Hippocampal proteome comparison of infant and adult Fmr1 deficiency mice reveals adult-related changes associated with postsynaptic density.

Deficiency in fragile X mental retardation 1 (Fmr1) leads to loss of its encoded protein FMRP and causes fragile X syndrome (FXS) by dysregulating its target gene expression in an age-related fashion. Using comparative proteomic analysis, this study identified 105 differentially expressed proteins (DEPs) in the hippocampus of postnatal day 7 (P7) Fmr1-/y mice and 306 DEPs of P90 Fmr1-/y mice. We found that most DEPs in P90 hippocampus were not changed in P7 hippocampus upon FMRP absence, and some P90 DEPs exhibited diverse proteophenotypes with abnormal expression of protein isoform or allele variants. Bioinformatic analyses showed that the P7 DEPs were mainly enriched in fatty acid metabolism and oxidoreductase activity and nutrient responses; whereas the P90 PEPs (especially down-regulated DEPs) were primarily enriched in postsynaptic density (PSD), neuronal projection development and synaptic plasticity. Interestingly, 25 of 30 down-regulated PSD proteins present in the most enriched protein to protein interaction network, and 6 of them (ANK3, ATP2B2, DST, GRIN1, SHANK2 and SYNGAP1) are both FMRP targets and autism candidates. Therefore, this study suggests age-dependent alterations in hippocampal proteomes upon loss of FMRP that may be associated with the pathogenesis of FXS and its related disorders. SIGNIFICANCE: It is well known that loss of FMRP resulted from Fmr1 deficiency leads to fragile X syndrome (FXS), a common neurodevelopmental disorder accompanied by intellectual disability and autism spectrum disorder (ASD). FMRP exhibits distinctly spatiotemporal patterns in the hippocampus between early development and adulthood, which lead to distinct dysregulations of gene expression upon loss of FMRP at the two age stages potentially linked to age-related phenotypes. Therefore, comparison of hippocampal proteomes between infancy and adulthood is valuable to provide insights into the early causations and adult-dependent consequences for FXS and ASD. Using a comparative proteomic analysis, this study identified 105 and 306 differentially expressed proteins (DEPs) in the hippocampi of postnatal day 7 (P7) and P90 Fmr1-/y mice, respectively. Few overlapping DEPs were identified between P7 and P90 stages, and the P7 DEPs were mainly enriched in the regulation of fatty acid metabolism and oxidoreduction, whereas the P90 DEPs were preferentially enriched in the regulation of synaptic formation and plasticity. Particularly, the up-regulated P90 proteins are primarily involved in immune responses and neurodegeneration, and the down-regulated P90 proteins are associated with postsynaptic density, neuron projection and synaptic plasticity. Our findings suggest that distinctly changed proteins in FMRP-absence hippocampus between infancy and adulthood may contribute to age-dependent pathogenesis of FXS and ASD.

Hippocampal proteomic changes in high-fat diet-induced obese mice associated with memory decline.

Substantial evidence suggest that chronic consumption of high-fat diets (HFDs) can lead to obesity, abnormal metabolism, as well as cognitive impairment. Molecular and cellular changes regarding hippocampal dysfunctions have been identified in multiple HFD animal models. Therefore, in-depth identification of expression changes of hippocampal proteins is critical for understanding the mechanism of HFD-induced cognitive deficits. In this study, we fed 3-week-old male mice with HFD for 3 months to generate obese mice who exhibit systemic metabolic abnormality and learning and memory decline. Using an iTRAQ-labeled proteomic analysis, we identified a total of 82 differentially expressed proteins (DEPs) in the hippocampus upon HFD with 35 up-regulated proteins and 47 down-regulated proteins. Functional enrichment indicated that these DEPs were predominantly enriched in regulation of catabolic process, dendritic shaft, neuron projection morphogenesis and GTPase regulator activity. Protein-protein interaction enrichment showed that the DEPs are mostly enriched in postsynaptic functions; and of them, six proteins (i.e., DLG3, SYNGAP1, DCLK1, GRIA4, GRIP1, and ARHGAP32) were involved in several functional assemblies of the postsynaptic density including G-protein signaling, scaffolding and adaptor, kinase and AMPA signaling, respectively. Collectively, our findings suggest that these DEPs upon HFD might contribute to memory decline by disturbing neuronal and postsynaptic functions in the hippocampus.

Proteome profiling of the prefrontal cortex of Fmr1 knockout mouse reveals enhancement of complement and coagulation cascades.

Fragile X mental retardation protein (FMRP) deficit resulted from mutations in its encoded fragile X mental retardation 1 (Fmr1) gene is a common inherited cause of Fragile X syndrome (FXS) characterized by intellectual disability and autism spectrum disorder (ASD). The FMRP absence-induced altered gene expression in prefrontal cortex (PFC) are associated with autistic behaviors. However, there lacks a large-scale protein profiling in the PFC upon loss of FMRP. This study used a TMT-labeled proteomic analysis to identify a protein profile of the PFC in the Fmr1 knockout mouse. A total of 5886 proteins were identified in the PFC with 100 differentially abundant proteins (DAPs) in response to FMRP deficiency. Bioinformatical analyses showed that these DAPs were mostly enriched in immune system, extracellular part and complement and coagulation cascades. The complement and coagulation cascades include 6 upregulated proteins (SERPING1, C1QA, C3, FGA, FGB and FGG), which are associated with fibrin degradation, cell lysis, degranulation chemotaxis and phagocytosis linked to activation of immune and inflammatory responses. Thus, our data provide an altered protein profile upon loss of FMRP in the PFC, and suggest that the enhancement of complement and coagulation cascades might contribute to etiological and pathogenic roles of ASD in FXS. SIGNIFICANCE: The etiology of autism spectrum disorder (ASD), a group of neurobiological disorders characterized by deficits in social interaction barriers and other abnormal behaviors, is still elusive. Autistic-like phenotypes are present in both Fragile X syndrome (FXS) patients and FMRP-deficiency FXS models. Given that prefrontal cortex is a critical brain area for social interaction, the FMRP absence induced-changes of a subset of proteins might contribute to ASD in FXS. Using a comprehensive proteomic analysis, this study provides a prefrontal protein profile of the FMRP-absent mouse with a total of 100 differentially abundant proteins (DAPs). Bioinformatic analyses suggest that these DAPs are mainly involved in the regulations of immune system and complement and coagulation cascades. We also show that 6 upregulated proteins (SERPING1, C1QA, C3, FGA, FGB and FGG) in the complement and coagulation cascades are associated with fibrin degradation, cell lysis, degranulation chemotaxis and phagocytosis regarding dysregulation of immune and inflammatory responses in the prefrontal cortex. Therefore, this study suggests that these FMRP-deficient DAPs in the prefrontal cortex might contribute to the etiology and pathogenesis of ASD in FXS.

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.

Lysine acetylome profiling in mouse hippocampus and its alterations upon FMRP deficiency linked to abnormal energy metabolism.

Loss of fragile X retardation protein (FMRP) leads to fragile X syndrome (FXS), a common cause of inherited intellectual disability. Protein lysine acetylation (K-ac), a reversible post-translational modification of proteins, is associated with the regulation of brain development and neuropathies. However, a comprehensive hippocampal K-ac protein profile in response to FMRP deficiency has not been reported until now. Using LC-MS/MS to analyze the enriched K-ac peptides, this study identified 1629 K-ac hits across 717 proteins in the mouse hippocampus, and these proteins were enriched in several metabolic processes. Of them, 51 K-ac hits across 45 proteins were significantly changed upon loss of FMRP. These altered K-ac proteins were enriched in energy metabolic processes including carboxylic acid metabolism process, aerobic respiration and citrate cycle, linking with several neurological disorders such as lactic acidosis, Lewy body disease, Leigh disease and encephalopathies. In the mouse hippocampus and the hippocampal HT-22 cells, FMRP deficiency could induce altered K-ac modification of several key enzymes, decrease in ATP and increase in lactate. Thus, this study identified a global hippocampal lysine acetylome and an altered K-ac protein profile upon loss of FMRP linked to abnormal energy metabolism, implicating in the pathogenesis of FXS. SIGNIFICANCE: Fragile X syndrome (FXS) is a common inherited neurodevelopment disorder characterized by intellectual disability and an increased risk for autism spectrum disorder. FXS is resulted from silencing of the FMR1 gene, which induces loss of its encoding protein FMRP. Molecular and metabolic changes of Fmr1-null animal models of FXS have been identified to potentially contribute to the pathogenesis of FXS. Here, we used a TMT-labeled quantitative proteomic analysis of the peptides enriched by anti-K-ac antibodies and identified a global K-ac protein profile in the mouse hippocampus with a total of 1629 K-ac peptides on 717 proteins. Of them, 51 K-ac peptides regarding 45 proteins altered in response to loss of FMRP, which were enriched in energy metabolic processes and were implicated in several neurological disorders. Thus this study for the first time provides a global hippocampal lysine acetylome upon FMRP deficiency linked to abnormal metabolic pathways, which may contribute to pathogenic mechanism of FXS.

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.

The ALOXE3 gene variants from patients with Dravet syndrome decrease gene expression and enzyme activity.

Aberrant expression or dysfunction of a number of genes in the brain contributes to epilepsy, a common neurological disorder characterized by recurrent seizures. Local overexpression of arachidonate lipoxygenase 3 (ALOXE3), a key enzyme for arachidonic acid (AA) metabolic pathway, alleviates seizure severities. However, the relationship between the ALOXE3 gene mutation and epilepsy has not been reported until now. Here we firstly characterized the promoter of human ALOXE3 gene and found that the ALOXE3 promoter could drive luciferase gene expression in the human HEK-293 and SH-SY5Y cells. We then screened the ALOXE3 promoter region and all coding exons from those patients with Dravet syndrome and identified 5 variants c.-163T > C, c.-50C > G, c.-37G > A, c. + 228G > A and c. + 290G > T in the promoter region and one missense variant c.1939A > G (p.I647 V) in the exon. Of these variants in the promoter region, only -50C > G was a novel variant located on the transcriptional factor NFII-I binding element. Luciferase reporter gene analyses indicated that the c.-50C > G could decrease gene expression by preventing the TFII-I's binding. In addition, the variant p.I647 V was conserved among all analyzed species and located within the ALOXE3 functional domain for catalyzing its substrate. In cultured cell lines, overexpression of ALOXE3 significantly decreased the cellular AA levels and overexpression of ALOXE3-I647 V could restore the AA levels, suggesting that the p.I647 V mutant led to a decrease in enzyme activity. Taken together, the present study proposes that the identified ALOXE3 variants potentially contribute to the AA-pathway-mediated epileptogenesis, which should provide a novel avenue for clinical diagnosis of epilepsy.

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.

Ilepcimide inhibited sodium channel activity in mouse hippocampal neurons.

Ilepcimide (ICM), a clinically effective antiepileptic drug, has been used in China for decades; however, its antiepileptic mechanism remains unclear. ICM is structurally similar to antiepileptic drug lamotrigine (LTG). LTG exerts its anticonvulsant effect by inhibiting voltage-gated Na+ channel (NaV) activity. Thus it is speculated that ICM also exert its antiepileptic activity by inhibiting sodium channel activity. We studied the inhibition of NaV activity by ICM in acutely isolated mouse hippocampal pyramidal neurons. We evaluated ICM-mediated tonic, concentration-dependent, and voltage-dependent inhibition of NaV, and the effects of ICM and LTG on NaV biophysical properties. Na+ currents in hippocampal pyramidal neurons were tonically inhibited by ICM in a concentration- and voltage-dependent manner. The half-maximal inhibitory concentration (IC50) of ICM at a holding potential (Vh) of -90 mV was higher than that at a Vh of -70 mV. Compared with the control groups, in the presence of 10 µM ICM, the current densities of Na+ channels were reduced, the half-maximal availability of the inactivation curve (V1/2) was shifted to more negative potentials, and the recovery from inactivation was delayed. These data can contribute to further investigation of the inhibitory effect of ICM on the sodium channel, suggesting that the main reason for the anticonvulsant effect of ICM is the small influx of sodium ions. ICM can prevent abnormal discharge of neurons, which may prevent epilepsy.

Valproate-Induced Epigenetic Upregulation of Hypothalamic Fto Expression Potentially Linked with Weight Gain.

Valproate (VPA), a widely-used antiepileptic drug, is a selective inhibitor of histone deacetylase (HDAC) that play important roles in epigenetic regulation. The patient with different diseases receiving this drug tend to exhibit weight gain and abnormal metabolic phenotypes, but the underlying mechanisms remain largely unknown. Here we show that VPA increases the Fto mRNA and protein expression in mouse hypothalamic GT1-7 cells. Interestingly, VPA promotes histone H3/H4 acetylation and the FTO expression which could be reversed by C646, an inhibitor for histone acetyltransferase. Furthermore, VPA weakens the FTO's binding and enhances the binding of transcription factor TAF1 to the Fto promoter, and C646 leads to reverse effect of the VPA, suggesting an involvement of the dynamic of histone H3/H4 acetylation in the regulation of FTO expression. In addition, the mice exhibit an increase in the food intake and body weight at the beginning of 2-week treatment with VPA. Simultaneously, in the hypothalamus of the VPA-treated mice, the FTO expression is upregulated and the H3/H4 acetylation is increased; further the FTO's binding to the Fto promoter is decreased and the TAF1's binding to the promoter is enhanced, suggesting that VPA promotes the assembly of the basal transcriptional machinery of the Fto gene. Finally, the inhibitor C646 could restore the effects of VPA on FTO expression, H3/H4 acetylation, body weight, and food intake; and loss of FTO could reverse the VPA-induced increase of body weight and food intake. Taken together, this study suggests an involvement of VPA in the epigenetic upregulation of hypothalamic FTO expression that is potentially associated with the VPA-induced weight gain.

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.

Hypothalamic proteome changes in response to nicotine and its withdrawal are potentially associated with alteration in body weight.

Nicotine, a major addictive component in tobacco, plays an important role in the changes of body weight upon smoking and its cessation. Here we showed that nicotine-treated mice exhibited weight loss and nicotine withdrawal led to weight gain. Using TMT-based proteomic analysis, we obtained the different hypothalamic protein profiles in response to nicotine and its withdrawal. A total of ~5000 proteins were identified from the hypothalamus with 50 altered proteins upon 28-day nicotine treatment and 28 altered proteins upon 15-day nicotine withdrawal. Of the altered proteins, CASP3, LCMT2, GRIN2D, CCNT2, FADS3 and MRPS18B were inversely changed in response to nicotine and withdrawal, coincidence with the change of body weight. Of them, CASP3, LCMT2, GRIN2D and CCNT2 were found to be associated with several GO terms and KEGG pathways linking with cell apoptosis, neurotransmission and metabolism. Further Western blot and RT-qPCR analyses confirmed that the levels of the 4 proteins CASP3, LCMT2, GRIN2D and CCNT2, instead of their mRNA transcripts, altered in response to nicotine and withdrawal. Thus this study provides nicotine- and withdrawal-induced hypothalamic protein profiles and suggests potential roles of these altered proteins in the change of body weight. SIGNIFICANCE: Cigarette smoking is one of important factors harming human health. Most smokers tend to have lower body weights and smoking cessation often lead to overweight or obesity, which is an important reason for smokers to insist on smoking. It is known that nicotine, a critical component in tobacco, is associated with the alteration in body weight by affecting hypothalamic function. Through TMT-based proteomic analysis, this study identified differential hypothalamic protein profiles in response to nicotine treatment and its withdrawal, and 4 nicotine- and withdrawal-induced contrary proteins CASP3, LCMT2, GRIN2D and CCNT2 are involved in several enriched GO terms and KEGG pathways, which are associated with cell apoptosis, neurotransmission and metabolism. Our study may provide novel targets for further investigation of the molecular mechanisms of nicotine- and withdrawal-induced alteration in body weight.

Optimization of in silico tools for predicting genetic variants: individualizing for genes with molecular sub-regional stratification.

Genes are unique in functional role and differ in their sensitivities to genetic defects, but with difficulties in pathogenicity prediction. This study attempted to improve the performance of existing in silico algorithms and find a common solution based on individualization strategy. We initiated the individualization with the epilepsy-related SCN1A variants by sub-regional stratification. SCN1A missense variants related to epilepsy were retrieved from mutation databases, and benign missense variants were collected from ExAC database. Predictions were performed by using 10 traditional tools with stepwise optimizations. Model predictive ability was evaluated using the five-fold cross-validations on variants of SCN1A, SCN2A, and KCNQ2. Additional validation was performed in SCN1A variants of damage-confirmed/familial epilepsy. The performance of commonly used predictors was less satisfactory for SCN1A with accuracy less than 80% and varied dramatically by functional domains of Nav1.1. Multistep individualized optimizations, including cutoff resetting, domain-based stratification, and combination of predicting algorithms, significantly increased predictive performance. Similar improvements were obtained for variants in SCN2A and KCNQ2. The predictive performance of the recently developed ensemble tools, such as Mendelian clinically applicable pathogenicity, combined annotation-dependent depletion and Eigen, was also improved dramatically by application of the strategy with molecular sub-regional stratification. The prediction scores of SCN1A variants showed linear correlations with the degree of functional defects and the severity of clinical phenotypes. This study highlights the need of individualized optimization with molecular sub-regional stratification for each gene in practice.

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.

FTO is a transcriptional repressor to auto-regulate its own gene and potentially associated with homeostasis of body weight.

Fat mass and obesity-associated (FTO) protein is a ferrous ion (Fe2+)/2-oxoglutarate (2-OG)-dependent demethylase preferentially catalyzing m6A sites in RNA. The FTO gene is highly expressed in the hypothalamus with fluctuation in response to various nutritional conditions, which is believed to be involved in the control of whole body metabolism. However, the underlying mechanism in response to different nutritional cues remains poorly understood. Here we show that ketogenic diet-derived ketone body ß-hydroxybutyrate (BHB) transiently increases FTO expression in both mouse hypothalamus and cultured cells. Interestingly, the FTO protein represses Fto promoter activity, which can be offset by BHB. We then demonstrate that FTO binds to its own gene promoter, and Fe2+, but not 2-OG, impedes this binding and increases FTO expression. The BHB-induced occupancy of the promoter by FTO influences the assembly of the basal transcriptional machinery. Importantly, a loss-of-function FTO mutant (I367F), which induces a lean phenotype in FTOI367F mice, exhibits augmented binding and elevated potency to repress the promoter. Furthermore, FTO fails to bind to its own promoter that promotes FTO expression in the hypothalamus of high-fat diet-induced obese and 48-h fasting mice, suggesting a disruption of the stable expression of this gene. Taken together, this study uncovers a new function of FTO as a Fe2+-sensitive transcriptional repressor dictating its own gene switch to form an auto-regulatory loop that may link with the hypothalamic control of body weight.

Autapses enhance bursting and coincidence detection in neocortical pyramidal cells.

Autapses are synaptic contacts of a neuron's axon onto its own dendrite and soma. In the neocortex, self-inhibiting autapses in GABAergic interneurons are abundant in number and play critical roles in regulating spike precision and network activity. Here we examine whether the principal glutamatergic pyramidal cells (PCs) also form functional autapses. In patch-clamp recording from both rodent and human PCs, we isolated autaptic responses and found that these occur predominantly in layer-5 PCs projecting to subcortical regions, with very few in those projecting to contralateral prefrontal cortex and layer 2/3 PCs. Moreover, PC autapses persist during development into adulthood. Surprisingly, they produce giant postsynaptic responses (∼5 fold greater than recurrent PC-PC synapses) that are exclusively mediated by AMPA receptors. Upon activation, autapses enhance burst firing, neuronal responsiveness and coincidence detection of synaptic inputs. These findings indicate that PC autapses are functional and represent an important circuit element in the neocortex.

Laminar Distribution of Neurochemically-Identified Interneurons and Cellular Co-expression of Molecular Markers in Epileptic Human Cortex.

Inhibitory GABAergic interneurons are fundamental elements of cortical circuits and play critical roles in shaping network activity. Dysfunction of interneurons can lead to various brain disorders, including epilepsy, schizophrenia, and anxiety. Based on the electrophysiological properties, cell morphology, and molecular identity, interneurons could be classified into various subgroups. In this study, we investigated the density and laminar distribution of different interneuron types and the co-expression of molecular markers in epileptic human cortex. We found that parvalbumin (PV) and somatostatin (SST) neurons were distributed in all cortical layers except layer I, while tyrosine hydroxylase (TH) and neuropeptide Y (NPY) were abundant in the deep layers and white matter. Cholecystokinin (CCK) neurons showed a high density in layers IV and VI. Neurons with these markers constituted ~7.2% (PV), 2.6% (SST), 0.5% (TH), 0.5% (NPY), and 4.4% (CCK) of the gray-matter neuron population. Double- and triple-labeling revealed that NPY neurons were also SST-immunoreactive (97.7%), and TH neurons were more likely to express SST (34.2%) than PV (14.6%). A subpopulation of CCK neurons (28.0%) also expressed PV, but none contained SST. Together, these results revealed the density and distribution patterns of different interneuron populations and the overlap between molecular markers in epileptic human cortex.

Long-term moderate exercise enhances specific proteins that constitute neurotrophin signaling pathway: A TMT-based quantitative proteomic analysis of rat plasma.

Physical exercise has been reported to increase neurotrophin in brain tissues as hippocampus as well as increased neurotrophic level peripherally in blood plasma and might have an effect on/or affect molecular processes of energy metabolism (and homeostasis). In this study, using quantitative proteomic analysis, we obtained a plasma protein profile from the rat with long-term moderate exercise. A total of 752 proteins were identified in the plasma. Among them, 54 proteins were significant up-regulated and 47 proteins were down-regulated in the plasma of exercise group compared with the control group. Bioinformatic analyses showed that these altered proteins are widely involved in multiple biological processes, molecular functions and cellular components, which connect with 11 signaling pathways. Interestingly, 5 up-regulated proteins Rap1b, PTPN11, ARHGDIA, Cdc42 and YWHAE, confirmed by Western blots, are involved in the neurotrophin signaling pathway which shows the lowest P value among the identified pathways. Further analyses showed that the 5 neurotrophin-signaling-pathway-related proteins participate in two important protein-protein interaction networks associated to cell survival and apoptosis, axonal development, synapse formation and plasticity. This study provides an exercise-induced plasma protein profile, suggesting that long-term exercise enhances the proteins involved in neurotrophin signaling pathway which may contribute to health benefit. SIGNIFICANCE: Physical activity contributes to myriad benefits on body health across the lifespan. The changes in plasma proteins after chronic moderate exercise may be used as biomarkers for health and may also play important roles in increase of cardiovascular fitness, enhancement of immune competence, prevention of obesity, decrease of risk for neurological disorders, cancer, stroke, diabetes and other metabolic disorders. Using a TMT-based proteomic method, this study identified 101 altered proteins in the plasma of rats after long-term moderate treadmill running, which may provide novel biomarkers for further investigation of the underlying mechanism of physical exercise. We confirmed that exercise enhances 5 proteins of the neurotrophin signaling pathway that may contribute to health benefits.

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.