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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.

Synapse formation analyses can be performed by imaging and quantifying fluorescent signals of synaptic markers. Traditionally, these analyses are done using simple or multiple thresholding and segmentation approaches or by labor-intensive manual analysis by a human observer. Here, we describeIntellicount, a high-throughput, fully-automated synapse quantification program which applies a novel machine learning (ML)-based image processing algorithm to systematically improve region of interest (ROI) identification over simple thresholding techniques. Through processing large datasets from both human and mouse neurons, we demonstrate that this approach allows image processing to proceed independently of carefully set thresholds, thus reducing the need for human intervention. As a result, this method can efficiently and accurately process large image datasets with minimal interaction by the experimenter, making it less prone to bias and less liable to human error. Furthermore,Intellicountis integrated into an intuitive graphical user interface (GUI) that provides a set of valuable features, including automated and multifunctional figure generation, routine statistical analyses, and the ability to run full datasets through nested folders, greatly expediting the data analysis process.

SUMMARYDown syndrome (DS) is a common neurodevelopmental disorder, and cognitive defects in DS patients may arise form imbalances in excitatory and inhibitory neurotransmission. Understanding the mechanisms underlying such imbalances may provide opportunities for therapeutic intervention. Here, we show that human induced pluripotent stem cells (hiPSCs) derived from DS patients overproduce OLIG2+ventral forebrain neural progenitors. As a result, DS hiPSC-derived cerebral organoids excessively produce specific subclasses of GABAergic interneurons and cause impaired recognition memory in neuronal chimeric mice. Increased OLIG2 expression in DS cells directly upregulates interneuron lineage-determining transcription factors. shRNA-mediated knockdown ofOLIG2largely reverses abnormal gene expression in early-stage DS neural progenitors, reduces interneuron production in DS organoids and chimeric mouse brains, and improves behavioral deficits in DS chimeric mice. Thus, altered OLIG2 expression may underlie neurodevelopmental abnormalities and cognitive defects in DS patients.

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.

Neurocircuits in the human brain govern complex behavior and involve connections from many different neuronal subtypes from different brain regions. Recent advances in stem cell biology have enabled the derivation of patient-specific human neuronal cells of various subtypes for the study of neuronal function and disease pathology. Nevertheless, one persistent challenge using these human-derived neurons is the ability to reconstruct models of human brain circuitry. To overcome this obstacle, we have developed a compartmentalized microfluidic device, which allows for spatial separation of cell bodies of different human-derived neuronal subtypes (excitatory, inhibitory and dopaminergic) but is permissive to the spreading of projecting processes. Induced neurons (iNs) cultured in the device expressed pan-neuronal markers and subtype specific markers. Morphologically, we demonstrate defined synaptic contacts between selected neuronal subtypes by synapsin staining. Functionally, we show that excitatory neuronal stimulation evoked excitatory postsynaptic current responses in the neurons cultured in a separate chamber.

Drug use disorders (DUDs) related to prescription opioids (narcotic pain medications) have a huge socioeconomical impact; however, the molecular and cellular mechanism of how opioids function in human is largely unknown. The Single Nucleotide Polymorphism (SNP) rs1799971 (OPRM1 A118G), produces a non-synonymous amino acid substitution, replacing Asparagine (N40) with Aspartate (D40), in the μ-opioid receptor (MOR), and is strongly associated with Drug use disorders (DUDs) and alcoholism. The impact of these genetic variants on neural function, including synaptic transmission and its molecular/cellular underpinnings, has not been established in humans. To lay the foundation for addressing these important and outstanding questions, we are using the induced neuronal (iN) cell technology that we have pioneered to generate human neurons carrying homozygous N40 or D40 MORs. Specifically, we have generated multiple induced pluripotent stem (iPS) cell lines carrying either homozygous MOR N40 or MOR D40 alleles from human subjects. Additionally, in order to mitigate the possible confounding issues related to the genetic background variations among these subjects, we also used CRISPR/Cas9 mediated gene targeting to create two isogenic pairs of human pluripotent stem cell lines: one introducing homozygous D40 alleles into a human embryonic stem (ES) cell line and the other converting one of the D40 homozygous iPS cell lines to homozygous N40. Using human neurons as a model system, our compelling preliminary data suggest that: 1) activation of both N40 and D40 MORs by agonist DAMGO in human neurons suppresses inhibitory synaptic release and the D40 MORs show stronger inhibitory effects; 2) exposure of human neurons to DAMGO for 1 day leads to loss of the DAMGO-induced suppression of synaptic release, possibly due to internalization of MORs or altered intracellular signaling; and 3) D40 human neurons show defective recovery from the loss of MOR regulation on synaptic transmission induced by prolonged agonist exposure. The objective of this proposal is to determine the functional impact and the underlying cellular/molecular mechanisms of OPRM1 allelic variants. The central hypothesis is that the N40 to D40 substitution leads to aberrant MOR expression and/or downstream signaling, resulting in altered cellular responses. The proposed research is innovative, because we will combine recent developments in stem cell biology with synaptic physiology to directly probe the impact of OPRM1 gene variants on synaptic function in human neurons. Our expectations are that cellular and synaptic function is altered in neurons carrying the MOR D40 variant. This study will provide fundamental biological insight into the regulatory functions of the MOR in human neurons.

The symptoms of ataxia-telangiectasia (A-T) include a progressive neurodegeneration caused by ATM protein deficiency. We previously found that nuclear accumulation of histone deacetylase-4, HDAC4, contributes to this degeneration; we now report that increased trimethylation of histone H3 on Lys27 (H3K27me3) mediated by polycomb repressive complex 2 (PRC2) is also important in the A-T phenotype. Enhancer of zeste homolog 2 (EZH2), a core catalytic component of PRC2, is a new ATM kinase target, and ATM-mediated phosphorylation of EZH2 on Ser734 reduces protein stability. Thus, PRC2 formation is elevated along with H3K27me3 in ATM deficiency. Chromatin immunoprecipitation and sequencing showed an increase in H3K27me3 'marks' and a dramatic shift in their location. The change of H3K27me3 chromatin-binding pattern is directly related to cell cycle reentry and cell death of ATM-deficient neurons. Lentiviral knockdown of EZH2 rescued Purkinje cell degeneration and behavioral abnormalities in Atm(-/-) mice, demonstrating that EZH2 hyperactivity is another key factor in A-T neurodegeneration.

After CNS injury, axonal regeneration is limited by myelin-associated inhibitors; however, this can be overcome through elevation of intracellular cyclic AMP (cAMP), as occurs with conditioning lesions of the sciatic nerve. This study reports that expression of secretory leukocyte protease inhibitor (SLPI) is strongly upregulated in response to elevation of cAMP. We also show that SLPI can overcome inhibition by CNS myelin and significantly enhance regeneration of transected retinal ganglion cell axons in rats. Furthermore, regeneration of dorsal column axons does not occur after a conditioning lesion in SLPI null mutant mice, indicating that expression of SLPI is required for the conditioning lesion effect. Mechanistically, we demonstrate that SLPI localizes to the nuclei of neurons, binds to the Smad2 promoter, and reduces levels of Smad2 protein. Adenoviral overexpression of Smad2 also blocked SLPI-induced axonal regeneration. SLPI and Smad2 may therefore represent new targets for therapeutic intervention in CNS injury.

Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 μM) with greater frequency and amplitude but they also exhibited rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. This study offers a proof-of-principle for utilizing human neurons to study gene variants contribution to addiction.

Summary Eukaryotic mRNAs generally possess a 5′ end N7 methyl guanosine (m 7 G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5′ end nicotinamide adenine dinucleotide (NAD + ) cap that, in contrast to the m 7 G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD + caps, and cocrystal structures of DXO/Rai1 with 3′-NADP + illuminate the molecular mechanism for how the "deNADding" reaction produces NAD + and 5′ phosphate RNA. Removal of DXO from cells increases NAD + -capped mRNA levels and enables detection of NAD + -capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD + caps can be added to 5′-processed termini. Our findings establish NAD + as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD + -capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5′ end modification distinct from the classical m 7 G cap that promotes rather than inhibits RNA decay.

Topoisomerase II beta (Top2b) is an enzyme that alters the topologic states of DNA during transcription. Top2b deletion in early retinal progenitor cells causes severe defects in neural differentiation and affects cell survival in all retinal cell types. However, it is unclear whether the observed severe phenotypes are the result of cell-autonomous/primary defects or non-cell-autonomous/secondary defects caused by alterations of other retinal cells. Using photoreceptor cells as a model, we first characterized the phenotypes in Top2b conditional knockout. Top2b deletion leads to malformation of photoreceptor outer segments (OSs) and synapses accompanied by dramatic cell loss at late-stage photoreceptor differentiation. Then, we performed mosaic analysis with shRNA-mediated Top2b knockdown in neonatal retina using in vivo electroportation to target rod photoreceptors in neonatal retina. Top2b knockdown causes defective OS without causing a dramatic cell loss, suggesting a Top2b cell-autonomous function. Furthermore, RNA-seq analysis reveals that Top2b controls the expression of key genes in the photoreceptor gene-regulatory network (e.g., Crx, Nr2e3, Opn1sw, Vsx2) and retinopathy-related genes (e.g., Abca4, Bbs7, Pde6b). Together, our data establish a combinatorial cell-autonomous and non-cell-autonomous role for Top2b in the late stage of photoreceptor differentiation and maturation. © 2017 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by an activation mutation of glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme responsible for the reversible interconversion between glutamate and α-ketoglutarate. The syndrome presents clinically with hyperammonemia, significant episodic hypoglycemia, seizures, and a frequent incidences of developmental and learning defects. Clinical research has implicated that although some of the developmental and neurological defects may be attributed to hypoglycemia, some characteristics cannot be ascribed to low glucose and as hyperammonemia is generally mild and asymptomatic, there exists the possibility that altered GDH1 activity within the brain leads to some clinical changes. GDH1 is allosterically regulated by many factors, and has been shown to be inhibited by the ADP-ribosyltransferase sirtuin 4 (SIRT4), a mitochondrially localized sirtuin. Here we show that SIRT4 is localized to mitochondria within the brain. SIRT4 is highly expressed in glial cells, specifically astrocytes, in the postnatal brain and in radial glia during embryogenesis. Furthermore, SIRT4 protein decreases in expression during development. We show that factors known to allosterically regulate GDH1 alter gliogenesis in CTX8 cells, a novel radial glial cell line. We find that SIRT4 and GDH1 overexpression play antagonistic roles in regulating gliogenesis and that a mutant variant of GDH1 found in HI/HA patients accelerates the development of glia from cultured radial glia cells.

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.

In contrast to mammals, adult zebrafish have the ability to regrow descending axons and gain locomotor recovery after spinal cord injury (SCI). In zebrafish, a decisive factor for successful spinal cord regeneration is the inherent ability of some neurons to regrow their axons via (re)expressing growth-associated genes during the regeneration period. The nucleus of the medial longitudinal fascicle (NMLF) is one of the nuclei capable of regenerative response after SCI. Using microarray analysis with laser capture microdissected NMLF, we show that cysteine- and glycine-rich protein (CRP)1a (encoded by the csrp1a gene in zebrafish), the function of which is largely unknown in the nervous system, was upregulated after SCI. In situ hybridization confirmed the upregulation of csrp1a expression in neurons during the axon growth phase after SCI, not only in the NMLF, but also in other nuclei capable of regeneration, such as the intermediate reticular formation and superior reticular formation. The upregulation of csrp1a expression in regenerating nuclei started at 3 days after SCI and continued to 21 days post-injury, the longest time point studied. In vivo knockdown of CRP1a expression using two different antisense morpholino oligonucleotides impaired axon regeneration and locomotor recovery when compared with a control morpholino, demonstrating that CRP1a upregulation is an important part of the innate regeneration capability in injured neurons of adult zebrafish. This study is the first to demonstrate the requirement of CRP1a for zebrafish spinal cord regeneration.

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.

Adult zebrafish have the ability to recover from spinal cord injury and exhibit re-growth of descending axons from the brainstem to the spinal cord. We performed gene expression analysis using microarray to find damage-induced genes after spinal cord injury, and found that Sox11b mRNA is up-regulated at 11 days after injury. However, the functional relevance of Sox11b for regeneration is not known. Here, we report that the up-regulation of Sox11b mRNA after spinal cord injury is mainly localized in ependymal cells lining the central canal and in newly differentiating neuronal precursors or immature neurons. Using an in vivo morpholino-based gene knockout approach, we demonstrate that Sox11b is essential for locomotor recovery after spinal cord injury. In the injured spinal cord, expression of the neural stem cell associated gene Nestin, and the proneural gene Ascl1a (Mash1a), which are involved in the self-renewal and cell fate specification of endogenous neural stem cells, respectively, is regulated by Sox11b. Our data indicate that Sox11b promotes neuronal determination of endogenous stem cells and regenerative neurogenesis following spinal cord injury in the adult zebrafish. Enhancing Sox11b expression to promote proliferation and neurogenic determination of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after spinal cord injury in mammals.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function as posttranscriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development, including neurogenesis, dendritogenesis, and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG–binding protein 2 ( MECP2 ). Although Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2 -null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dysregulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA-methylated and are bound directly by Mecp2. Functional analysis demonstrates that the 3′ UTR of messenger RNA encoding Brain-derived neurotrophic factor ( Bdnf ) can be targeted by multiple miRNAs aberrantly up-regulated in the absence of Mecp2. Taken together, these results suggest that dysregulation of miRNAs may contribute to RTT pathoetiology and also may provide a valuable resource for further investigations of the role of miRNAs in RTT.

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.

A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2-oxoglutarate-dependent dioxygenases, known as the HIF prolyl 4-hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF-1 in some of the salutary effects of iron chelation-induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development of HIF activators that do not bind iron a high priority. Here we report the development of a high throughput screen to develop novel HIF activators and/or PHD inhibitors for therapeutic use in the central nervous system (CNS). We show that tilorone, a low-molecular weight, antiviral, immunomodulatory agent is the most effective activator of the HIF pathway in a neuronal line. We also show that tilorone enhances HIF protein levels and increases the expression of downstream target genes independent of iron chelation and HIF PHD inhibition in vitro. We further demonstrate that tilorone can activate an HIF-regulated reporter gene in the CNS. These studies confirm that tilorone can penetrate the blood–brain barrier to activate HIF in the CNS. As expected from these findings, we show that tilorone provides effective prophylaxis against permanent ischemic stroke and traumatic spinal cord injury in male rodents. Altogether these findings identify tilorone as a novel and potent modulator of HIF-mediated gene expression in neurons with neuroprotective properties.

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.

Forkhead-box domain (Fox) containing family members are known to play a role in neocorticogenesis and have also been associated with disorders on the autism spectrum. Here we show that a single RNA-binding protein, Hu antigen R (HuR), dictates translation specificity of bound mRNAs and is sufficient to define distinct Foxp-characterized subpopulations of neocortical projection neurons. Furthermore, distinct phosphorylation states of HuR differentially regulate translation of Foxp mRNAs in vitro. This demonstrates the importance of RNA binding proteins within the framework of the developing brain and further confirms the role of mRNA translation in autism pathogenesis.

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.

Activation of macrophages via toll-like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non-pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFkappaB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2-/-) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild-type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin-1beta mRNA. In TLR2-/- mice, locomotor recovery also was impaired relative to SCI wild-type controls and novel patterns of myelin pathology existed within ventromedial white matter--an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post-injury sequelae and perhaps in regulating inflammation and gliosis after SCI.

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.

Glutamate in excessive amounts is a major contributor to neuronal degeneration, and its removal is attributed mainly to astrocytes. Traumatic injury to the central nervous system (CNS) is often accompanied by disappearance of astrocytes from the lesion site and failure of the remaining cells to withstand the ensuing toxicity. Microglia that repopulate the lesion site are the usual suspects for causing redox imbalance and inflammation and thus further exacerbating the neurotoxicity. However, our group recently demonstrated that early post-injury activation of microglia as antigen-presenting cells correlates with an ability to withstand injurious conditions. Moreover, we found that T cells reactive to CNS-specific self-antigens protected neurons against glutamate toxicity. Here, we show that antigen-specific autoimmune T cells, by tailoring the microglial phenotype, can increase the ability of microglia-enriched cultures to remove glutamate. This T-cell-mediated effect could not be achieved by the potent microglia-activating agent lipopolysaccharide (LPS), but was dose-dependently reproduced by the Th1 cytokine interferon (IFN)-gamma and significantly reduced by neutralizing anti-IFN-gamma antibodies. Under the same conditions, IFN-gamma had no effect on cultured astrocytes. Up-regulation of glutamate uptake induced by IFN-gamma activation was not accompanied by the acute inflammatory response seen in LPS-activated cultures. These findings suggest that T cells or their cytokines can cause microglia to adopt a phenotype that facilitates rather than impairs glutamate clearance, possibly contributing to restoration of homeostasis.

Inflammatory responses contribute to secondary tissue damage following spinal cord injury (SCI). A potent anti-inflammatory glucocorticoid, methylprednisolone (MP), is the only currently accepted therapy for acute SCI but its efficacy has been questioned. To search for additional anti-inflammatory compounds, we combined microarray analysis with an explanted spinal cord slice culture injury model. We compared gene expression profiles after treatment with MP, acetaminophen, indomethacin, NS398, and combined cytokine inhibitors (IL-1ra and soluble TNFR). Multiple gene filtering methods and statistical clustering analyses were applied to the multi-dimensional data set and results were compared. Our analysis showed a consistent and unique gene expression profile associated with NS398, the selective cyclooxygenase-2 (COX-2) inhibitor, in which the overall effect of these upregulated genes could be interpreted as neuroprotective. In vivo testing demonstrated that NS398 reduced lesion volumes, unlike MP or acetaminophen, consistent with a predicted physiological effect in spinal cord. Combining explanted spinal cultures, microarrays, and flexible clustering algorithms allows us to accelerate selection of compounds for in vivo testing.

To capture all pertinent data during spinal cord injury animal experimentation, the authors have designed and implemented a database that is available for use under a public license. Their goals were to record all daily medical care of paraplegic animals, including unexpected complications; to store all injury parameters and/or therapeutic procedures; to track locomotor scores and other measures of functional recovery; to allow planning and management of experiments; and to serve as an externally linkable, SQL-queryable data mining source. Ultimately, the use of databases such as this will allow multiple neurotrauma laboratories to compare animal data through web meta-analysis.

Radial glia are a polarized cell type that in most neural regions appear only transiently during development. They have long been recognized as glia or glial progenitors that support neuronal migration. Recent evidence indicates that radial glia also give rise to neurons and appear to be a major population of dividing precursor cells in the embryonic cortical ventricular zone. Radial glia extend long processes from the ventricular zone to the pial surface that provide guides for neuronal migration. We reasoned that the unique morphology of radial glia is due to the composition and organization of their cytoskeleton. In this present study, we have used C6-R, a radial glial-like cell line and isolated perinatal cerebellar radial glia to ask what are the critical cytoskeletal elements in radial glial cells and how they are regulated. Treatments with nocodazole and cytochalasin D showed that microtubules, but not microfilaments, are critical for the polarized morphology of radial glia. In addition, quantitative real-time PCR indicated that certain mRNAs specific for microtubule-associated proteins (MAPs) are selectively expressed in radial glia. These results together with the known ability of microtubule affinity-regulating kinases to regulate microtubule organization suggest that microtubules and MAPs are critical for the morphology of radial glia.

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.

Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA- binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

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.

We have completed the first large-scale gene expression study of acute spinal cord injury (SCI) in rat. Oligonucleotide microarrays containing 1,200 gene-specific probes were used to quantify mRNA levels, relative to uninjured controls, in spinal cords injured using a standard contusion model. Our results revealed a marked loss of neuron-specific mRNAs at the injury site. The surviving cells showed a characteristic inflammatory response that started at the injury site and spread to the distal cord. Changes in several mRNA levels were associated with putative regenerative responses in the spinal cord. Notably, phosphodiesterase 4, nestin, glia-derived neurite promoting factor, and GAP-43 mRNAs increased significantly. Other mRNAs clustered temporally and spatially with these regeneration-associated genes. Thus we have described global patterns of gene expression following acute SCI, and we have identified targets for future study and possible therapeutic intervention.

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.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr[superscript −/−] neonates, whereas linc–Brn1b[superscript −/−] mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules., National Science Foundation (U.S.) (Postdoctoral Fellowship in Biology)

Interleukin-1 (IL-1) is a multifunctional cytokine with profound effects on ovarian function. The effects of IL-1 on ovarian steroidogenesis have been demonstrated in several species. IL-1 mRNA levels are increased in the thecal layer of the ovulating follicle and IL-1β has been shown to induce ovulations in vitro. In this study we have investigated the presence and distribution of the mRNAs for type I IL-1 receptor (IL-1RtI) and for the naturally occurring IL-1 receptor antagonist (IL-1ra) in ovaries of adult cycling rats, to elucidate the target cells for IL-1 action. We have demonstrated the presence of mRNA for both substances by in situ hybridisation and reverse transcription PCR. mRNA for IL-1RtI was not found in primordial follicles but was abundant in the granulosa and thecal layer in developing follicles with stronger signals in the granulosa layer. In the preovulatory and ovulatory follicles, there was a further increase in the signal for IL-1RtI mRNA in the thecal layer compared with the granulosa layer. Corpora lutea were weakly positive at all stages and atretic follicles were largely negative. No mRNA was detected in oocytes of any stage. mRNA for IL-1ra showed a similar distribution to that of IL-1RtI. The changes in distribution suggest an action of IL-1 on rat granulosa cells during follicular development and on thecal cells during ovulation. Journal of Endocrinology (1997) 152, 11–17

The mature neocortex is a unique six-layered mammalian brain region. It is composed of morphologically and functionally distinct subpopulations of primary projection neurons that form complex circuits across the central nervous system. The precisely-timed generation of projection neurons from neural stem cells governs their differentiation, postmitotic specification, and signaling, and is critical for cognitive and sensorimotor ability. Developmental perturbations to the birthdate, location, and connectivity of neocortical neurons are observed in neurological and psychiatric disorders. These facts are highlighting the importance of the precise spatiotemporal development of the neocortex regulated by intricate transcriptional, but also complex post-transcriptional events. Indeed, mRNA transcripts undergo many post-transcriptional regulatory steps before the production of functional proteins, which specify neocortical neural stem cells and subpopulations of neocortical neurons. Therefore, particular attention is paid to the differential post-transcriptional regulation of key transcripts by RNA-binding proteins, including splicing, localization, stability, and translation. We also present a transcriptome screen of candidate molecules associated with post-transcriptional mRNA processing that are differentially expressed at key developmental time points across neocortical prenatal neurogenesis.

Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain.

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.

Axotomy of sympathetic superior cervical ganglia (SCG) causes Schwann cells to induce mRNA encoding leukemia inhibitory factor (LIF), a neuropoietic cytokine that has been shown to promote sympathetic neuron survival and peptide gene regulation. LIF mRNA is virtually undetectable in uninjured SCG, but is induced by the inflammatory cytokine interleukin-1 (IL-1). The SC1 Schwann cell line was used to study this regulatory mechanism. LIF mRNA increased five-to-tenfold in SC1 cells when IL-1 receptors were stimulated with IL-1. The action of IL-1 is thought to be mediated by the type I IL-1 receptor (IL-1RI), which has been suggested to stimulate a ceramide-dependent protein kinase pathway, much like tumor necrosis factor-alpha. However, stimulation of the ceramide-dependent protein kinase pathways in SC1 cells with either 2-acetylceramide or sphingomyelinase treatment does not induce LIF mRNA accumulation, but 2-acetylceramide addition induces cyclooxygenase-2 mRNA in parallel experiments. Inhibition of phosphotidylcholine-phospholipase C activity, endosomal acidification, or activity of atypical protein kinase C reduce LIF induction by IL-1. These results are consistent with IL-1 regulation of LIF mRNA through stimulation of the endosomal, acidic sphingomyelinase pathway, leading to ceramide activation of protein kinase C zeta. Utilization of this branch of the ceramide signaling pathway may be cell type specific or may be specific for the LIF mRNA response.

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.

Without appropriate response to traumatic injury, the nervous system cannot survive. The superior cervical (sympathetic) ganglion of the neonatal rat has been a uniquely valuable model system in which to study neuronal response to traumatic injury. Explantation of the ganglion into culture involves severing both presynaptic fibers (compromising preganglionic input) and axotomy (damaging efferent axons of ganglionic neurons). Since traumatic injury of any kind involves the elaboration of inflammatory cytokines, we have been interested in examining the action of these inflammatory cytokines on neurotransmitter gene expression in injured sympathetic ganglia.