizures and partial seizures occur in both GEFS+ and SMEI; sporadic onset and seizure aggravation by antiepileptic drugs (AEDs) are features of SMEI. We thus searched gene mutations in isolated cases of partial epilepsy with antecedent FS (PEFS+) that showed seizure aggravations by AEDs. METHODS: Genomic DNA from four patients was screened for mutations in SCN1A, SCN2A, SCN1B, and GABRG2 using denaturing high-performance liquid chromatography (dHPLC) and sequencing. Whole-cell patch clamp analysis was used to characterize biophysical properties of two newly defined mutants of Na(v) 1.1 in tsA201 cells. RESULTS: Two heterozygous de novo mutations of SCN1A (R946H and F1765L) were detected, which were proven to cause loss of function of Na(v) 1.1. When the functional defects of mutants reported previously are compared, it is found that all mutants from PEFS+ have features of loss of function, whereas GEFS+ shows mild dysfunction excluding loss of function, coincident with mild clinical manifestations. PEFS+ is similar to SMEI clinically with possible AED-induced seizure aggravation and biophysiologically with features of loss of function, and different from SMEI by missense mutation without changes in hydrophobicity or polarity of the residues. CONCLUSIONS: Isolated milder PEFS+ may associate with SCN1A mutations and loss of function of Na(v) 1.1, which may be the basis of seizure aggravation by sodium channel-blocking AEDs. This study characterized phenotypes biologically, which may be helpful in understanding the pathophysiologic basis, and further in management of the disease.