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Circular RNAs (circRNAs) play important roles in the pathogenesis of age-related cataract (ARC). CircRNA zinc finger protein 292 (circZNF292, hsa_circ_0004058) is downregulated in ARC lens capsules. Here, we focused on its precise roles in oxidative stress underlying the pathogenesis of ARC. CircZNF292, microRNA (miR)-222-3p, and E2F transcription factor 3 (E2F3) were quantified by quantitative real-time polymerase chain reaction or western blot. Cell viability was assessed by the cell counting kit-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The activities of superoxide dismutase, catalase, and malondialdehyde were measured using the corresponding assay kit. Targeted correlations among circZNF292, miR-222-3p, and E2F3 were verified by the dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Our data showed that circZNF292 was downregulated in ARC tissues and H 2 O 2 -treated human lens epithelial B3 (HLE-B3) cells. Increased expression of circZNF292 alleviated H 2 O 2 -induced cell viability suppression, apoptosis promotion, and oxidative stress enhancement. Mechanistically, circZNF292 directly targeted miR-222-3p, and circZNF292 regulated E2F3 expression through miR-222-3p. MiR-222-3p was a functional mediator of circZNF292 in modulating H 2 O 2 -induced injury in HLE-B3 cells. Furthermore, reduced level of miR-222-3p ameliorated H 2 O 2 -induced HLE-B3 cell damage by upregulating E2F3. Our present study demonstrated that increased expression of circZNF292 ameliorated H 2 O 2 -induced injury in HLE-B3 cells at least in part through the miR-222-3p/E2F3 axis, highlighting a novel insight into the involvement of circRNAs in the pathogenesis of ARC., (© 2021 International Federation for Cell Biology.)

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3 , a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Aims: The recent failures of HDL-raising therapies have underscored our incomplete understanding of HDL biology. Therefore there is an urgent need to comprehensively investigate HDL metabolism to enable the development of effective HDL-centric therapies. To identify novel regulators of HDL metabolism, we performed a joint analysis of human genetic, transcriptomic, and plasma HDL-cholesterol (HDL-C) concentration data and identified a novel association between trafficking protein, kinesin binding 2 (TRAK2) and HDL-C concentration. Here we characterize the molecular basis of the novel association between TRAK2 and HDL-cholesterol concentration., Methods and Results: Analysis of lymphocyte transcriptomic data together with plasma HDL from the San Antonio Family Heart Study (n = 1240) revealed a significant negative correlation between TRAK2 mRNA levels and HDL-C concentration, HDL particle diameter and HDL subspecies heterogeneity. TRAK2 siRNA-mediated knockdown significantly increased cholesterol efflux to apolipoprotein A-I and isolated HDL from human macrophage (THP-1) and liver (HepG2) cells by increasing the mRNA and protein expression of the cholesterol transporter ATP-binding cassette, sub-family A member 1 (ABCA1). The effect of TRAK2 knockdown on cholesterol efflux was abolished in the absence of ABCA1, indicating that TRAK2 functions in an ABCA1-dependent efflux pathway. TRAK2 knockdown significantly increased liver X receptor (LXR) binding at the ABCA1 promoter, establishing TRAK2 as a regulator of LXR-mediated transcription of ABCA1., Conclusion: We show, for the first time, that TRAK2 is a novel regulator of LXR-mediated ABCA1 expression, cholesterol efflux, and HDL biogenesis. TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Endosomal maturation and transport constitutes a complex trafficking system present in all cell types. Neurons have adapted their endosomal system to meet their unique and complex needs. These adaptations include repurposing existing proteins to diversify endocytosis and trafficking, as well as preferential expression of certain regulators more highly in neurons than other cell types. These neuronal regulators include the family of Neuron-Specific Gene family members (Nsg), NEEP21 (Nsg1), and P19 (Nsg2). NEEP21/Nsg1 plays a role in the trafficking of multiple receptors, including the cell adhesion molecule L1/NgCAM, the neurotransmitter receptor GluA2, and β-APP. Recently, we showed that NEEP2/Nsg1 and P19/Nsg2 are not expressed in all neuronal cell types in vitro. However, it is not known where and when NEEP21/Nsg1 and P19/Nsg2 are expressed in vivo, and whether both proteins are always coexpressed. Here, we show that NEEP21/Nsg1 and P19/Nsg2 are present in both overlapping and distinct cell populations in the hippocampus, neocortex, and cerebellum during development. NEEP21/Nsg1 and P19/Nsg2 levels are highest during embryonic development, and expression persists in the juvenile mouse brain. In particular, a subset of layer V cortical neurons retains relatively high expression of both NEEP21/Nsg1 and P19/Nsg2 at postnatal day 16 as well as in the CA1-3 regions of the hippocampus. In the cerebellum, NEEP21/Nsg1 expression becomes largely restricted to Purkinje neurons in adulthood whereas P19/Nsg2 expression strikingly disappears from the cerebellum with age. This divergent and restricted expression likely reflects differential needs for this class of trafficking regulators in different neurons during different stages of maturation., (© 2017 Wiley Periodicals, Inc.)

Tripartite motif (TRIM) 8 functions as an E3 ubiquitin ligase, interacting with and ubiquitinating diverse substrates, and is implicated in various pathological processes. However, the function of TRIM8 in the heart remains largely uncharacterized. This study aims to explore the role of TRIM8 in the development of pathological cardiac hypertrophy. Mice and isolated neonatal rat cardiomyocytes overexpressing or lacking TRIM8 were examined in several experiments. The effect of aortic banding-induced cardiac hypertrophy was analyzed by echocardiographic, pathological and molecular analyses. Our results indicated that the TRIM8 overexpression in hearts exacerbated the cardiac hypertrophy triggered by aortic banding. In contrast, the development of pathological cardiac hypertrophy was profoundly blocked in TRIM8-deficient hearts. Mechanistically, our study suggests that TRIM8 may elicit cardiodetrimental effects by promoting the activation of transforming growth factor β-activated kinase 1 (TAK1)-p38/JNK signaling pathways. Similar results were observed in cultured neonatal rat cardiomyocytes treated with angiotensin II. The rescue experiments using the TAK1-specific inhibitor 5z-7-ox confirmed the requirement of TAK1 activation in TRIM8-mediated pathological cardiac hypertrophy. Furthermore, TRIM8 contributed to TAK1 activation by binding to and promoting TAK1 ubiquitination. In conclusion, our study demonstrates that TRIM8 plays a deleterious role in pressure overload-induced cardiac hypertrophy by accelerating the activation of TAK1-dependent signaling pathways., (© 2016 American Heart Association, Inc.)

Background: Autism spectrum disorders (ASDs) represent a heterogeneous group of disorders with a wide range of behavioral impairments including social and communication deficits. Apart from these core symptoms, a significant number of ASD individuals display higher levels of anxiety, and some studies indicate that a subset of ASD individuals have a reduced ability to be fear conditioned. Deciphering the molecular basis of ASD has been considerably challenging and it currently remains poorly understood. In this study we examined the molecular basis of autism-like impairments in an environmentally induced animal model of ASD, where pregnant rats are exposed to the known teratogen, valproic acid (VPA), on day 12.5 of gestation and the subsequent progeny exhibit ASD-like symptoms. We focused our analysis on the basal and lateral nucleus of the amygdala (BLA), a region of the brain found to be associated with ASD pathology., Methods: We performed whole genome gene expression analysis on the BLA using DNA microarrays to examine differences in gene expression within the amygdala of VPA-exposed animals. We validated one VPA-dysregulated candidate gene (Homer1a) using both quantitative PCR (qRT-PCR) and western blot. Finally, we overexpressed Homer1a within the basal and lateral amygdala of naïve animals utilizing adeno-associated viruses (AAV) and subsequently examined these animals in a battery of behavioral tests associated with ASD, including auditory fear conditioning, social interaction and open field., Results: Our microarray data indicated that Homer1a was one of the genes which exhibited a significant upregulation within the amygdala. We observed an increase in Homer1a messenger RNA (mRNA) and protein in multiple cohorts of VPA-exposed animals indicating that dysregulation of Homer1a levels might underlie some of the symptoms exhibited by VPA-exposed animals. To test this hypothesis, we overexpressed Homer1a within BLA neurons utilizing a viral-mediated approach and found that overexpression of Homer1a impaired auditory fear conditioning and reduced social interaction, while having no influence on open-field behavior., Conclusions: This study indicates that dysregulation of amygdala Homer1a might contribute to some autism-like symptoms induced by VPA exposure. These findings are interesting in part because Homer1a influences the functioning of Shank3, metabotropic glutamate receptors (mGluR5), and Homer1, and these proteins have previously been associated with ASD, indicating that these differing models of ASD may have a similar molecular basis.

Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform-specific primers and exon-specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α-synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphilin-1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over-expressed in the striatum and cerebellar cortex, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal control brains. We have focused on brain regions that are severely affected by α-synuclein pathology and neurodegeneration in MSA. The reported changes of isoform expression profiles suggest alterations in the regulation of transcription that may be important for aggregation of α-synuclein in the brain., (© 2015 International Society for Neurochemistry.)

Background: Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma., Methods: TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays., Results: We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient's glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3' UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8., Conclusions: Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients.

SH2B1 is an adaptor protein known to enhance neurite outgrowth. In this study, we provide evidence suggesting that the SH2B1 level is increased during in vitro culture of hippocampal neurons, and the β isoform (SH2B1β) is the predominant isoform. The fact that formation of filopodia is prerequisite for neurite initiation suggests that SH2B1 may regulate filopodium formation and thus neurite initiation. To investigate whether SH2B1 may regulate filopodium formation, the effect of SH2B1 and a membrane and actin regulator, IRSp53 (insulin receptor tyrosine kinase substrate p53), is investigated. Overexpressing both SH2B1β and IRSp53 significantly enhances filopodium formation, neurite outgrowth, and branching. Both in vivo and in vitro data show that SH2B1 interacts with IRSp53 in hippocampal neurons. This interaction depends on the N-terminal proline-rich domains of SH2B1. In addition, SH2B1 and IRSp53 co-localize at the plasma membrane, and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These findings suggest that SH2B1-IRSp53 complexes promote the formation of filopodia, neurite initiation, and neuronal branching., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A hopeful spinal cord repairing strategy involves the activation of neural precursor cells. Unfortunately, their ability to generate neurons after injury appears limited. Another process promoting functional recovery is synaptic plasticity. We have previously studied some mechanisms of spinal plasticity involving BDNF, Shh, Notch-1, Numb, and Noggin, by using a mouse model of motoneuron depletion induced by cholera toxin-B saporin. TDP-43 is a nuclear RNA/DNA binding protein involved in amyotrophic lateral sclerosis. Interestingly, TDP-43 could be localized at the synapse and affect synaptic strength. Here, we would like to deepen the investigation of this model of spinal plasticity. After lesion, we observed a glial reaction and an activity-dependent modification of Shh, Noggin, and Numb proteins. By using multivariate regression models, we found that Shh and Noggin could affect motor performance and that these proteins could be associated with both TDP-43 and Numb. Our data suggest that TDP-43 is likely an important regulator of synaptic plasticity, probably in collaboration with other proteins involved in both neurogenesis and synaptic plasticity. Moreover, given the rapidly increasing knowledge about spinal cord plasticity, we believe that further efforts to achieve spinal cord repair by stimulating the intrinsic potential of spinal cord will produce interesting results.

The impairment of mitochondrial function is an important pathogenic factor in glaucoma and other optic neuropathies in which retinal ganglion cell (RGC) death is the fundamental pathology. Syntaphilin was recently discovered as a docking protein that affects mitochondrial mobility. However, no reports have investigated the involvement of syntaphilin in the visual system. We investigated the expression of syntaphilin in the rat retina, optic nerve and brain. The expression of syntaphilin exhibited varying patterns in the visual system. Syntaphilin was expressed in retinal ganglion cells in the retina, in the cell bodies of neurons in the superior colliculus and was abundant in the astrocytes of rat optic nerves (similar to the findings that syntaphilin is expressed in human optic nerves). After optic nerve transection, which caused RGC death and axonal degeneration, quantitative real-time RT-PCR was used to assess changes in gene expression in the rat retina and optic nerve. Syntaphilin gene and protein expression in the optic nerve was downregulated 3 and 7 days after optic nerve transection. Our study suggests that syntaphilin expression in astrocytes at the optic nerve might be involved in axonal injury., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The expression of two adjacent imprinted genes, Peg3 and Zim1, is inversely correlated: down-regulation of Peg3 coinciding with up-regulation of Zim1. The current study characterized this inverse correlation using a mutant allele targeting Peg3. According to the results, the mutation on the paternal allele of Peg3 resulted in a dramatic increase in the transcription levels of the maternal allele of Zim1, suggesting the involvement of unknown trans factors in this trans-allelic event. Subsequent ChIP experiments revealed that the protein encoded by Peg3 itself binds to the zinc finger exon of Zim1, which is modified with the repression mark H3K9me3. Interestingly, the levels of H3K9me3 on Zim1 are also reduced in the mutant cells lacking the protein PEG3, suggesting potential roles for PEG3 in establishing H3K9me3 on Zim1. Reintroducing PEG3 into the mutant cell restored down-regulation of Zim1, confirming the predicted repressor role for Peg3 on Zim1. Overall, these results demonstrated that paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor. The current study also provides the first case for the trans-allelic interaction of two oppositely imprinted genes through their gene products.

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H2O2) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H2O2-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.

Dysbindin, the product of the DTNBP1 gene, was identified by yeast two hybrid assay as a binding partner of dystrobrevin, a cytosolic component of the dystrophin protein complex. Although its functional role has not yet been completely elucidated, the finding that dysbindin assembles into the biogenesis of lysosome related organelles complex 1 (BLOC-1) suggests that it participates in intracellular trafficking and biogenesis of organelles and vesicles. Dysbindin is ubiquitous and in brain is expressed primarily in neurons. Variations at the dysbindin gene have been associated with increased risk for schizophrenia. As anomalies in retinal function have been reported in patients suffering from neuropsychiatric disorders, we investigated the expression of dysbindin in the retina. Our results show that differentially regulated dysbindin isoforms are expressed in rat retina during postnatal maturation. Interestingly, we found that dysbindin is mainly localized in Müller cells. The identification of dysbindin in glial cells may open new perspectives for a better understanding of the functional involvement of this protein in visual alterations associated to neuropsychiatric disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-D-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.

Thioredoxin-interacting protein (TXNIP) has emerged as an important factor in pancreatic beta cell biology, and tight regulation of TXNIP levels is necessary for beta cell survival. However, the mechanisms regulating TXNIP expression have only started to be elucidated. The forkhead boxO1 transcription factor (FOXO1) has been reported to up-regulate TXNIP expression in neurons and endothelial cells but to down-regulate TXNIP in liver, and the effects on beta cells have remained unknown. We now have found that FOXO1 binds to the TXNIP promoter in vivo in human islets and INS-1 beta cells and significantly decreases TXNIP expression. TXNIP promoter deletion analyses revealed that an E-box motif conferring carbohydrate response element-binding protein (ChREBP)-mediated, glucose-induced TXNIP expression is necessary and sufficient for this effect, and electromobility shift assays confirmed FOXO1 binding to this site. Moreover, FOXO1 blocked glucose-induced TXNIP expression and reduced glucose-induced ChREBP binding at the TXNIP promoter without affecting ChREBP expression or nuclear localization, suggesting that FOXO1 may compete with ChREBP for binding to the TXNIP promoter. In fact, a FOXO1 DNA-binding mutant (FOXO1-H215R) failed to inhibit TXNIP transcription, and the effects were not restricted to TXNIP as FOXO1 also inhibited transcription of other ChREBP target genes such as liver pyruvate kinase. Together, these results demonstrate that FOXO1 inhibits beta cell TXNIP transcription and suggest that FOXO1 confers this inhibition by interfering with ChREBP DNA binding at target gene promoters. Our findings thereby reveal a novel gene regulatory mechanism and a previously unappreciated cross-talk between FOXO1 and ChREBP, two major metabolic signaling pathways.

Two theories have influenced our understanding of cortical development: the integrated network theory, where synaptic development is coordinated across areas; and the cascade theory, where the cortex develops in a wave-like manner from sensory to non-sensory areas. These different views on cortical development raise challenges for current studies aimed at comparing detailed maturation of the connectome among cortical areas. We have taken a different approach to compare synaptic development in rat visual, somatosensory, and frontal cortex by measuring expression of pre-synaptic (synapsin and synaptophysin) proteins that regulate vesicle cycling, and post-synaptic density (PSD-95 and Gephyrin) proteins that anchor excitatory or inhibitory (E-I) receptors. We also compared development of the balances between the pairs of pre- or post-synaptic proteins, and the overall pre- to post-synaptic balance, to address functional maturation and emergence of the E-I balance. We found that development of the individual proteins and the post-synaptic index overlapped among the three cortical areas, but the pre-synaptic index matured later in frontal cortex. Finally, we applied a neuroinformatics approach using principal component analysis and found that three components captured development of the synaptic proteins. The first component accounted for 64% of the variance in protein expression and reflected total protein expression, which overlapped among the three cortical areas. The second component was gephyrin and the E-I balance, it emerged as sequential waves starting in somatosensory, then frontal, and finally visual cortex. The third component was the balance between pre- and post-synaptic proteins, and this followed a different developmental trajectory in somatosensory cortex. Together, these results give the most support to an integrated network of synaptic development, but also highlight more complex patterns of development that vary in timing and end point among the cortical areas.

The Prdm (PRDI-BF1-RIZ1 homologous) family is involved in cell differentiation, and several Prdms have been reported to methylate histone H3 by intrinsic or extrinsic pathways. Here, we report that Prdm12 recruits G9a to methylate histone H3 on lysine 9 through its zinc finger domains. Because of the expression of Prdm12 in the developmental nervous system, we investigated the role of Prdm12 on P19 embryonic carcinoma cells as a model system for neurogenesis. In P19 cells, Prdm12 is induced by Retinoic acid (RA). Overproduction of Prdm12 in P19 cells impairs cell proliferation and increases the G1 population accompanied by the upregulation of p27. In contrast, the knockdown of Prdm12 increases the number of cells in a suspension culture of RA-induced neural differentiation. Both the PR domain and zinc finger domains are required for the anti-proliferative activity of Prdm12. While the data in this study is based on in vitro models, the results suggest that Prdm12 is induced by the RA signaling in vivo, and may regulate neural differentiation during animal development.

The brain's noradrenergic system is involved in the development of behaviours induced by drugs of abuse, e.g. dependence and withdrawal, and also reward or psychomotor effects. To investigate how noradrenergic system activity is controlled in the context associated with drug-induced behaviours, we generated a Cre/loxP mouse model in which the essential glutamate NMDA receptor subunit NR1 is ablated in cells expressing dopamine β-hydroxylase (Dbh). As a result, the noradrenergic cells in NR1DbhCre mice lack the NMDA receptor-dependent component of excitatory post-synaptic currents. The mutant mice displayed no obvious behavioural alterations, had unchanged noradrenaline content and mild increase in dopamine levels in the nucleus accumbens. Interestingly, NR1DbhCre animals did not develop morphine-induced psychomotor sensitization. However, when the morphine injections were preceded by treatment with RX821002, an antagonist of α2-adrenergic receptors, the development of sensitization was restored. Conversely, pretreatment with clonidine, an agonist of α2-adrenergic receptors, blocked development of sensitization in wild-type mice. We also found that while the development of tolerance to morphine was normal in mutant mice, withdrawal symptoms were attenuated. These data reveal that NMDA receptors on noradrenergic neurons regulate development of opiate dependence and psychomotor sensitization, by controlling drug-induced noradrenaline signalling.

The expression of Arc and Homer 1a (H1a) depends on neural activity. This study was designed to determine hippocampal Arc and H1a mRNA expression levels after spatial learning with differing behavioral task demands. Forty-four male rats were distributed into 11 groups of four. One group received no training or trial sessions. Of the ten remaining groups, three were tested on the 8-arm maze, three on the 12-arm maze, two on the 8-arm maze and then the 12-arm maze, and two on the 12-arm maze and then the 8-arm maze. Each animal was sacrificed 30 min after the last session of maze testing and its hippocampus was immediately dissected and stored at -80°C. The level of mRNA expression at different stages of maze learning was determined using real-time reverse-transcription polymerase chain reaction (qRT-PCR). Significantly elevated expression of both Arc and H1a was observed. The orchestrated expression levels of both genes were correlated with the behavioral task demand level and behavioral performance., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

STOX1A is a transcription factor which is functionally and structurally similar to the forkhead box protein family. STOX1A has been shown to be associated with pre-eclampsia, a pregnancy associated disease, and to have potential implications in late onset Alzheimer's disease. However, the exact function of STOX1A and its target genes are still largely unknown. Therefore, in this study we performed chromatin immunoprecipitation coupled to shotgun cloning to discover novel STOX1A target genes. Our results show that CNTNAP2, a member of the neurexin family, is directly downregulated by STOX1A. Additionally, we show that CNTNAP2 expression is downregulated in the hippocampus of Alzheimer's disease patients where STOX1A expression has been shown to be upregulated. In conclusion, these results further indicate the potential involvement of STOX1A and its target genes in the etiology of Alzheimer's disease.

Nolz-1/Zfp503, a zinc finger-containing gene, is a mammalian member of the SP1-related nocA/elb/tlp-1 gene family. Previous studies have shown that Nolz-1 homologs are important for patterning the rhombomeres in zebrafish hindbrain. We therefore studied the expression pattern of Nolz-1 in the developing mouse hindbrain. Nolz-1 mRNA expression was detected in the prospective rhombomere 3, 5 and caudal regions as early as E8.75. After E11.5, Nolz-1-positive cells were organized as distinct cell clusters, and they were largely non-overlapped with either Pax2-positive or Phox2b-positive domains. Most interestingly, we found that Nolz-1 was specifically expressed by Phox2b-negative/Isl1/2-positive somatic motor neurons, but not by Phox2b-positive/Isl1/2-positive branchial and visceral motor neurons, suggesting that Nolz-1 may regulate development of somatic motor neurons in the hindbrain. In addition to be expressed in differentiating post-mitotic neurons, Nolz-1 was also expressed by progenitor cells in the ventricular zone located in the dorsal part of aqueduct and the alar plates of hindbrain, which suggests a regulatory role of Nolz-1 in the germinal zone. Taken together, based on its domain- and cell type-selective pattern, Nolz-1 may involve in regulation of various developmental processes, including regional patterning and cell-type specification and differentiation in the developing mouse hindbrain., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Several lines of evidence indicate that remyelination represents one of the most effective mechanisms to achieve axonal protection. For reasons that are not yet understood, this process is often incomplete or fails in multiple sclerosis (MS). Activated astrocytes appear to be able to boost or inhibit endogenous repair processes. A better understanding of remyelination in MS and possible reasons for its failure is needed. Using the well-established toxic demyelination cuprizone model, we created lesions with either robust or impaired endogenous remyelination capacity. Lesions were analyzed for mRNA expression levels by Affymetrix GeneChip® arrays. One finding was the predominance of immune and stress response factors in the group of genes which were classified as remyelination-supporting factors. We further demonstrate that lesions with impaired remyelination capacity show weak expression of the radial-glia cell marker brain lipid binding protein (BLBP, also called B-FABP or FABP7). The expression of BLBP in activated astrocytes correlates with the presence of oligodendrocyte progenitor cells. BLBP-expressing astrocytes are also detected in experimental autoimmune encephalomyelitis during the remission phase. Furthermore, highest numbers of BLBP-expressing astrocytes were evident in lesions of early MS, whereas significantly less are present at the rim of (chronic)-active lesions from patients with long disease duration. Transfection experiments show that BLBP regulates growth factor expression in U87 astrocytoma cells. In conclusion, we provide evidence that expression of BLBP in activated astrocytes negatively correlates with disease duration and in parallel with remyelination failure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Microarray technology was used to explore differences in brain gene expression under basal conditions in two strains of psychogenetically selected rats which differ in anxiety/stress responses, the inbred Roman High-(RHA-I) and Roman Low-(RLA-I) Avoidance rats. Microarray analysis detected 14 up-regulated and 24 down-regulated genes in RLA-I vs. RHA-I rats functionally related to neurobiological processes. The differentially expressed genes CAMKK2, CRHBP, EPHX2, HOMER3, NDN, PRL and RPL6 were selected for microarray validation using qRT-PCR. EPHX2, CAMKK2 (both up-regulated in RLA-I vs. RHA-I rats) and HOMER3 (down-regulated in RLA-I vs. RHA-I rats) showed a similar tendency and fold-change both in microarray and RT-PCR analyses; PRL (up-regulated in RLA-I vs. RHA-I rats), CRHBP and RPL6 (both down-regulated in RLA-I vs. RHA-I animals) showed a similar tendency but a different order of magnitude of change among experiments; finally, NDN was validated neither in tendency nor in magnitude of change., (Published by Elsevier Ireland Ltd.)

Experimental cerebral ischemia has been reportedly alleviated by the immunosuppressive agent cyclosporin A (CsA). Cyclophilin C-associated protein (CyCAP) was proposed to be an endogenous equivalent of CsA; CsA- and CyCAP-targeting protein cyclophilin C have attracted extensive attention regarding their ischemia-alleviating mechanisms. In this study we have introduced the specific CyCAP antibody for evaluating its distribution in the rat ischemic brain after middle cerebral artery occlusion. During the recovery of cerebral ischemia in rats, CyCAP was highly expressed in the activated microglia/macrophages in the ischemic lesion. However, it remains unknown what roles CyCAP plays in the activation of macrophage phagocytosis. Thus, we studied CyCAP function using a RAW264.7 macrophage cell line. When we expressed CyCAP-GFP and cyclophilin C-FLAG in RAW264.7 cells, we found that CyCAP and cyclophilin C make a complex, which is competitively inhibited by CsA. Consistently, in immunoprecipitates by anti-calcineurin antibody, cyclophilin C and CyCAP were detected, and CyCAP pulled down NFATc1, suggesting that both CyCAP and cyclophilin C form a complex with calcineurin and NFATc1. When CyCAP was adenovirally overexpressed in RAW cells, NFAT staining increased over the nucleus. Furthermore, calcineurin and IL-2 were increased with time. Thus, CyCAP appears to control macrophage functions by activating NFAT and the resultant IL-2 production. With a protein phosphatase inhibitor PhoSTOP, NFAT was localized more to the cytoplasm, and phagocytosis was decreased strikingly. Thus, we suggest that in a CyCAP-cyclophilin C pathway for macrophage activation, calcineurin phosphatase activity is essential for the phagocytosis activity via dephosphorylation of NFATc1., (Copyright © 2011 Elsevier B.V. All rights reserved.)

X-linked intellectual disability (XLID), also known as X-linked mental retardation, is a highly genetically heterogeneous condition for which mutations in >90 different genes have been identified. In this study, we used a custom-made sequencing array based on the Affymetrix 50k platform for mutation screening in 17 known XLID genes in patients from 135 families and found eight single-nucleotide changes that were absent in controls. For four mutations affecting ATRX (p.1761M>T), PQBP1 (p.155R>X) and SLC6A8 (p.390P>L and p.477S>L), we provide evidence for a functional involvement of these changes in the aetiology of intellectual disability.

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.

The Nob1 gene is assumed to be associated with transcription regulation and may play important roles in mediating some physiological and pathological functions. Here, the rats were randomized equally into experimental group and control group. In experimental group, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL, 8 h per day for 7 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 1 h after the cessation of noise exposure. Then, we investigated for the first time the expression of Nob1 in noise-exposed and noise-unexposed rats by quantitative polymerase chain reaction. The distribution of Nob1 in rat cochlea was further examined by immunohistochemistry. The results indicated that the hearing threshold was significantly higher in the noise-injured group than in the uninjured group after noise exposure. Nob1 mRNA was present at higher levels in regions of the noise-injured cochlea. As for noise-exposed rats, Nob1 expression was positive in the inner and outer hair cells of the organ of Corti and spiral ganglion neurons, but it undetectable in the uninjured cochlea. Therefore, Nob1 may play an important role in auditory function following acoustic trauma and can be used as a new target for the treatment of noise-induced hearing loss., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Some heavy metals, or aluminium, could participate in the development of Alzheimer disease (AD). Depleted uranium (DU), another heavy metal, modulates the cholinergic system and the cholesterol metabolism in the brain of rats, but without neurological disorders. The aim of this study was to determine what happens in organisms exposed to DU that will/are developing the AD. This study was thus performed on a transgenic mouse model for human amyloid precursor protein (APP), the Tg2576 strain. The possible effects of DU through drinking water (20 mg/L) over an 8-month period were analyzed on acetylcholine and cholesterol metabolisms at gene level in the cerebral cortex. The mRNA levels of choline acetyl transferase (ChAT) vesicular acetylcholine transporter (VAChT) and ATP-binding cassette transporter A1 (ABC A1) decreased in control Tg2576 mice in comparison with wild-type mice (respectively -89%, -86% and -44%, p < 0.05). Chronic exposure of Tg2576 mice to DU increased mRNA levels of ChAT (+189%, p < 0.05), VAChT (+120%, p < 0.05) and ABC A1 (+52%, p < 0.05) compared to control Tg2576 mice. Overall, these modifications of acetylcholine and cholesterol metabolisms did not lead to increased disturbances that are specific of AD, suggesting that chronic DU exposure did not worsen the pathology in this experimental model., (Copyright © 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Objective: Chromosome 13 deletions (del[13]), detected by metaphase cytogenetics, predict poor outcomes in multiple myeloma (MM), but the gene(s) responsible have not been conclusively identified. We sought to identify tumor-suppressor genes on chromosome 13 using a novel array comparative genomic hybridization (aCGH) strategy., Materials and Methods: We identified DNA copy number losses on chromosome 13 using genomic DNA isolated from CD138-enriched bone marrow cells (tumor) from 20 patients with MM, monoclonal gammopathy of undetermined significance, or amyloidosis. We used matched skin biopsy (germline) genomic DNA to control for copy number polymorphisms and a novel aCGH array dedicated to chromosome 13 to map somatic DNA gains and losses at ultra-high resolution (>385,000 probes; median probe spacing 60 bp). We analyzed microarray expression data from an additional 262 patient samples both with and without del[13]., Results: Two distinct minimally deleted regions at 13q14 and 13q13 were defined that affected the RB1 and NBEA genes, respectively. RB1 is a canonical tumor suppressor previously implicated in MM. NBEA is implicated in membrane trafficking in neurons, protein kinase A binding, and has no known role in cancer. Noncoding RNAs on chromosome 13 were not affected by interstitial deletions. Both the RB1 and NBEA genes were deleted in 40% of cases (8 of 20; 5 patients with del[13] detected by traditional methods and 3 patients with interstitial deletions detected by aCGH). Forty-one additional MM patient samples were used for complete exonic sequencing of RB1, but no somatic mutations were found. Along with RB1, NBEA gene expression was significantly reduced in cases with del[13]., Conclusions: The NBEA gene at 13q13, and its expression are frequently disrupted in MM. Additional studies are warranted to evaluate the role of NBEA as a novel candidate tumor-suppressor gene.

Background: Reelin and Notch-1 signaling pathways have been recently found to be necessary to induce the expression of brain lipid binding protein (BLBP) and to promote the process extension and the maturation of the neuronal progenitors, the radial glial cells. In this study, we report the cross talk between these two pathways., Results: Both in vitro Reelin treatment and overexpression of Notch-1 intracellular domain (NICD) induced BLBP expression and a radial glial phenotype in an immortalized human neural progenitor (HNP) cell line, isolated from the cortex of 14 weeks old fetus. Reelin treatment increased the level of NICD, indicating that Reelin signaling directly activates Notch-1. In addition, reducing NICD release, by inhibiting gamma-secretase activity, inhibited the Reelin-induced radial glial phenotype in human neural progenitor cells. Furthermore, we found that Dab-1, an adaptor protein downstream of Reelin, was co-immunoprecipitated with Notch-1 and NICD., Conclusion: These data indicate that Reelin signaling induces BLBP expression and a radial glial phenotype in human neural progenitor cells via the activation of Notch-1. This study suggest that Reelin signaling may act to fine tune Notch-1 activation to favor the induction of a radial glial phenotype prenataly and would thus offer an insight into how Notch-1 signaling leads to different cellular fates at different developmental stages.

Background: Nanog, nucleostemin (NS) and musashi1 (Msi1) are proteins that are highly expressed in undifferentiated embryonic stem (ES) cells and have been shown to be essential in maintaining the pluripotency and regulating the proliferation and asymmetric division of ES cells and several nervous system tumor cells. The roles of Nanog, NS and Msi1 in development and progression of cervical carcinoma have, until now, not been well documented., Methods: In this study, expression of Nanog, NS and Msi1 was detected by immunohistochemistry analysis in 235 patients with various degrees of cervical epithelial lesions, including 49 with normal cervical epithelia, 31 with mild dysplasia (CIN I), 77 with moderate-severe dysplasia (CIN II-III) and 78 with squamous cervical carcinomas (SCCs). Associations with various clinical pathological prognostic variables were analyzed in 50 early-stage SCC patients., Results: Nanog, NS and Msi1 expression levels were significantly higher in SCC patients compared with CIN patients, and were higher in CIN patients compared with those with normal cervical epithelia. Nanog expression levels showed significantly differences according to different tumor sizes (P < 0.05), whereas there were no differences in NS and Msi1 expression levels according to different clinical pathological parameters., Conclusion: Our findings indicate that Nanog, NS and Msi1 may be involved in carcinogenesis of the cervix and progression of cervical carcinoma.

Hypoxia is a hallmark of respiratory, neurological, or hematological diseases as well as life at high altitude. For example, chronic constant hypoxia (CCH) occurs in chronic lung diseases or at high altitude, whereas chronic intermittent hypoxia (CIH) occurs in diseases such as sleep apnea or sickle cell disease. Despite the fact that such conditions are frequent, the cellular and molecular mechanisms underlying the effect of hypoxia, whether constant or intermittent, are not well understood. In this study, we first determined the effect of CCH and CIH on global gene expression in different regions of mouse brain using microarrays and then investigated the biological role of genes of interest. We found that: 1) in the cortical region, the expression level of 80 genes was significantly altered by CIH (16 up- and 64 downregulated), and this number increased to 137 genes following CCH (34 up- and 103 downregulated); 2) a similar number of gene alterations was identified in the hippocampal area, and the majority of the changes in this region were upregulations; 3) two genes (Sspn and Ttc27) were downregulated in both brain regions and following both treatments; and 4) RNA interference-mediated knockdown of Sspn increased cell death in hypoxia in a cell culture system. We conclude that CIH or CCH induced significant and distinguishable alterations in gene expression in cortex and hippocampus and that Sspn seems to play a critical role in inducing cell death under hypoxic conditions.

We describe the isolation and embryonic pattern of expression of mouse Zfp462, a novel zinc finger protein gene. Zfp462 was isolated in a microarray-based search to identify genes differentially expressed in the late embryonic mouse cerebral cortex. The mouse Zfp462 deduced amino acid sequence is 2500 residues in length and contains 23 C(2)H(2)-type zinc finger motifs. Zfp462 shares low homology (30%) with non-vertebrate zinc finger proteins, suggesting that it is vertebrate-specific. However, domains found only in vertebrate-specific zinc finger proteins, such as the KRAB and SCAN domains, were not identified in Zfp462. We show that Zfp462 is expressed in the developing central nervous system, branchial arches, otic vesicles, sensory ganglia, somites and limbs. In the central nervous system, Zfp462 is expressed at the headfold stage and is later expressed in the developing forebrain, brainstem and spinal cord. Interestingly, beginning at E13.5, Zfp462 is expressed in a graded pattern in the mouse cerebral cortex. As predicted by the microarray analysis, Zfp462 is expressed at its highest levels in the caudal and medial cerebral cortex. Its expression in cortical layers is greatest in the marginal zone, the cortical plate and the subventricular zone. Zfp462 thus belongs to a growing family of molecular determinants that are expressed in a graded pattern in the developing cerebral cortex. This family includes transcription factor-encoding genes such as COUP-TF1, Emx2 and Pax6, which have been implicated in the process of cortical arealization.

Recently, the expression of the human peptide/histidine transporter (hPHT1, SLC15A4) mRNA was observed in the GI tract and in Caco-2 cells, suggesting that it may participate in the intestinal absorption of peptide-based agents. This study aims to elucidate the: (i) protein expression pattern of hPHT1 (SLC15A4) in human small intestine; (ii) cloning of the hPHT1 full-length sequence; (iii) functional characterization of hPHT1 in transiently transfected COS-7 cells. The expression of hPHT1 was measured using Western blot and immunohistochemical analysis. The hPHT1 full-sequence was amplified from BeWo cells, inserted into the pcDNA3.1-V5/His TOPO plasmid and transiently transfected into COS-7 cells to investigate the uptake kinetics of [3H]histidine and [3H]carnosine. Time, pH and sodium-dependent uptake studies were performed in mock (empty vector) and hPHT1-COS-7 cells. Results demonstrated hPHT1 protein expression in different intestinal regions. Histidine and carnosine uptake was linear in hPHT1-COS-7 cells over 15 min and was found to be pH-dependent. These substrates and valacyclovir showed significantly higher uptake at pH 5.0 in the hPHT1 transients when contrasted to the mock COS-7 cells, whereas glycylsarcosine uptake was significantly lower and unaffected by pH. Other di- and tripeptides also showed affinity for hPHT1. This study presents the initial functional characterization, the protein expression of the hPHT1 transporter and provides insight into a potentially different route for increasing peptide and peptide-based drug transport.

Rab11 family interacting protein 4 (Rab11-FIP4) was initially identified in humans as an Rab11-binding protein, but its biological function has remained unknown. We cloned the zebrafish orthologue of Rab11-FIP4 (zRab11-FIP4) and analyzed its function in vivo by using antisense morpholino. zRab11-FIP4 was expressed as 2 alternative transcripts, i.e., the longer A-form predominantly expressed in neural tissues and the shorter B-form expressed ubiquitously; and in situ hybridization revealed that the A-form was the dominant form. In the developing retina, zRab11-FIP4 was expressed in progenitors throughout the retina at early stages; and then, along with the differentiation, the expression became gradually restricted to the ganglion cell layer and ciliary marginal zone. zRab11-FIP4A knockdown embryos exhibited eye phenotypes similar to those of the shh mutant, such as a small eye with impaired cell proliferation and the delay in cell-cycle exit and differentiation of retinal progenitors. The lack of induction of p57kip2 and enhanced expression of cyclin D1 were observed in the morphant retina. Importantly, the delay in cell-cycle exit was rescued by ectopic expression of either p57Kip2 or dominant-negative PKA, suggesting that Rab11-FIP4A plays pivotal roles in retinal development by regulating Shh signaling and a mechanism acting in parallel with Shh signaling in the control of cell-cycle exit.

Extracellular matrix (ECM) remodeling is achieved by both production and degradation of ECM molecules during bone development. ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) constitutes a family of extracellular proteases which are implicated in cleaving the protein versican. The present study was designed to investigate the expression of versican and ADAMTS1, 4, and 5 mRNA during bone development in rat mandibles and hind limbs by RT-PCR and in situ hybridization. Versican was localized by immunohistochemistry. The process of bone development from day 14 postcoitum through week 6 postnatum was divided into the beginning of osteogenesis, woven bone, and lamellar bone stages. Versican protein was abundant in the woven bone matrix, but decreased in the lamellar bone matrix. Versican mRNA was prominent in some osteoblasts with corresponding localization of the cognate protein. The temporal and spatial mRNA expression pattern of ADAMTS1, 4, and 5 was comparable to that of versican. These results suggest that woven bone rich in versican alters into lamellar bone containing little versican during bone development in both mandibles and hind limbs, where some osteoblasts may be involved in production as well as degradation of versican by secreting ADAMTS1, 4, and 5.

Malignant gliomas are deadly brain tumors characterized by diffuse invasion into the surrounding brain tissue. Understanding the mechanisms involved in glioma invasion could lead to new therapeutic strategies. We have previously shown that BEHAB/brevican, an extracellular matrix protein in the central nervous system, plays a role in the invasive ability of gliomas. The mechanisms that underlie BEHAB/brevican function are not yet understood, due in part to the existence of several isoforms that may have different functions. Here we describe for the first time the expression of BEHAB/brevican in human brain and characterize two novel glioma-specific isoforms, B/b(sia) and B/b(Deltag), which are generated by differential glycosylation and are absent from normal adult brain and other neuropathologies. B/b(sia) is an oversialylated isoform expressed by about half the high- and low-grade gliomas analyzed. B/b(Deltag) lacks most of the carbohydrates typically present on BEHAB/brevican and is the major up-regulated isoform of this protein in high-grade gliomas but is absent in a specific subset of low-grade, indolent oligodendrogliomas. B/b(Deltag) is detected on the extracellular surface, where it binds to the membrane by a mechanism distinct from the other BEHAB/brevican isoforms. The glioma-specific expression of B/b(Deltag), its restricted membrane localization, and its expression in all high-grade gliomas tested to date suggest that it may play a significant role in glioma progression and make it an important new potential therapeutic target. In addition, its absence from benign gliomas prompts its use as a diagnostic marker to distinguish primary brain tumors of similar histology but different pathologic course.

The small GTP-binding protein ADP ribosylation factor 6 (ARF6) has recently been implicated in the internalization of G protein-coupled receptors (GPCRs), although its precise molecular mechanism in this process remains unclear. We have recently identified centaurin alpha(1) as a GTPase activating protein (GAP) for ARF6. In the current study, we characterized the effects of centaurin alpha(1) on the agonist-induced internalization of the beta(2)-adrenoceptor transiently expressed in human embryonic kidney (HEK) 293 cells. Using an enzyme-linked immunosorbent assay as well as confocal imaging of cells, we found that expression of centaurin alpha(1) strongly inhibited the isoproterenol-induced internalization of beta(2)-adrenoceptor. On the other hand, expression of functionally inactive versions of centaurin alpha(1), including an R49C mutant, which has no catalytic activity, and a double pleckstrin homology (PH) mutant (DM; R148C/R273C), which has mutations in both the PH domains of centaurin alpha(1), rendering it unable to translocate to the cell membrane, were unable to inhibit beta(2)-adrenoceptor internalization. In addition, a constitutively active version of ARF6, ARF6Q67L, reversed the ability of centaurin alpha(1) to inhibit beta(2)-adrenoceptor internalization. Finally, expression of centaurin alpha(1) also inhibited the agonist-induced internalization of beta(2)-adrenoceptor endogenously expressed in HEK 293 cells, whereas the R49C and DM mutant versions of centaurin alpha(1) had no effect. Together, these data indicate that by acting as an ARF6 GAP, centaurin alpha(1) is able to switch off ARF6 and so inhibit its ability to mediate beta(2)-adrenoceptor internalization. Thus, ARF6 GAPs, such as centaurin alpha(1), are likely to play a crucial role in GPCR trafficking by modulating the activity of ARF6.

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the mu-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.

Tomosyn was previously identified as a syntaxin-binding protein that inhibits soluble NSF (n-ethylmaleimide-sensitive fusion protein) attachment protein receptor (SNARE)-mediated secretion. We set out to investigate the distribution of tomosyn mRNA in the mammalian brain and found evidence for the presence of two paralogous genes designated tomosyn-1 and -2. In a collection of tomosyn-2 cDNA clones, we observed four splice variants (named xb-, b-, m- and s-tomosyn-2) derived from the skipping of exons 19 and 21. This feature is conserved with tomosyn-1 that encodes three splice variants. To compare the expression pattern of tomosyn-1 and -2, we performed in situ hybridization experiments with gene-specific probes. Both genes were expressed in the nervous system, clearly following distinct spatial and developmental expression patterns. Real-time quantitative PCR experiments indicated that tomosyn-1 expression was up-regulated less than threefold between developmental stages E10 and P12, whereas tomosyn-2 expression increased 31-fold. Not only the transcription level, but also the splice composition of tomosyn-2 mRNA shifted during development. We conclude that two distinct genes drive expression of seven tomosyn isoforms. Their expression patterns support a role in regulating neuronal secretion. All isoforms share conserved WD40 and SNARE domains separated by a hypervariable module, the function of which remains to be clarified.

Precursor cells have the capacity to repopulate the demyelinated brain, but the molecular mechanisms that facilitate their recruitment are largely unknown. The low-affinity neurotrophin receptor, p75(NTR), may be one of these regulators; however, its expression profile by oligodendroglia within the multiple sclerosis (MS) brain remains uncertain. We therefore assessed the expression profile of this receptor within 8 MS and 4 control brains. We found no evidence of expression of p75(NTR) by mature oligodendrocytes. Instead, we demonstrated the presence of p75(NTR) on a subgroup of NG2-positive oligodendroglial progenitors in a periventricular plaque in one MS sample. Notably, p75(NTR)-expressing cells were also detected within the subventricular zone (SVZ) of this brain, adjacent to the periventricular plaque. In animals with experimental demyelination we observed similar patterns of p75(NTR) expression, initially confined to precursor cells within the SVZ, followed at later stages in the disease course by its expression amongst a subset of oligodendroglial progenitors within the corpus callosum. These data suggest that a population of precursor cells within the SVZ can be induced to express p75(NTR) and to subsequently assume an oligodendroglial progenitor phenotype in response to demyelination in the adjacent white matter.

We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is gamma-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with gamma-aminobutyric acid, type A, receptor (GABA(A)R) clusters than with alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA(A)R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce inflammation and as analgesics by inhibition of cyclooxygenase-2. At higher concentrations, some NSAIDs inhibit proliferation and induce apoptosis of cancer cells. Although several molecular mechanisms have been postulated to explain the anticancer effects of NSAIDs, they do not involve merely the inhibition of cyclooxygenase-2, and a more proximate initiator molecule may be regulated by NSAIDs to inhibit growth. The p75 neurotrophin receptor (p75NTR) is a proximate cell membrane receptor glycoprotein that has been identified as a tumor and metastasis suppressor. We observed that NSAID treatment of cell lines from bladder and other organs induced expression of the p75NTR protein. Of the different types of NSAIDs examined, ibuprofen was more efficacious than aspirin and acetaminophen and comparable with (R)-flurbiprofen and indomethacin in induction of p75NTR protein expression. This rank order NSAID induction of the p75NTR protein correlated with the ability of these NSAIDs to reduce cancer cell survival. To examine a mechanistic relationship between ibuprofen induction of p75NTR protein and inhibition of survival, bladder cancer cells were transfected with ponasterone A-inducible vectors that expressed a death domain-deleted (DeltaDD) or intracellular domain-deleted (DeltaICD) p75NTR product that acts as a dominant negative antagonist of the intact p75NTR protein. Expression of DeltaDD and DeltaICD rescued cells from ibuprofen inhibition of growth. These observations suggest that p75NTR is an important upstream modulator of the anticancer effects of NSAIDs and that ibuprofen induction of the p75NTR protein establishes an alternate mechanism by which ibuprofen may exert an anticancer effect.

The N-methyl-D-aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK-801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.

A gene for the zebrafish brain-type fatty acid-binding protein (fabp7b) was identified and its structure defined. The zebrafish fabp7b gene spans 1479 bp and consists of four exons encoding 24, 58, 34 and 16 amino acids, respectively, which is identical to the structure of the fabp7a gene previously described. The complete fabp7b cDNA was isolated by 5' and 3' RACE and its nucleotide sequence determined. The deduced amino acid sequence of FABP7B encoded by the zebrafish fabp7b gene shares 82% identity with that of FABP7A encoded by the zebrafish fabp7a gene. A single transcription start site for the fabp7b gene was mapped by 5' RNA ligase-mediated RACE. Phylogenetic analysis indicated that the duplication of the fabp7 genes occurred in the fish lineage after their divergence from mammals. The zebrafish fabp7b gene was assigned to linkage group 20 by radiation hybrid mapping. Reverse transcription-polymerase chain reaction detected fabp7b transcripts in the same adult tissues as fabp7a transcripts. In the brain, levels of fabp7b transcripts were lower than fabp7a transcripts. Whole-mount in situ hybridization showed that the zebrafish fabp7a transcripts were distributed in the early developing central nervous system. In addition to being expressed in the developing brain and retina, zebrafish fabp7b mRNA was also detected in the swim bladder and pharynx during the embryonic to larval transitory phase.

To elucidate the anatomical distribution of the serotonergic neurotransmitter system, we identified serotonin transporter (5-HTT) in the hippocampus of rats and monkeys by immunohistochemistry. A widespread and heterogeneous distribution of 5-HTT-immunoreactive fine fibers was noted in the rat brain. However, in monkeys, punctuate 5-HTT-immunoreactive deposits and fewer fibers were observed. The species difference in 5-HTT immunohistochemical staining pattern may be caused by differences in localization of 5-HTT between species., (Copyright 2004 Elsevier B.V.)

In immuno-light and -electron microscopy, brain-type fatty acid binding protein (B-FABP) is localized in the sustentacular cells enclosing the chromaffin cells in the adrenal medulla. This represents another new feature commonly shared by the sustentacular cells and ganglionic satellite cells, the latter of which has already been reported to localize this molecule, and suggests a common feature in lipid metabolism shared by the two cells enclosing peripheral neurons and paraneurons. On the other hand, epidermal-type fatty acid binding protein (E-FABP) is localized in two discrete cells in the adrenal gland: the one is a subpopulation of intra-adrenal macrophages which are intensely immunoreactive for F4/80, a marker of macrophages, and are rich in pleomorphic lysosomes. Because of their direct apposition to adjacent cortical endocrine cells and medullary chromaffin cells, the macrophages may be involved not only in phagocytosis of degenerating adrenal cells but also in exertion of some yet unknown effects on the endocrine function of the cortical and medullary cells via humoral factors such as cytokines which have recently been known to be secreted by macrophages. The other is a population of cells having scanty perikaryal cytoplasm poor in organneles and several thinny extended processes in the cortex and exhibiting weak immunoreactivity for E-FABP. The possible natures of these cells immunoreactive for E-FABP are discussed in view of a subpopulation of endothelial cells or the dendritic cells of antigen-presenting property.

Cytokine signaling is negatively regulated by a set of SH2 domain-containing proteins, the Suppressors of Cytokine Signaling (SOCS) acting as intracellular modulators. Experimental evidence indicates that SOCS gene expression is induced by cytokines and pro-inflammatory stimuli and is highly controlled both at transcription and translation level. Furthermore, SOCS proteins appear rapidly degraded inside the cells, mostly controlling their stability by interacting with specific molecules such as elongin B and C. It has been shown that SOCS-1/JAB, a member of the SOCS family, interacts with TRIM-8/Gerp, a new ring protein specifically binding SOCS-1 recombitant polypeptide in-vitro and in-vivo. Trim-8/Gerp, transcribes IFN-gamma in epithelial and lymphoid cells and is expressed mostly ubiquitously in murine and human tissues. Here in this report we present the genomic organization of this new SOCS-1 interactor, and we add new tools for extending investigation of the complex mechanism that undergoes negatively regulation of cytokine signaling.

We report a random disruption in the mouse genome that resulted in lethal paralysis in homozygous newborns. The disruption blocked expression of neurobeachin, a protein containing a BEACH (beige and Chediak-Higashi) domain implicated in synaptic vesicle trafficking and an AKAP (A-kinase anchor protein) domain linked to localization of cAMP-dependent protein kinase activity. nbea-null mice demonstrated a complete block of evoked synaptic transmission at neuromuscular junctions, whereas nerve conduction, synaptic structure, and spontaneous synaptic vesicle release were completely normal. These findings support an essential role for neurobeachin in evoked neurotransmitter release at neuromuscular junctions and suggest that it plays an important role in synaptic transmission.