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Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain.Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons that corresponded to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C-mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASDs.

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium (GIRK2) channel subunit 2, have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants (‘Affected: AF’) and four control subjects without variants (‘Unaffected: UN’). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 A resolution provides insights into the molecular basis of the deNADding activity in the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells, and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism. Structural and biochemical analyses identify Nudt12 as a novel mRNA deNADing enzyme in mammalian cells, which has a different substrate preference compared to the deNADing enzyme DXO.

Specific deletion of the tumor suppressor TRAF3 from B lymphocytes in mice leads to the prolonged survival of mature B cells and expanded B cell compartments in secondary lymphoid organs. In the current study, we investigated the metabolic basis of TRAF3-mediated regulation of B cell survival by employing metabolomic, lipidomic, and transcriptomic analyses. We compared the polar metabolites, lipids, and metabolic enzymes of resting splenic B cells purified from young adult B cell–specific Traf3−/− and littermate control mice. We found that multiple metabolites, lipids, and enzymes regulated by TRAF3 in B cells are clustered in the choline metabolic pathway. Using stable isotope labeling, we demonstrated that phosphocholine and phosphatidylcholine biosynthesis was markedly elevated in Traf3−/− mouse B cells and decreased in TRAF3-reconstituted human multiple myeloma cells. Furthermore, pharmacological inhibition of choline kinase α, an enzyme that catalyzes phosphocholine synthesis and was strikingly increased in Traf3−/− B cells, substantially reversed the survival phenotype of Traf3−/− B cells both in vitro and in vivo. Taken together, our results indicate that enhanced phosphocholine and phosphatidylcholine synthesis supports the prolonged survival of Traf3−/− B lymphocytes. Our findings suggest that TRAF3-regulated choline metabolism has diagnostic and therapeutic value for B cell malignancies with TRAF3 deletions or relevant mutations.

The OPRM1 A118G single nucleotide polymorphism (SNP rs1799971) gene variant encoding the N40D µ-opioid receptor (MOR) has been associated with dependence on opiates and other drugs of abuse but its mechanism is unknown. The frequency of G-allele carriers is ~40% in Asians, ~16% in Europeans, and ~3% in African-Americans. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Here we generated homozygous N40D subject-specific induced inhibitory neuronal cells (iNs) from seven human-induced pluripotent stem (iPS) cell lines from subjects of European descent (both male and female) and probed the impact of N40D MOR regulation on synaptic transmission. We found that D40 iNs exhibit consistently stronger suppression (versus N40) of spontaneous inhibitory postsynaptic currents (sIPSCs) across multiple subjects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were recapitulated in two sets of independently engineered isogenic N40D iNs. In addition, we employed biochemical analysis and observed differential N-linked glycosylation of human MOR N40D. This study identifies neurophysiological and molecular differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

Tuberous Sclerosis Complex (TSC) is a disease caused by autosomal dominant mutations in the TSC1 or TSC2 genes, and is characterized by tumor susceptibility, brain lesions, seizures and behavioral impairments. The TSC1 and TSC2 genes encode proteins forming a complex (TSC), which is a major regulator and suppressor of mammalian target of rapamycin complex 1 (mTORC1), a signaling complex that promotes cell growth and proliferation. TSC1/2 loss of heterozygosity (LOH) and the subsequent complete loss of TSC regulatory activity in null cells causes mTORC1 dysregulation and TSC-associated brain lesions or other tissue tumors. However, it is not clear whether TSC1/2 heterozygous brain cells are abnormal and contribute to TSC neuropathology. To investigate this issue, we generated induced pluripotent stem cells (iPSCs) from TSC patients and unaffected controls, and utilized these to obtain neural progenitor cells (NPCs) and differentiated neurons in vitro. These patient-derived TSC2 heterozygous NPCs were delayed in their ability to differentiate into neurons. Patient-derived progenitor cells also exhibited a modest activation of mTORC1 signaling downstream of TSC, and a marked attenuation of upstream PI3K/AKT signaling. We further show that pharmacologic PI3K or AKT inhibition, but not mTORC1 inhibition, causes a neuronal differentiation delay, mimicking the patient phenotype. Together these data suggest that heterozygous TSC2 mutations disrupt neuronal development, potentially contributing to the disease neuropathology, and that this defect may result from dysregulated PI3K/AKT signaling in neural progenitor cells.

The adult CNS does not spontaneously regenerate after injury, due in large part to myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo-A, and oligodendrocyte-myelin glycoprotein. All three inhibitors can interact with either the Nogo receptor complex or paired immunoglobulin-like receptor B. A conditioning lesion of the sciatic nerve allows the central processes of dorsal root ganglion (DRG) neurons to spontaneously regenerate in vivo after a dorsal column lesion. After a conditioning lesion, DRG neurons are no longer inhibited by myelin, and this effect is cyclic AMP (cAMP)- and transcription-dependent. Using a microarray analysis, we identified several genes that are up-regulated both in adult DRGs after a conditioning lesion and in DRG neurons treated with cAMP analogues. One gene that was up-regulated under both conditions is metallothionein (MT)-I. We show here that treatment with two closely related isoforms of MT (MT-I/II) can overcome the inhibitory effects of both myelin and MAG for cortical, hippocampal, and DRG neurons. Intrathecal delivery of MT-I/II to adult DRGs also promotes neurite outgrowth in the presence of MAG. Adult DRGs from MT-I/II-deficient mice extend significantly shorter processes on MAG compared with wild-type DRG neurons, and regeneration of dorsal column axons does not occur after a conditioning lesion in MT-I/II-deficient mice. Furthermore, a single intravitreal injection of MT-I/II after optic nerve crush promotes axonal regeneration. Mechanistically, MT-I/II ability to overcome MAG-mediated inhibition is transcription-dependent, and MT-I/II can block the proteolytic activity of α-secretase and the activation of PKC and Rho in response to soluble MAG.

Background DNA methylation is an epigenetic modification that is studied at a single-base resolution with bisulfite treatment followed by high-throughput sequencing. After alignment of the sequence reads to a reference genome, methylation counts are analyzed to determine genomic regions that are differentially methylated between two or more biological conditions. Even though a variety of software packages is available for different aspects of the bioinformatics analysis, they often produce results that are biased or require excessive computational requirements. Results DMRfinder is a novel computational pipeline that identifies differentially methylated regions efficiently. Following alignment, DMRfinder extracts methylation counts and performs a modified single-linkage clustering of methylation sites into genomic regions. It then compares methylation levels using beta-binomial hierarchical modeling and Wald tests. Among its innovative attributes are the analyses of novel methylation sites and methylation linkage, as well as the simultaneous statistical analysis of multiple sample groups. To demonstrate its efficiency, DMRfinder is benchmarked against other computational approaches using a large published dataset. Contrasting two replicates of the same sample yielded minimal genomic regions with DMRfinder, whereas two alternative software packages reported a substantial number of false positives. Further analyses of biological samples revealed fundamental differences between DMRfinder and another software package, despite the fact that they utilize the same underlying statistical basis. For each step, DMRfinder completed the analysis in a fraction of the time required by other software. Conclusions Among the computational approaches for identifying differentially methylated regions from high-throughput bisulfite sequencing datasets, DMRfinder is the first that integrates all the post-alignment steps in a single package. Compared to other software, DMRfinder is extremely efficient and unbiased in this process. DMRfinder is free and open-source software, available on GitHub (github.com/jsh58/DMRfinder); it is written in Python and R, and is supported on Linux. Electronic supplementary material The online version of this article (10.1186/s12859-017-1909-0) contains supplementary material, which is available to authorized users.

Friedreich ataxia (FRDA) is the most common inherited ataxia caused primarily by an intronic GAA.TTC triplet repeat expansion in the frataxin ( FXN ) gene. FXN RNA and protein levels are reduced in patients leading to progressive gait and limb ataxia, sensory loss, reduced tendon reflexes, dysarthria, absent lower limb reflexes, and loss of position and vibration sense. Neurological manifestations ensue from primary loss of dorsal root ganglia neurons and their associated axons ascending centrally in the spinal cord and peripherally in large myelinated nerves. Small noncoding RNAs such as microRNAs have been shown to be dysregulated in neurodegenerative diseases such as Alzheimer's and Huntington's disease. Here we report that hsa-miR-886-3p (miR-886-3p) was increased in patient cells as well as peripheral patient blood samples. Selective reduction in miR-886-3p by an anti-miR led to elevation of FXN message and protein levels without associated changes in histone marks at the FXN locus. Nevertheless, derepression of frataxin by a histone deacetylase inhibitor leads to a decrease in miR-886-3p. These results outline involvement of a small RNA, miR-886-3p in FRDA and a novel therapeutic approach to this disease using an anti-miR-886-3p.

MicroRNAs (miRNAs) are endogenous single-stranded RNA molecules of about 21 nucleotides in length that are fundamental post-transcriptional regulators of gene expression. Although the transcriptional and processing events involved in the generation of miRNAs have been extensively studied, very little is known pertaining to components that regulate the stability of individual miRNAs. All RNAs have distinct inherent half-lives that dictate their level of accumulation and miRNAs would be expected to follow a similar principle. Here we demonstrate that although most miRNA appear to be stable, like mRNAs, miRNAs possess differential stability in human cells. In particular, we found that miR-382, a miRNA that contributes to HIV-1 provirus latency, is unstable in cells. To determine the region of miR-382 responsible for its rapid decay, we developed a cell-free system that recapitulated the observed cell-based-regulated miR-382 turnover. The system utilizes in vitro-processed mature miRNA derived from pre-miRNA and follows the decay of the processed miRNA. Using this system, we demonstrate that instability of miR-382 is driven by sequences outside its seed region and required the 3′ terminal seven nucleotides where mutations in this region increased the stability of the RNA. Moreover, the exosome 3′–5′ exoribonuclease complex was identified as the primary nuclease involved in miR-382 decay with a more modest contribution by the Xrn1 and no detectable contribution by Xrn2. These studies provide evidence for an miRNA element essential for rapid miRNA decay and implicate the exosome in this process. The development of a biochemically amendable system to analyze the mechanism of differential miRNA stability provides an important step in efforts to regulate gene expression by modulating miRNA stability.

We have generated clones (L2.3 and RG3.6) of neural progenitors with radial glial properties from rat E14.5 cortex that differentiate into astrocytes, neurons, and oligodendrocytes. Here, we describe a different clone (L2.2) that gives rise exclusively to neurons, but not to glia. Neuronal differentiation of L2.2 cells was inhibited by bone morphogenic protein 2 (BMP2) and enhanced by Sonic Hedgehog (SHH) similar to cortical interneuron progenitors. Compared with L2.3, differentiating L2.2 cells expressed significantly higher levels of mRNAs for glutamate decarboxylases (GADs), DLX transcription factors, calretinin, calbindin, neuropeptide Y (NPY), and somatostatin. Increased levels of DLX-2, GADs, and calretinin proteins were confirmed upon differentiation. L2.2 cells differentiated into neurons that fired action potentials in vitro, and their electrophysiological differentiation was accelerated and more complete when cocultured with developing astroglial cells but not with conditioned medium from these cells. The combined results suggest that clone L2.2 resembles GABAergic interneuron progenitors in the developing forebrain.

Radial glia are neural stem cells that exist only transiently during central nervous system (CNS) development, where they serve as scaffolds for neuronal migration. Their instability makes them difficult to study, and therefore we have isolated stabilized radial glial clones from E14.5 cortical progenitors (e.g., L2.3) after expression of v-myc. Activated Notch1 intracellular region (actNotch1) promotes radial glia in the embryonic mouse forebrain (Gaiano et al., (2000), and when it was introduced into E14.5 cortical progenitors or radial glial clone L2.3, the cells exhibited enhanced radial morphology and increased expression of the radial glial marker BLBP. A representative clone of L2.3 cells expressing actNotch1 called NL2.3-4 migrated more extensively than L2.3 cells in culture and in white matter of the adult rat spinal cord. Microarray and RT-PCR comparisons of mRNAs expressed in these closely related clones showed extensive similarities, but differed significantly for certain mRNAs including several cell adhesion molecules. Cell adhesion assays demonstrated significantly enhanced adhesion to laminin of NL2.3-4 by comparison to L2.3 cells. The laminin binding protein nidogen was the most highly induced adhesion molecule in NL2.3-4, and immunological analyses indicated that radial glia synthesize and secrete nidogen. Adhesion of NL2.3-4 cells to laminin was inhibited by anti-nidogen antibodies and required the nidogen binding region in laminin, indicating that nidogen promotes cell adhesion to laminin. The combined results indicate that persistent expression of activated Notch1 maintains the phenotype of radial glial cells, inhibits their differentiation, and promotes their adhesion and migration on a laminin/nidogen complex. © 2008 Wiley-Liss, Inc.

Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.

Removal of the 5′-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.

Background Alcohol abuse produces an enormous impact on health, society, and the economy. Currently, there are very limited therapies available, largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. Results Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3+ (LC3B+) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover, a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. Conclusions This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models.

Poor regeneration of severed axons in the central nervous system (CNS) limits functional recovery. Regeneration failure involves an interplay of inhibitory environmental elements and the growth state of the neuron. To find internal changes in gene expression that might overcome inhibitory environmental cues, we compared several paradigms that allow growth in the inhibitory environment. Conditions that allow axon growth by axotomized and cultured dorsal root ganglion (DRG) neurons on CNS myelin include immaturity (the first few postnatal days), high levels of cyclic adenosine mono phosphate (cAMP), and conditioning with a peripheral nerve lesion before explant. This shift from inhibition to growth depends on transcription. Seeking to understand the transcriptome changes that allow axon growth in the CNS, we collaborated with the Marie Filbin laboratory to identify several mRNAs that are functionally relevant, as determined by gain- and loss-of-function studies. In this Perspective, we review evidence from these experiments and discuss the merits of comparing multiple regenerative paradigms to identify a core transcriptional program for CNS axon regeneration.

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.

Spinal cord injury (SCI)-induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post-traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI-induced N-methyl-D-aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with -(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate [MK-801]) reversed the effect of SCI on about 50% of the SCI-affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up-regulation of inflammatory factors. Therefore, SCI-induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK-801 effects on the SCI-induced mRNA changes reported here are novel. Additionally, we found that the MK-801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.

Aims Ultraviolet irradiation and carcinogens have been reported to induce epigenetic alterations, which potentially contribute to the development of skin cancer. We aimed to study the genome-wide DNA methylation profiles of skin cancers induced by ultraviolet B (UVB) irradiation and 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-1,3-acetate (TPA). Main methods Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was utilized to ascertain the DNA methylation profiles in the following common mouse skin cancer models: SKH-1 mice treated with UVB irradiation and CD-1 mice treated with DMBA/TPA. Ingenuity® Pathway Analysis (IPA) software was utilized to analyze the data and to identify gene interactions among the different pathways. Key findings 6003 genes in the UVB group and 5424 genes in the DMBA/TPA group exhibited a greater than 2-fold change in CpG methylation as mapped by the IPA software. The top canonical pathways identified by IPA after the two treatments were ranked were pathways related to cancer development, cAMP-mediated signaling, G protein-coupled receptor signaling and PTEN signaling associated with UVB treatment, whereas protein kinase A signaling and xenobiotic metabolism signaling were associated with DMBA/TPA treatment. In addition, the mapped IL-6-related inflammatory pathways displayed alterations in the methylation profiles of inflammation-related genes linked to UVB treatment. Significance Genes with altered methylation were ranked in the UVB and DMBA/TPA models, and the molecular interaction networks of those genes were identified by the IPA software. The genome-wide DNA methylation profiles of skin cancers induced by UV irradiation or by DMBA/TPA will be useful for future studies on epigenetic gene regulation in skin carcinogenesis.

Background Identification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells. Methods We used microarray analysis to identify genes differentially expressed in TRAF3−/− mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing. Results We identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3−/− B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells. Conclusions Our results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM. Electronic supplementary material The online version of this article (doi:10.1186/s13045-014-0056-6) contains supplementary material, which is available to authorized users.

Since recent work has identified an apoptotic pathway in sympathetic neurons that is mediated by autocrine interleukin-1 (IL-1), we investigated whether cultured sympathetic neurons possess functional IL-1 receptors. Cultured sympathetic neurons express levels of IL-1RI and IL-1RAcP mRNAs consistent with signal transduction. Neurons stimulated with IL-1 demonstrate enhanced p65 NF-kappaB nuclear translocation and enhanced NF-kappaB DNA binding activity, with at least p65 and p50 subunits participating in the DNA binding activity. RNA differential display identified several neuronal mRNAs regulated by IL-1, including a member of the reticulon family. We conclude that IL-1 stimulates a potential component of a neuronal secretory pathway.

Interleukin-1 beta (IL-1 beta) induces leukemia inhibitor factor (LIF) expression in a number of cell types including non-neuronal cells of the sympathetic superior cervical ganglion (SCG). Upregulation of LIF by inflammatory cytokines is usually associated with injury response. We characterized the molecular mechanism of LIF mRNA regulation by IL-1 beta in explanted neonatal rat SCG and a Schwann cell line. IL-1 beta increases LIF mRNA levels by interacting with IL-1 receptors in SCG, since this induction could be diminished by inclusion of either soluble IL-1 receptors or IL-1 receptor antagonist. The antiinflammatory glucocorticoid dexamethasone also inhibits LIF mRNA induction by IL-1 beta. LIF mRNA encodes a 3' AU-rich mRNA stability control sequence, but IL-1 beta does not appear to regulate the decay of LIF mRNA by this mechanism. IL-1 beta does not raise LIF gene transcription rate in cultured SCG 6 or 24 h after addition of IL-1 beta as measured by nuclear run-on assays. LIF gene transcription is induced repidly and transiently in an immortalized Schwann cell line, returning to uninduced rates by 1 h after induction. These results suggest that the IL-1 beta induction of LIF gene expression is at least partially transcriptional, but that LIF mRNA increases to a greater extent than LIF transcription, suggesting the possibility of posttranscriptional regulation as well.

Two types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of approximately 55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1beta as the ligand and shown to have an affinity for human 125I-IL-1beta (KD approximately 2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have approximately 95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5' end identical to the IL-1RII, the 200 base pairs at the 3' end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125I-IL-1beta binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125I-IL-1beta binding (Ki approximately 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced.

Substance P (SP), one of the best characterized neurogenic mediators of immune hyperactivity, stimulates the production of a variety of cytokines, and acts as a general proinflammatory agent inducing proliferation and activation of immune cells Neurokinin 1 receptor (NK-1R), a G-protein-coupled receptor with high affinity for SP, has been cloned from nonlymphoid tissues. Although previous binding studies indicated the presence of high affinity SP receptors on immune cells, recent studies questioned the existence of specific NK-1R on lymphocytes and monocytes. In this study we investigate the expression of the NK-1R gene in murine T lymphocytes and T cell lines. The expression of NK-1R mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR), with two separate sets of specific primers, and by nuclease protection assays. The NK-1R nature of the lymphocytic amplified fragments was confirmed by Southern blots with specific murine brain NK-1R probes. Both T cells lines (EL-4.IL-2 and LBRM-T6G) which were previously shown to respond to SP by increased IL-2 production express NK-1R mRNA constitutively. Normal spleen cells and purified CD4+ T lymphocytes which also responded to SP by increased IL-2 production express NK-1R mRNA. In addition to CD4+ T cells, NK-1R message is also expressed in murine splenic B lymphocytes. Sequencing of the RT-PCR amplified fragments from EL-4.IL-2 cells, purified CD4+ T lymphocytes, and murine brain showed complete identity to the published murine NK-1R sequence. This study indicates that murine CD4+ T lymphocytes possess the molecular template for the production of NK-1R. Together with previous binding studies, and with pharmacological studies regarding the effect of SP and of selective NK-1R antagonists on cytokine production, the present study supports the hypothesis that the response of CD4+ T cells to SP and related tachykinins is mediated through specific NK-1 receptors.

We have constructed expression clones for mature rat interleukin-1 beta (IL-1 beta). Using these clones, rat recombinant IL-1 beta (rrIL-1 beta) was prepared and purified by a single-step cation-exchange chromatography method. In addition, synthetic rrIL-1 beta mRNA was transcribed and used to synthesize radiolabeled 35S-rrIL-1 beta for binding studies. The rrIL-1 beta produced was demonstrated to have IL-1 bioactivity in a mouse thymocyte proliferation assay, in which it could be blocked by inclusion of human recombinant IL-1 receptor antagonist (hrIL-1Ra). Similarly, rrIL-1 beta induced LIF mRNA expression in cultured rat sympathetic ganglia. In each case, the maximal dose of rrIL-1 beta was similar to that of human IL-1 beta. However, the peak response in rat tissue was twofold higher with rat IL-1 beta than with human IL-1 beta. The expression of rrIL-1 beta provides a useful tool for studying IL-1 receptors in rat, in which human and mouse IL-1 beta have been found to bind poorly. Species-specific activity of rat IL-1 beta may correlate with compensatory ionic properties of rat IL-1 receptor.

In recent years, microRNAs or miRNAs have been proposed to target neuronal mRNAs localized near the synapse, exerting a pivotal role in modulating local protein synthesis, and presumably affecting adaptive mechanisms such as synaptic plasticity. In the present study we have characterized the distribution of miRNAs in five regions of the adult mammalian brain and compared the relative abundance between total fractions and purified synaptoneurosomes (SN), using three different methodologies. The results show selective enrichment or depletion of some miRNAs when comparing total versus SN fractions. These miRNAs were different for each brain region explored. Changes in distribution could not be attributed to simple diffusion or to a targeting sequence inside the miRNAs. In silico analysis suggest that the differences in distribution may be related to the preferential concentration of synaptically localized mRNA targeted by the miRNAs. These results favor a model of co-transport of the miRNA-mRNA complex to the synapse, although further studies are required to validate this hypothesis. Using an in vivo model for increasing excitatory activity in the cortex and the hippocampus indicates that the distribution of some miRNAs can be modulated by enhanced neuronal (epileptogenic) activity. All these results demonstrate the dynamic modulation in the local distribution of miRNAs from the adult brain, which may play key roles in controlling localized protein synthesis at the synapse.

Neurons of the medullary raphe nuclei contain multiple neurotransmitters including the monoamine neurotransmitter serotonin (5-HT) and the tachykinin substance P (SP). Previously, we showed that inhibition of serotonin synthesis increased levels of mRNA coding for preprotachykinin (PPT), the prohormone precursor of SP (P. D. Walker et al. , 1990, Mol. Brain Res. 8: 113–119). To determine whether augmented serotonin transmission would have the predictably opposite effect, we have examined the effects of a 5-HT uptake blocker (zimelidine) and a monoamine oxidase inhibitor (clorgyline) on levels of PPT mRNA. We looked also at the effect of zimelidine and clorgyline on levels of ventral spinal cord substance P-like immunoreactivity (SP-LI) of the same animals. Both zimelidine and clorgyline decreased levels of PPT mRNA in medullary raphe neurons and SP-LI in the ventral spinal cord. The effect on message was transient with levels returning to control by 14 days of drug treatment. These results show that increases in synaptic 5-HT cause transient decreases in PPT mRNA and SP-LI

We have cloned a segment of the rat tryptophan hydroxylase (TPH) gene and used it to investigate differences in mRNA levels in two tissues: brain stem and pineal gland. The cloned, 13-kb genomic region contains five exons corresponding to cloned TPH cDNA sequence. Most intron/axon junctions are homologous to those found in the closely related genes encoding tyrosine and phenylalanine hydroxylase. The gene segment contains a region of low complexity that is repeated in the genome, but the remainder of the segment is single-copy sequence. TPH mRNAs can be detected by RNase protection assays and demonstrate a large difference in steady-state mRNA levels between pineal gland and brain stem dissections, agreeing with the results of Dumas and collaborators (1989, J. Neurosci. Res. 24:537–547). However, nuclear run-on assays of TPH gene transcription rates reveal similar levels of gene expression for pineal and brain stem. Control of TPH mRNA levels, therefore, appears to be post-transcriptional.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a widely expressed, secreted protein that functions primarily to inhibit members of a large family of metalloproteinases (MPs). Because of the ability of TIMP-1 to inhibit MPs, it functions in many of the same pathophysiological processes as these enzymes, e.g. wound healing, ovulation, angiogenesis, and cancer cell metastasis. TIMP-1 can also stimulate proliferation ([3H]thymidine incorporation) and cellular anabolic processes (Alamar Blue reduction). This stimulation has been shown to be dependent on the MP-inhibitory ability of TIMP-1 in the human breast cancer cell line MDA-MB-435 (Porter et al., Br J Cancer 90: 463, 2004). To shed light on the mechanism by which TIMP-1 stimulates cellular anabolic processes, an oligonucleotide microarray analysis was performed over a time course of TIMP-1 treatment of MDA-MB-435 cells. Fifteen genes whose mRNAs were differentially regulated were identified. Six (Importin-7, MGC10471, FOXC1, subunit p20 of Arp2/3 complex, mitochondrial ribosomal protein L32, and the serine/threonine kinase-4 (MST1)) of these genes were confirmed by quantitative real time PCR. These same mRNAs were shown to be regulated by the synthetic hydroxamate MP-inhibitor GM6001 but not by its inactive derivative GM6001*, suggesting that the differential regulation occurs through the MP-inhibitory ability of TIMP-1. These results suggest a complex action of TIMP-1 on cancer cells mediated by constitutively active cell surface metalloproteinases that release factors regulating cell signaling pathways; they may account for the paradoxical observation that elevated levels of TIMP-1 in tumors can correlate with an adverse prognosis.

Sulforaphane (SUL) is one member of the isothiocyanate class of cancer chemopreventive compounds that has been shown to be effective in blocking initiation and progression of carcinogenesis. Previously, many studies have shown that SUL can potently induce phase II detoxifying enzymes, which contributes to its chemopreventive functions. In this study, we used 4967 oligonucleotides microarray to assess the genes that are modulated by SUL in in vivo rat livers, as well as time course of expression of these genes. The pharmacokinetics of SUL was assessed after oral dose of 50 micromol of SUL. The plasma concentration occurred at 1 h and peaked around 20 microM at 4 h after dosing and declined with a half-life of about 2.2 h. Analysis of the gene expression data found various clusters of genes that are important in cellular defense mechanisms and cell cycle regulation. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which are increased up to 10-fold by 2 to 4 h after SUL dosing. The second cluster of genes is the glutathione S-transferase-A3-like genes, which include aflatoxin B1 aldehyde reductase and aldehyde oxidase. These genes are increased slightly by 4 h and peaked at 12 h. Real-time polymerase chain reaction was performed to authenticate the mRNA expression of some of these genes. In summary, this in vivo study of SUL provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes. The results from this study may yield better insights for its chemopreventive functions.

TRAF3 is a novel tumor suppressor identified in human non-Hodgkin lymphoma and multiple myeloma. We recently reported that TRAF3 deletion causes vastly prolonged survival of mature B cells, which eventually leads to B lymphoma development in mice. The long latency of B lymphoma development observed in B-TRAF3−/− mice suggest that additional oncogenic pathways are required for B lymphomagenesis. To delineate such oncogenic pathways in TRAF3−/− B lymphomas, we performed microarray analyses and identified Sox5 as a gene recurrently up-regulated in B lymphomas spontaneously developed in different individual B-TRAF3−/− mice. We confirmed the striking up-regulation of Sox5 expression in TRAF3−/− B lymphomas at both the mRNA and protein levels by quantitative real time PCR and Western blot analyses, respectively. We further cloned the full-length cDNA of Sox5 from B lymphomas derived from 4 different individual B-TRAF3−/− mice. Surprisingly, we found that the sequence of Sox5 expressed in TRAF3−/− B lymphomas represents a novel isoform of Sox5, which has not been previously reported. This new isoform of Sox5 contains a 35 aa deletion in the N-terminal region in front of the leucine zipper domain. When transduced into human multiple myeloma cells, the cloned Sox5 cDNA was expressed into a protein of 80 kDa, a size identical to that detected in TRAF3−/− B lymphomas. Taken together, our findings identified a novel isoform of Sox5 as a candidate oncogene in B lymphoma. Our ongoing experiments will further elucidate the roles and mechanisms of Sox5 in TRAF3 deficiency-initiated B cell malignant transformation. This study is supported by a seed grant from the New Jersey Commission on Cancer Research (10-1066-CCR-EO, P. Xie), a Faculty Research Grant (P. Xie), and the Arthur Herrmann Endowed Cancer Research Fund (P. Xie); supported in part by a grant from NCI (R. Hart) and an Aresty Research Grant (A. Desai). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-272. doi:1538-7445.AM2012-LB-272

Interleukin-1 (IL-1) induces substance P (SP) gene expression in cultured rat superior cervical ganglion (SCG) explants. In order to study the molecular mechanism of this action of IL-1, the presence of an interleukin-1 receptor (IL-1R) activity and the identity of an mRNA homologous to known IL-1R sequence was determined in SCG. The SP increase is blocked by recombinant IL-1 receptor antagonist protein, so IL-1 must be interacting with a specific receptor. We have cloned cDNA homologous to IL-1R Type I from rat SCG using a reverse transcription-polymerase chain reaction (RT-PCR). The resulting cDNA sequence is strongly homologous with mouse and human IL-1R cDNA of the T cell and fibroblast type (type I; encoding an 80-kDa protein). mRNA specific for IL-1R can be readily detected in intact SCG by quantitative RT-PCR and S1 hybridization. However, the level of IL-1R mRNA increases 3–6-fold by 2 days in culture. This increase is independent of the presence of dexamethasone, IL-1β or IL-1 receptor antagonist protein ligands. The increase of IL-1R following explantation, a model of nerve injury, may provide a mechanism linking inflammatory signalling to neuronal phenotypic changes.

The serotonergic neurons of the medullary raphe also contain the peptide neurotransmitters substance P (SP) and thyrotropin releasing hormone (TRH). In this study we asked whether the manipulation of serotonin levels alters the levels of mRNA coding for pre-proTRH. Just like the mRNA coding for the precursor of SP (preprotachykinin, PPT), levels of TRH mRNA are increased when serotonin synthesis is inhibited by para-chlorophenylalanine (pCPA) and decreased when serotonin reuptake is blocked by zimelidine. However, subtle differences suggest that the mechanisms behind these changes are different. Levels of TRH mRNA are still decreased after 14 days of zimelidine treatment, a time when levels of PPT mRNA have returned to control values. In addition, the serotonin reuptake blocker fluoxetine lowers levels of TRH but not PPT mRNA. Finally, the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induces a transient decrease in levels of PPT mRNA similar to that induced by zimelidine34, but does not decrease levels of TRH mRNA even when 10-fold higher doses are administered. These results suggest that while some pharmacological manipulations appear to alter TRH and PPT mRNA levels coordinately, the mechanisms regulating the synthesis of these two colocalized neurotransmitters are different.

In order to determine whether dopamine mediates the effects of serotonin on tachykinin biosynthesis in the neostriatum, serotonin neurotransmission was altered following depletion of dopamine. Neonatal rats received intracisternal injections of saline or the dopamine neurotoxin 6-hydroxydopamine (6HD). This lesion caused significant reductions in the neostriatum of substance P-like immunoreactivity as well as levels of mRNA coding for preprotachykinin (PPT; the prohormone precursor to tachykinins substance P, neurokinin A and related peptides). Two months later, rats were treated for 5–6 days with saline or the serotonin-uptake inhibitor, zimelidine. Zimelidine treatment of unlesioned animals significantly increased PPT mRNA levels in the neostriatum. However, zimelidine treatment failed to increase PPT mRNA content in 6HD-treated animals. By contrast, neostriatal substance P-like immunoreactivity was restored by zimelidine treatment of 6HD-lesioned animals. These results suggest that an intact nigrostriatal pathway may be required for serotonin neurotransmission to alter PPT mRNA levels in the neostriatum. However, neostriatal tachykinins may be regulated by direct serotonin innervation.

Serotonin (5-HT) neurotransmission was altered to determine its role in regulating the biosynthesis of tachykinins in the neostriatum (NS). Depletion of 5-HT with subchronic p -chlorophenylalanine> ( p CPA) treatment decreased preprotachykinin (PPT, the prohormone precursor to SP) mRNA levels in the NS. By contrast, raising extracellular 5-HT levels with zimelidine (a 5-HT uptake inhibitor) or clorgyline (a monoamine oxidase inhibitor) resulted in increased levels of PPT mRNA. To determine whether 5-HT receptors played a role in mediating the changes in PPT mRNA, animals were treated with the 5-HT 2 agonist DOI. This drug significantly increased both PPT mRNA and SP-like immunoreactivity in the NS. These results together indicate that neostriatal tachykinin biosynthesis is sensitive to alterations in 5-HT neurotransmission.

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self-renewal and differentiation. We propose that specific intracellular signaling pathways modulate gene expression during differentiation by regulating microRNA expression. Materials and Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with platelet-derived growth factor (PDGF) signaling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells, such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted toward specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signaling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signaling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways.

In order to study the regulation of co-localized monoamine and peptide neurotransmitters in the medullary raphe nuclei (MRN), we determined whether inhibition of serotonin (5-HT) synthesis affected levels of preprotachykinin (PPT; the prohormone precursor of substance P) mRNA in the MRN. Adult rats received p-chlorophenylalanine (pCPA), an irreversible inhibitor of tryptophan hydroxylase (TPH), via Alzet minipumps. TPH activity was inhibited by 70-80% for 3 weeks following pump implantation. During this period Northern mRNA analysis revealed that PPT mRNA levels in the MRN were increased 1.5-2-fold. The pCPA-induced increase was specific for PPT mRNA since no change was detected in mRNA coding for neuron-specific enolase (NSE; a constitutive neuronal protein) or 28 S ribosomal RNA. To determine whether fetal inhibition of 5-HT synthesis affected development of PPT mRNA in the MRN, pregnant rats were administered pCPA via Alzet minipump implanted on embryonic day 8. In pCPA-treated litters TPH activity was decreased by 60-70% from E16 to postnatal day 3 (P3), returning to control levels by P8. Northern mRNA analysis revealed that PPT mRNA levels increased 2.4-fold of control levels at P1. Infusion of pCPA for one week resulted in an earlier increase in PPT mRNA levels, suggesting that birth was not required to elicit the surge in PPT message. These results support the hypothesis that alterations in 5-HT metabolism have regulatory consequences for co-localized substance P formation in the MRN.

Background RNA amplification is required for incorporating laser-capture microdissection techniques into microarray assays. However, standard oligonucleotide microarrays contain sense-strand probes, so traditional T7 amplification schemes producing anti-sense RNA are not appropriate for hybridization when combined with conventional reverse transcription labeling methods. We wished to assess the accuracy of a new sense-strand RNA amplification method by comparing ratios between two samples using quantitative real-time PCR (qPCR), mimicking a two-color microarray assay. Results We performed our validation using qPCR. Three samples of rat brain RNA and three samples of rat liver RNA were amplified using several kits (Ambion messageAmp, NuGen Ovation, and several versions of Genisphere SenseAmp). Results were assessed by comparing the liver/brain ratio for 192 mRNAs before and after amplification. In general, all kits produced strong correlations with unamplified RNAs. The SenseAmp kit produced the highest correlation, and was also able to amplify a partially degraded sample accurately. Conclusion We have validated an optimized sense-strand RNA amplification method for use in comparative studies such as two-color microarrays.

We have cloned and partially characterized Bam HI fragments of Syrian hamster DNA containing most of the ribosomal RNA-coding region. Several restriction site polymorphisms within the transcribed portions of the hamster rDNA repeats have been noted. Approximately three-fifths of the repeats contain a Bam HI site upstream of the 18 S coding sequences. Approximately four-fifths of the repeats contain a Bam HI site very close to the 5' end of the 28 S coding sequences. This microheterogeneity has been maintained in the DNA of baby hamster kidney (BHK)-21 cells, a cell line established nearly 20 years ago. R-loop analysis with homologous hamster rRNAs has established the size of the coding regions and the internal transcribed spacer. Heterologous R-loop analyses with cloned hamster rDNAs and human rRNAs reveal several well-defined regions within the 28 S gene where the homology between human and hamster RNAs is greatly reduced. These regions are not detectable in heteroduplexes of hamster and human rDNAs. Sequences encoding the 18 S gene do not exhibit such reduced homology.

Several recent studies have shown that a functional poly(A) site consists of both an AAUAAA element as well as sequences downstream of the cleavage site. Two downstream regions were analyzed in an attempt to accurately locate and define the critical sequences. Chemically synthesized oligonucleotides of sequence from the early SV40 and the adenovirus E2A poly(A) sites were able to restore efficient cleavage to a deleted SV40 poly(A) site. Inversion of the sequence completely abolished poly(A) site function. A series of base substitution mutants were generated in each downstream sequence. Certain single base changes drastically altered poly(A) site function. Thus, it is concluded that a defined downstream sequence of limited complexity is important for efficient processing of the primary transcript at the poly(A) site. The position of the downstream elements relative to the AAUAAA and cleavage site was found to be critical since moving either the E2 element or the SV40 element an additional 40 nucleotides downstream abolished function. There were differences, however, in the effect of spacing on the function of the two elements. This observation, along with the fact that the two sequences are clearly different, indicates that there are at least two distinct genetic elements that direct efficient cleavage at the poly(A) site.

We have cloned and characterized a G+C-rich 2.2-kilobase pair Bam HI fragment which contains a single Syrian hamster 5 S rRNA gene. The cloned fragment directs the synthesis in vitro of 5 S RNA catalyzed by RNA polymerase III. Southern hybridization analysis of genomic hamster DNA indicates that the sequences present in the clone are tandemly repeated with a redundancy of 2700 copies per diploid cell.

Previous studies have shown that a sequence element downstream of the poly(A) addition site is required for efficient cleavage in vivo. We tested a group of downstream element point mutations in an in vitro reaction using HeLa cell nuclear extract as a source of cleavage activity. In close agreement with earlier studies (M. A. McDevitt, R. P. Hart, W. W. Wong, and J. R. Nevins, EMBO J. 5:2907-2913, 1986), a downstream element from the adenovirus E2a gene directed a higher level of cleavage activity than one from the simian virus 40 early gene. Furthermore, a single-base change in the downstream element could result in a decrease in cleavage activity of about 50-fold. That these mutations have similar effects in vivo and in vitro indicates that the HeLa cell nuclear extract system contains all of the factors required to study the mechanism of sequence recognition.

Using a chloramphenicol acetyltransferase gene under the control of a series of adenovirus E2 promoter mutants, we have determined the sequence requirements for E1A-induced as well as uninduced expression of the E2 promoter. Fully induced expression requires 79 nucleotides of upstream sequence, and the same sequences are required for uninduced expression. A promoter containing 70 nucleotides of upstream sequence is inducible, although to a lower level than wild type, while a promoter containing 59 nucleotides of upstream sequence is not inducible. The sequences required for induction as well as for uninduced promoter activity map between 21 and 79 nucleotides upstream of the transcriptional initiation site and function in either orientation and at a distance. We suggest that E1A-mediated induction involves the effective increase in a factor utilizing this upstream site and that the factor can functionally interact with the site irrespective of its location relative to the start of transcription.

Background Our previous studies of Alzheimer’s disease (AD) suggested that glutamine broadly improves cellular readiness to respond to stress and acts as a neuroprotectant both in vitro and in AD mouse models. We now expand our studies to a second neurodegenerative disease, ataxia-telangiectasia (A-T). Unlike AD, where clinically significant cognitive decline does not typically occur before age 65, A-T symptoms appear in early childhood and are caused exclusively by mutations in the ATM (A-T mutated) gene. Results Genetically ATM-deficient mice and wild type littermates were maintained with or without 4 % glutamine in their drinking water for several weeks. In ATM mutants, glutamine supplementation restored serum glutamine and glucose levels and reduced body weight loss. Lost neurophysiological function assessed through the magnitude of hippocampal long term potentiation was significantly restored. Glutamine supplemented mice also showed reduced thymus pathology and, remarkably, a full one-third extension of lifespan. In vitro assays revealed that ATM-deficient cells are more sensitive to glutamine deprivation, while supra-molar glutamine (8 mM) partially rescued the reduction of BDNF expression and HDAC4 nuclear translocation of genetically mutant Atm−/− neurons. Analysis of microarray data suggested that glutamine metabolism is significantly altered in human A-T brains as well. Conclusion Glutamine is a powerful part of an organism’s internal environment. Changes in its concentrations can have a huge impact on the function of all organ systems, especially the brain. Glutamine supplementation thus bears consideration as a therapeutic strategy for the treatment of human A-T and perhaps other neurodegenerative diseases.

We have used RNA differential display to identify specific mRNA species that are regulated by interferon-gamma (IFN gamma) treatment of enriched astrocyte cultures. Following a 24-hour treatment with IFN, mRNA was subjected to differential display using 15 different random primers. 105 upregulated and 5 downregulated cDNAs were selected for further sequencing and identification. Northern blot analysis confirmed the upregulation of 13 genes identical or highly similar to: intercrine adrenomedullin, H-rev 107, CAP-like protein, ATP synthase epsilon-subunit, complement C3, S-100 beta, Ca2+ ATPase, mg11, IFN-upregulated 56-kD protein mRNA, laminin receptor-like protein, protein tyrosine phosphatase, and zic. These data suggest that exposure to IFN gamma results in a complex change in the pattern of astrocyte gene expression.

Our previous studies of Alzheimer's disease (AD) suggested that glutamine broadly improves cellular readiness to respond to stress and acts as a neuroprotectant both in vitro and in AD mouse models. We now expand our studies to a second neurodegenerative disease, ataxia-telangiectasia (A-T). Unlike AD, where clinically significant cognitive decline does not typically occur before age 65, A-T symptoms appear in early childhood and are caused exclusively by mutations in the ATM (A-T mutated) gene.Genetically ATM-deficient mice and wild type littermates were maintained with or without 4 % glutamine in their drinking water for several weeks. In ATM mutants, glutamine supplementation restored serum glutamine and glucose levels and reduced body weight loss. Lost neurophysiological function assessed through the magnitude of hippocampal long term potentiation was significantly restored. Glutamine supplemented mice also showed reduced thymus pathology and, remarkably, a full one-third extension of lifespan. In vitro assays revealed that ATM-deficient cells are more sensitive to glutamine deprivation, while supra-molar glutamine (8 mM) partially rescued the reduction of BDNF expression and HDAC4 nuclear translocation of genetically mutant Atm(-/-) neurons. Analysis of microarray data suggested that glutamine metabolism is significantly altered in human A-T brains as well.Glutamine is a powerful part of an organism's internal environment. Changes in its concentrations can have a huge impact on the function of all organ systems, especially the brain. Glutamine supplementation thus bears consideration as a therapeutic strategy for the treatment of human A-T and perhaps other neurodegenerative diseases.