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.

Chronotypes and their association with sleep quality among Chinese college students of Anhui Province: a cross-sectional study.

OBJECTIVES: To describe the prevalence of chronotype and sleep quality among Chinese college students and explore the relationship between chronotype and sleep quality. DESIGN: A cross-sectional study. SETTING: Four colleges and universities in Anhui, China, between November and December 2020. PARTICIPANTS: A total of 4768 college students were recruited using a stratified, multistage, cluster sampling survey. OUTCOME MEASURES: Morningness-Eveningness Questionnaire 19 was used to determine the chronotype of the students and the Pittsburgh Sleep Quality Index (PSQI) was used to measure their sleep quality. The multiple logistic regression model was used to explore the potential association between chronotype and sleep quality. RESULTS: The self-reported proportions of evening-type (E-type), neutral-type and morning-type among college students were 51.17%, 45.14% and 3.69%, respectively. The mean PSQI score was 4.97±2.82 and the prevalence of poor sleep quality was 18.2%. After adjusting the covariates by multiple logistic regression analysis, E-type was positively associated with subjective sleep quality (OR=1.671, 95% CI 1.414 to 1.975), sleep latency (OR=1.436, 95% CI 1.252 to 1.647), sleep duration (OR=2.149, 95% CI 1.506 to 3.067), habitual sleep efficiency (OR=1.702, 95% CI 1.329 to 2.180), daytime dysfunction (OR=1.602, 95% CI 1.412 to 1.818) and overall poor sleep quality (OR=1.866, 95% CI 1.586 to 2.196). CONCLUSIONS: College students mainly exhibited E-type, and an elevated prevalence of poor sleep quality existed among these students. The E-type was positively associated with poor sleep quality.

Curriculum vitae of HDAC6 in solid tumors.

Histone deacetylase 6 (HDAC6) is the only member of the HDAC family that resides primarily in the cytoplasm with two catalytic domains and a ubiquitin-binding domain. HDAC6 is highly expressed in various solid tumors and participates in a wide range of biological activities, including hormone receptors, the p53 signaling pathway, and the kinase cascade signaling pathway due to its unique structural foundation and abundant substrate types. Additionally, HDAC6 can function as an oncogenic factor in solid tumors, boosting tumor cell proliferation, invasion and metastasis, drug resistance, stemness, and lowering tumor cell immunogenicity, so assisting in carcinogenesis. Pan-HDAC inhibitors for cancer prevention are associated with potential cardiotoxicity in clinical investigations. It's interesting that HDAC6 silencing didn't cause any significant harm to normal cells. Currently, the use of HDAC6 specific inhibitors, individually or in combination, is among the most promising therapies in solid tumors. This review's objective is to give a general overview of the structure, biological functions, and mechanism of HDAC6 in solid tumor cells and in the immunological milieu and discuss the preclinical and clinical trials of selective HDAC6 inhibitors. These endeavors highlight that targeting HDAC6 could effectively kill tumor cells and enhance patients' immunity during solid tumor therapy.

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.

Clinical and Functional Features of Epilepsy-Associated In-Frame Deletion Variants in SCN1A.

Objective: Naturally occurring in-frame deletion is a unique type of genetic variations, causing the loss of one or more amino acids of proteins. A number of in-frame deletion variants in an epilepsy-associated gene SCN1A, encoding voltage gated sodium channel alpha unit 1.1 (Nav1.1), have been reported in public database. In contrast to the missense and truncation variants, the in-frame deletions in SCN1A remains largely uncharacterized. Methods: We summarized the basic information of forty-four SCN1A in-frame deletion variants and performed further analysis on six variants identified in our cases with epilepsy. Mutants of the six in-frame deletions and one truncating variant used as comparison were generated and co-transfected with beta-1 and -2 subunits in tsA201 cells, followed by patch clamp recordings. Results: Reviewing all the in-frame deletions showed that they spread over the entire Nav1.1 protein, without obvious "hot spots." The dominant type (54%) was single residue loss. There was no obvious relationship between the length or locations of deletions and their clinical phenotypes. The six in-frame deletions were two single residue deletions (p.M400del and p.I1772del), one microdeletion (p.S128_F130del) and three macrodeletions (p.T303_R322del, p.T160_Y202del, and p.V1335_V1428del). They scatter and affect different functional domains, including transmembrane helices, pore region, and P-loop. Electrophysiological recordings revealed no measurable sodium current in all of the six mutants. In contrast, the truncating mutant p.M1619Ifs*7 that loses a long stretch of peptides retains partial function. Significance: The complete loss-of-function in these shortened, abnormal mutants indicates that Nav1.1 protein is a highly accurate structure, and many of the residues have no redundancy to ion conductance. In-frame deletions caused particularly deleterious effect on protein function possibly due to the disruption of ordered residues.

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.

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.

[Discussion on safety of Xanthii Fructus and consideration on its rational use].

Xanthii Fructus is a traditional Chinese medicine for the treatment of sinusitis and headache,rich in medicinal materials and is widely used for more than 1 800 years. Modern pharmacological studies have showed that Xanthii Fructus has anti-inflammatory,analgesic,anti-tumor,anti-bacterial,hypoglycemic,anti-allergic,immunomodulatory and other pharmacological effects,which can be commonly used in the treatment of diseases relating to immune abnormalities,such as rheumatoid arthritis,acute and chronic rhinitis,allergic rhinitis,and skin diseases,with a high medicinal value. Toxicological studies have shown that Xanthii Fructus poisoning can cause substantial damage to organs,such as the liver,kidney,and gastrointestinal tract,especially to liver. Because of the coexisting of its efficacy and toxicity,Xanthii Fructus often leads to a series of safety problems in the clinical application process. This study attempts to summarize its characteristics of adverse reactions,analyze the root cause of the toxicity of Xanthii Fructus from such aspects as processing,dose,course of treatment and eating by mistake,discuss the substance of its efficacy/toxicity from chemical compositions,and put forward exploratory thinking about how to promote its clinical rational application from the aspects such as strict processing,reasonable compatibility,medication information,contraindication,strict control of the dose,and course of treatment,so as to promote the safe and reasonable application of Xanthii Fructus.

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.

Epigenetic Downregulation of Scn3a Expression by Valproate: a Possible Role in Its Anticonvulsant Activity.

Upregulation of sodium channel SCN3A expression in epileptic tissues is known to contribute to enhancing neuronal excitability and the development of epilepsy. Therefore, certain strategies to reduce SCN3A expression may be helpful for seizure control. Here, we reveal a novel role of valproate (VPA) in the epigenetic downregulation of Scn3a expression. We found that VPA, instead of carbamazepine (CBZ) and lamotrigine (LTG), could significantly downregulate Scn3a expression in mouse Neuro-2a cells. Luciferase assays and CpG methylation analyses showed that VPA induced the methylation at the -39C site in Scn3a promoter which decreased the promoter activity. We further showed that VPA downregulated the expression of methyl-CpG-binding domain protein 2 (MBD2) at the posttranscriptional level and knockdown of MBD2 increased Scn3a expression. In addition, we found that VPA induced the expression of fat mass and obesity-associated (FTO) protein and FTO knockdown abolished the repressive effects of VPA on MBD2 and Nav1.3 expressions. Furthermore, VPA, instead of other two anticonvulsant drugs, induced the expressions of Scn3a and Mbd2 and reduced Fto expression in the hippocampus of VPA-treated seizure mice. Taken together, this study suggests an epigenetic pathway for the VPA-induced downregulation of Scn3a expression, which provides a possible role of this pathway in the anticonvulsant action of VPA.

Involvement of FMRP in Primary MicroRNA Processing via Enhancing Drosha Translation.

Fragile X mental retardation protein (FMRP), associated with fragile X syndrome, is known as an RNA-binding protein to regulate gene expression at post-transcriptional level in the brain. FMRP is also involved in microRNA (miRNA) biogenesis during the process of precursor miRNA (pre-miRNA) into mature miRNA. However, there is no description of the effect of FMRP on primary miRNA (pri-miRNA) processing. Here, we uncover a novel role of FMRP in pri-miRNA processing via controlling Drosha translation. We show that the expression of DROSHA protein, instead of its messenger RNA (mRNA) transcripts, is downregulated in both the hippocampus of Fmr1-knockout mice and the FMRP-knockdown Neuro-2a cells. Overexpression or knockdown FMRP does not alter Drosha mRNA stability. Immunoprecipitation and polysome analyses demonstrate that FMRP binds to the Drosha mRNA and enhances its translation. Additionally, we show that loss of FMRP in Fmr1-deficient mice results in the accumulation of three in six analyzed pri-miRNAs and the reduction of the corresponding pre-miRNAs and mature miRNAs. Thus, our data suggest that FMRP is involved in pri-miRNA processing via enhancing DROSHA expression that may play an important role in fragile X syndrome.