Syt1 Expression Research Articles

MiRNA-363-3p Hinders Proliferation, Migration, Invasion and Autophagy of Thyroid Cancer Cells by Controlling SYT1 Transcription to affect NF-κB.

Thyroid cancer (TC) is a frequent endocrine malignant tumor with various pathologic types. miRNA-363-3p plays a pivotal part in the occurrence, development, prognosis, and treatment of cancer. To explore the mechanism of miRNA-363-3p in TC and provide a new idea for targeted therapy of TC. Differential miRNAs and downstream target mRNAs in TC tissues were predicted with bioinformatics analysis. Expression levels of miRNA-363-3p and Synaptotagmin I (SYT1) in TC cells were ascertained by qRT-PCR. Cell migration, invasion, and proliferation were detected by wound healing assay, transwell assay, colony formation assay, CCK-8, and BrdU fluorescence experiment, respectively. Flow cytometry was utilized to detect the levels of apoptosis and necrosis. Immunofluorescence assay was used for detecting autophagosome formation in cells, and the expression levels of autophagy-related proteins, as well as NF-κB related proteins, were measured by western blot. Dual-luciferase reporter gene assay was applied for detecting the interaction between miRNA-363-3p and SYT1. miRNA-363-3p was prominently down-regulated in TC cells. miRNA-363-3p overexpression suppressed migration, invasion, and proliferation, promoting apoptosis and necrosis of TC cells. As the downstream target of miRNA-363-3p, SYT1 was up-regulated in TC cells. SYT1 overexpression reversed the inhibition of TC cell proliferation, invasion, migration, and autophagy mediated by miRNA-363-3p overexpression. In addition, miRNA-363-3p overexpression inhibited the activation of the NF-κB pathway in cells, while further overexpression of SYT1 weakened the inhibition of miRNA-363-3p overexpression on the NF-κB pathway. miRNA-363-3p affected the NF-κB signaling pathway by down-regulating SYT1 expression to inhibit the malignant progression of TC cells, providing theoretical support for the treatment of TC.

MiR-128 regulates epilepsy sensitivity in mice by suppressing SNAP-25 and SYT1 expression in the hippocampus

Epilepsy is accompanied by abnormal neurotransmission, and microRNAs, as versatile players in the modulation of gene expression, are important in epilepsy pathology. Here, we found that miR-128 expression was elevated in the acute seizure phase and decreased during the recurrent seizure phase after status epilepticus in mice. Both SNAP-25 and SYT1 are regulated by miR-128 in vitro and in vivo. Overexpressing miR-128 in cultured neurons decreased neurotransmitter released by suppressing SNAP-25 and SYT1 expression. Anti-miR-128 injection before kainic acid (KA) injection increased the sensitivity of mice to KA-induced seizures, while overexpressing miR-128 at the latent and recurrent phases had a neuroprotective effect in KA-induced seizures. Our study shows for the first time that miR-128, a key regulator of neurotransmission, plays an important role in epilepsy pathology and that miR-128 might be a potential candidate molecular target for epilepsy therapy.

Hepatic Synaptotagmin 1 is involved in the remodelling of liver plasma- membrane lipid composition and gene expression in male Apoe-deficient mice consuming a Western diet

Background and aims.The molecular mechanisms by which the liver develops steatotic disease still remain unclear. Previous studies using nutritional and genetic models of hepatic steatosis in mice showed that liver synaptotagmin 1 (Syt1) expression was associated with lipid droplet area. Hepatic Syt1 overexpression was used as a tool to explore its effect on hepatic and plasma lipids. Methods and resultsTo find out a cause-effect, hepatic mouse Syt1 mRNA was cloned into a vector driving hepatocyte-specific expression and administered by hydrodynamic injection to male Apoe-deficient mice fed on a Western diet, the latter as a model of rapid spontaneous steatosis development. Hepatic microsomal, large vesicle, lysosomal and plasma membrane fractions were enriched in SYT1 protein following gene overexpression. In these conditions, very low density lipoprotein esterified cholesterol increased. Likewise, the transgene caused an alteration in lipid droplet surface and a positive correlation between Syt1 expression and hepatic total cholesterol content. A lipidomic approach evidenced a decrease in lysophosphatidylcholine, phosphatidylcholine and triglycerides in isolated plasma membrane fraction. Expressions of genes involved in biosynthesis of bile acids, fatty acid metabolism, lipoprotein dynamics and vesicular transport were modified by the increased SYT1 expression. ConclusionsThese results indicate that this protein is involved in hepatic management of lipids and in the regulation of genes involved in lipid metabolism.

Effects of Prenatal Exposure to Inflammation Coupled With Stress Exposure During Adolescence on Cognition and Synaptic Protein Levels in Aged CD-1 Mice.

Age-associated impairment of spatial learning and memory (AISLM) presents substantial challenges to our health and society. Increasing evidence has indicated that embryonic exposure to inflammation accelerates the AISLM, and this can be attributable, at least partly, to changed synaptic plasticity associated with the activities of various proteins. However, it is still uncertain whether social psychological factors affect this AISLM and/or the expression of synaptic protein-associated genes. Synaptotagmin-1 (Syt1) and activity-regulated cytoskeleton-associated protein (Arc) are two synaptic proteins closely related to cognitive functions. In this study, pregnant CD-1 mice received daily intraperitoneal injections of lipopolysaccharide (LPS) (50 μg/kg) or normal saline at days 15–17 of gestation, and half of the offspring of each group were then subjected to stress for 28 days in adolescence. The Morris water maze (MWM) test was used to separately evaluate spatial learning and memory at 3 and 15 months of age, while western blotting and RNAscope assays were used to measure the protein and mRNA levels of Arc and Syt1 in the hippocampus. The results showed that, at 15 months of age, control mice had worse cognitive ability and higher protein and mRNA levels of Arc and Syt1 than their younger counterparts. Embryonic exposure to inflammation or exposure to stress in adolescence aggravated the AISLM, as well as the age-related increase in Arc and Syt1 expression. Moreover, the hippocampal protein and mRNA levels of Arc and Syt1 were significantly correlated with the performance in the learning and memory periods of the MWM test, especially in the mice that had suffered adverse insults in early life. Our findings indicated that prenatal exposure to inflammation or stress exposure in adolescence exacerbated the AISLM and age-related upregulation of Arc and Syt1 expression, and these effects were linked to cognitive impairments in CD-1 mice exposed to adverse factors in early life.

An Epilepsy-Associated SV2A Mutation Disrupts Synaptotagmin-1 Expression and Activity-Dependent Trafficking.

The epilepsy-linked gene SV2A, has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity.SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.

Synaptotagmin 1 Is Involved in Neuropathic Pain and Electroacupuncture-Mediated Analgesic Effect.

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral “Sanjinjiao” and “Zusanli” acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations

Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV–neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.

The Schizophrenia Risk Gene Product Mir-137 Alters Presynaptic Plasticity

Noncoding variants in the human MIR137 gene locus increase schizophrenia risk with genome-wide significance. However, the functional consequence of these risk alleles is unknown. Here we examined induced human neurons harboring the minor alleles of four disease-associated single nucleotide polymorphisms in MIR137. We observed increased MIR137 levels compared to those in major allele-carrying cells. microRNA-137 gain of function caused downregulation of the presynaptic target genes complexin-1 (Cplx1), Nsf and synaptotagmin-1 (Syt1), leading to impaired vesicle release. In vivo, miR-137 gain of function resulted in changes in synaptic vesicle pool distribution, impaired induction of mossy fiber long-term potentiation and deficits in hippocampus-dependent learning and memory. By sequestering endogenous miR-137, we were able to ameliorate the synaptic phenotypes. Moreover, reinstatement of Syt1 expression partially restored synaptic plasticity, demonstrating the importance of Syt1 as a miR-137 target. Our data provide new insight into the mechanism by which miR-137 dysregulation can impair synaptic plasticity in the hippocampus.

Diet and sexual hormones regulate hepatic synaptotagmin 1 mRNA in mice.

The expression of Synaptotagmin 1 (Syt1) has been found to be associated with the lipid droplets in liver. Here, we studied the expression of Syt1 in Apoe-deficient mice receiving cholesterol, Western diet, squalene, and oleanolic acid. We also studied the influence of sex and impact of surgical castration. Dietary cholesterol increased hepatic Syt1 expression, an effect that was enhanced when cholesterol was combined with saturated fat present in a Western diet. This potentiation was modified by the administration of 10 mg/kg oleanolic acid or 1 g/kg squalene. Females fed chow or Western diet showed higher levels of hepatic Syt1 expression as compared to male mice on the same diet. Surgical castration of males did not modify the Syt1 expression; however, ovariectomy led to decreased levels. The data show that hepatic Syt1 expression is influenced by diet and hormonal milieu.

Synaptotagmins interact with APP and promote Aβ generation.

BackgroundAccumulation of the β-amyloid peptide (Aβ) is a major pathological hallmark of Alzheimer’s disease (AD). Recent studies have shown that synaptic Aβ toxicity may directly impair synaptic function. However, proteins regulating Aβ generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate Aβ generation.ResultsAffinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, −2 and −9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted Aβ levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a ~ 50 % reduction in secreted Aβ generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both Aβ40 and Aβ42 generation. As secreted sAPPβ levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aβ generation by modulating BACE1-mediated cleavage of APP.ConclusionAltogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote Aβ generation and thus may play an important role in the pathogenesis of AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0028-5) contains supplementary material, which is available to authorized users.

The schizophrenia risk gene product miR-137 alters presynaptic plasticity.

Non-coding variants in the human MIR137 gene locus increase schizophrenia risk at a genome-wide significance level. However, the functional consequence of these risk alleles is unknown. Here, we examined induced human neurons harboring the minor alleles of four disease-associated single nucleotide polymorphisms (SNPs) in MIR137, and observed increased MIR137 levels compared to major allele-carrying cells. We found that miR-137 gain-of-function causes downregulation of the presynaptic target genes, Complexin-1 (Cplx1), Nsf, and Synaptotagmin-1 (Syt1), leading to impaired vesicle release. In vivo, miR-137 gain-of-function results in changes in synaptic vesicle pool distribution, impaired mossy fiber-LTP induction and deficits in hippocampus-dependent learning and memory. By sequestering endogenous miR-137, we were able to ameliorate the synaptic phenotypes. Moreover, reinstatement of Syt1 expression partially restored synaptic plasticity, demonstrating the importance of Syt1 as a miR-137 target. Our data provide new insight into the mechanism by which miR-137 dysregulation can impair synaptic plasticity in the hippocampus.

Abnormal expression of synaptophysin, SNAP-25, and synaptotagmin 1 in the hippocampus of kainic acid-exposed rats with behavioral deficits.

Temporal lobe epilepsy is characterized by spontaneous recurrent seizures (SRS) and associated with behavioral problems. However, the molecular mechanisms underlying these problems are not yet clear. In this study, kainic acid (KA) was systemically administered to immature male Wistar rats to induce SRS. The behavior of the immature rats was evaluated with a water maze, elevated-plus mazes, and open field tests. The expression patterns of synaptophysin, SNAP-25, and synaptotagmin 1 (Syt 1) were examined by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. KA-treated rats with SRS demonstrated learning and memory deficits, reduced anxiety, and increased locomotor activity, compared with placebo-treated rats and KA-treated rats without SRS. No neuronal cell loss was observed in the hippocampus 6 weeks after exposure to KA. However, RT-PCR and Western blot analyses revealed decreased synaptophysin, SNAP-25, and Syt 1 expression in KA-treated rats with SRS. Synaptophysin, SNAP-25, and Syt1 expression levels were found to be positively correlated with learning and memory but negatively correlated with anxiety and locomotor activity. These data suggested that SRS may induce changes in synaptophysin, SNAP-25, and Syt1 expression and may be functionally related to SRS-induced behavioral deficits.

Retinal Glycoprotein Enrichment by Concanavalin A Enabled Identification of Novel Membrane Autoantigen Synaptotagmin-1 in Equine Recurrent Uveitis

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

Synaptotagmin1 is required for spindle stability and metaphase-to-anaphase transition in mouse oocytes

Synaptotagmin1, a calcium sensor for exocytosis, forms the 7S complex, or so-called SNARE protein complex, together with SNAP -25, syntaxin and synaptobrevin to mediate docking and fusion of synaptic vesicles to the plasma membrane of the nerve terminal. Here, we identified the unique localization, expression and function of Syt1 during mouse oocyte meiotic maturation by using confocal microscopy, western blotting, Morpholino-based knockdown and time-lapse live cell imaging. We showed that Syt1 expression was gradually increased during oocyte maturation. Syt1 was localized at the oocyte cortex from GV to MII stages and at the spindle poles in MI and MII phases, with one third of a signal-free zone at the oocyte cortex, where the chromosomes are located, which is similar to the distribution pattern of CGs from the pro-MI to MII stages. Knockdown of Syt1 resulted in pro-MI/MI arrest and PB1 extrusion decrease, with severely disrupted spindles and misaligned chromosomes. Knockdown of Syt1 also caused abnormal localization of γ-tubulin, which became redistributed into the cytoplasm. Chromosome spreading showed failure of homologous chromosome segregation. The spindle assembly checkpoint protein Bub3 was detected at the kinetochores even after 10 h of oocyte culture. Live cell imaging analysis revealed that knockdown of Syt1 resulted in abnormal spindles with various morphologies and chromosomes arrested at the pro-MI/MI stage. Defective spindles failed to support chromosome alignment along microtubules, which led to repetitive unsuccessful metaphase-anaphase transitions and failure of PB1 extrusion after extended culture. Taken together, we suggest that Syt1 may act as a MTOC-associated protein to play important roles in mouse oocyte spindle organization/stability, and that it is indispensable for the metaphase-anaphase transition to promote mouse oocyte meiotic maturation.

Alcohol induces synaptotagmin 1 expression in neurons via activation of heat shock factor 1

Many synapses within the central nervous system are sensitive to ethanol. Although alcohol is known to affect the probability of neurotransmitter release in specific brain regions, the effects of alcohol on the underlying synaptic vesicle fusion machinery have been little studied. To identify a potential pathway by which ethanol can regulate neurotransmitter release, we investigated the effects of acute alcohol exposure (1-24 h) on the expression of the gene encoding synaptotagmin 1 (Syt1), a synaptic protein that binds calcium to directly trigger vesicle fusion. Syt1 was identified in a microarray screen as a gene that may be sensitive to alcohol and heat shock. We found that Syt1 mRNA and protein expression are rapidly and robustly up-regulated by ethanol in mouse cortical neurons, and that the distribution of Syt1 protein along neuronal processes is also altered. Syt1 mRNA up-regulation is dependent on the activation of the transcription factor heat shock factor 1 (HSF1). The transfection of a constitutively active Hsf1 construct into neurons stimulates Syt1 transcription, while transfection of Hsf1 small interfering RNA (siRNA) or a constitutively inactive Hsf1 construct into neurons attenuates the induction of Syt1 by ethanol. This suggests that the activation of HSF1 can induce Syt1 expression and that this may be a mechanism by which alcohol regulates neurotransmitter release during brief exposures. Further analysis revealed that a subset of the genes encoding the core synaptic vesicle fusion (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; SNARE) proteins share this property of induction by ethanol, suggesting that alcohol may trigger a specific coordinated adaptation in synaptic function. This molecular mechanism could explain some of the changes in synaptic function that occur following alcohol administration and may be an important step in the process of neuronal adaptation to alcohol.

The Heterohexameric Complex Structure, a Component in the Non-classical Pathway for Fibroblast Growth Factor 1 (FGF1) Secretion

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis, and migration. FGFs are essential for early embryonic development, organ formation, and angiogenesis. FGF1 also plays an important role in inflammation, wound healing, and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase fibroblast growth factor receptors in the presence of heparin sulfate proteoglycans and, therefore, require the release of the protein into the extracellular space. FGF1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex. The protein constituents of this complex include FGF1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). Because FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multiprotein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both complexes possibly formed during the non-classical pathway of FGF1 release.

A comparison of molecular alterations in environmental and genetic rat models of ADHD: A pilot study

Attention deficit hyperactivity disorder (ADHD) is the most common neurobehavioral disorder in school-aged children. In addition to genetic factors, environmental influences or gene x environmental interactions also play an important role in ADHD. One example of a well studied environmental risk factor for ADHD is exposure to polychlorinated biphenyls (PCBs). In this study, we investigated whether the well-established genetic model of ADHD based on the spontaneously hypertensive rat (SHR) and a well established PCB-based model of ADHD exhibited similar molecular changes in brain circuits involved in ADHD. The brains from 28 male rats (8 SHR, 8 Sprague-Dawley (SD) controls, 8 Wistar/Kyoto (WKY) controls, and 4 PCB-exposed SD rats) were harvested at postnatal days (PNDs) 55-65 and RNA was isolated from six brain regions of interest. The RNA was analyzed for differences in expression of a set of 308 probe sets interrogating 218 unique genes considered highly relevant to ADHD or epigenetic gene regulation using the Rat RAE230 2.0 GeneChip (Affymetrix). Selected observations were confirmed by real-time quantitative RT-PCR. The results show that the expression levels of genes Gnal, COMT, Adrbk1, Ntrk2, Hk1, Syt11, and Csnk1a1 were altered in both the SHR rats and the PCB-exposed SD rats. Arrb2, Stx12, Aqp6, Syt1, Ddc, and Pgk1 expression levels were changed only in the PCB-exposed SD rats. Genes with altered expression only in the SHRs included Oprm1, Calcyon, Calmodulin, Lhx1, and Hes6. The epigenetic genes Crebbp, Mecp2, and Hdac5 are significantly altered in both models. The data provide strong evidence that genes and environment can affect different set of genes in two different models of ADHD and yet result in the similar disease-like symptoms.

Pdx-1 enables insulin secretion by regulating synaptotagmin 1 gene expression

Pdx-1 plays important roles both in the development of the pancreas and in maintaining pancreatic β cell function. However, the role of Pdx-1 in the regulation of insulin release is not well established. We previously demonstrated that Pdx-1 overcomes the defect in insulin release from the insulin-producing cells derived from small hepatocytes (SHCs). Insulin secretion is regulated in vivo by the sequential events triggered by the increase of intracellular Ca 2+-concentration in response to high glucose concentration. In the present study, we identified a new target of Pdx-1 involved in insulin release. Pdx-1 positively regulates the transcription of the gene encoding synaptotagmin 1 (Syt1) (a Ca 2+-sensor that plays a central role in insulin release) through Pdx-1-binding sites within the 3 ′ regulatory region of the Syt1 gene. We further demonstrated the essential role of Pdx-1 in insulin secretion by the gene knock-down strategy. Small interfering RNA (siRNA) directed against Pdx-1 specifically reduced the levels of Pdx-1 protein and Syt1 transcript in insulinoma lines. Our data indicate that Pdx-1 might contribute to the regulation of insulin release by promoting Syt1 expression in vivo, and provide useful information for future therapy of diabetes mellitus.

Variations of Synaptotagmin I, Synaptotagmin IV, and Synaptophysin mRNA Levels in Rat Hippocampus during the Estrous Cycle

Periodic changes in ovarian steroid levels during fertility cycles affect learning both in humans and in rats in parallel with electrophysiological and morphological fluctuations in selective neuronal populations. In particular, during the estrous cycle of the female rat, hippocampal CA1 region undergoes cyclic modifications in synaptic density. To investigate the molecular mechanisms involved in synaptic remodeling during the estrous cycle, we analyzed the expression of three presynaptic markers, synaptotagmin I, synaptotagmin IV, and synaptophysin, in the female adult rat brain by in situ hybridization. Relative abundance in mRNA for these three markers was quantified at four phases of the estrous cycle: diestrus, proestrus (AM and PM), and estrus. mRNA levels for syt1 exhibited cyclic variations in pyramidal neurons of the CA3 region of hippocampus during the estrous cycle, while mRNA levels for syt4 and SYN were relatively invariant in this or other regions of the hippocampus. Because CA3 pyramidal neurons make synaptic contacts in CA1, modulation of syt1 expression in CA3 may participate in the changes in synaptic density observed in CA1 during the estrous cycle. Furthermore, both syt1 and SYN mRNA varied cyclically in layer II, but not in layer III of entorhinal cortex, while syt4 remained unchanged throughout the cycle. These data suggest that regular variations in steroid hormone levels during fertility cycles may alter the properties of several networks involved in information processing and learning and memory through altered levels of presynaptic proteins.