Olga Ponomareva

Positively correlated miRNA-mRNA regulatory networks in mouse frontal cortex during early stages of alcohol dependence

BackgroundAlthough the study of gene regulation via the action of specific microRNAs (miRNAs) has experienced a boom in recent years, the analysis of genome-wide interaction networks among miRNAs and respective targeted mRNAs has lagged behind. MicroRNAs simultaneously target many transcripts and fine-tune the expression of genes through cooperative/combinatorial targeting. Therefore, they have a large regulatory potential that could widely impact development and progression of diseases, as well as contribute unpredicted collateral effects due to their natural, pathophysiological, or treatment-induced modulation. We support the viewpoint that whole mirnome-transcriptome interaction analysis is required to better understand the mechanisms and potential consequences of miRNA regulation and/or deregulation in relevant biological models. In this study, we tested the hypotheses that ethanol consumption induces changes in miRNA-mRNA interaction networks in the mouse frontal cortex and that some of the changes observed in the mouse are equivalent to changes in similar brain regions from human alcoholics.ResultsmiRNA-mRNA interaction networks responding to ethanol insult were identified by differential expression analysis and weighted gene coexpression network analysis (WGCNA). Important pathways (coexpressed modular networks detected by WGCNA) and hub genes central to the neuronal response to ethanol are highlighted, as well as key miRNAs that regulate these processes and therefore represent potential therapeutic targets for treating alcohol addiction. Importantly, we discovered a conserved signature of changing miRNAs between ethanol-treated mice and human alcoholics, which provides a valuable tool for future biomarker/diagnostic studies in humans. We report positively correlated miRNA-mRNA expression networks that suggest an adaptive, targeted miRNA response due to binge ethanol drinking.ConclusionsThis study provides new evidence for the role of miRNA regulation in brain homeostasis and sheds new light on current understanding of the development of alcohol dependence. To our knowledge this is the first report that activated expression of miRNAs correlates with activated expression of mRNAs rather than with mRNA downregulation in an in vivo model. We speculate that early activation of miRNAs designed to limit the effects of alcohol-induced genes may be an essential adaptive response during disease progression.

Regulation of the Intermediate Filament Protein Nestin at Rodent Neuromuscular Junctions by Innervation and Activity

The intermediate filament nestin is localized postsynaptically at rodent neuromuscular junctions. The protein forms a filamentous network beneath and between the synaptic gutters, surrounds myofiber nuclei, and is associated with Z-discs adjacent to the junction. In situ hybridization shows that nestin mRNA is synthesized selectively by synaptic myonuclei. Although weak immunoreactivity is present in myelinating Schwann cells that wrap the preterminal axon, nestin is not detected in the terminal Schwann cells (tSCs) that cover the nerve terminal branches. However, after denervation of muscle, nestin is upregulated in tSCs and in SCs within the nerve distal to the lesion site. In contrast, immunoreactivity is strongly downregulated in the muscle fiber. Transgenic mice in which the nestin neural enhancer drives expression of a green fluorescent protein (GFP) reporter show that the regulation in SCs is transcriptional. However, the postsynaptic expression occurs through enhancer elements distinct from those responsible for regulation in SCs. Application of botulinum toxin shows that the upregulation in tSCs and the loss of immunoreactivity in muscle fibers occurs with blockade of transmitter release. Extrinsic stimulation of denervated muscle maintains the postsynaptic expression of nestin but does not affect the upregulation in SCs. Thus, a nestin-containing cytoskeleton is promoted in the postsynaptic muscle fiber by nerve-evoked muscle activity but suppressed in tSCs by transmitter release. Nestin antibodies and GFP driven by nestin promoter elements serve as excellent markers for the reactive state of SCs. Vital imaging of GFP shows that SCs grow a dynamic set of processes after denervation.

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4.

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation.

Dynamin-1 co-associates with native mouse brain BKCa channels: Proteomics analysis of synaptic protein complexes

MINT‐7543476: Vamp‐2 (uniprotkb:P63044) physically interacts (MI:0914) with Syntaxin‐7 (uniprotkb:O70439), Neuronal membrane glycoprotein M6‐a (uniprotkb:P35802), Syntaxin‐1B (uniprotkb:P61264), Beta‐soluble NSF attachment protein (uniprotkb:P28663), Guanine nucleotide‐binding protein G(I)/G(S)/G(T) subunit beta‐3 (uniprotkb:Q61011), Guanine nucleotide‐binding protein G(I)/G(S)/G(T) subunit beta‐1 (uniprotkb:P62874), Guanine nucleotide‐binding protein G(o) subunit alpha (uniprotkb:P18872), V‐type proton ATPase subunit d 1 (uniprotkb:P51863), Zinc transporter 3 (uniprotkb:P97441), Sodium/potassium‐transporting ATPase subunit alpha‐2 (uniprotkb:Q6PIE5), Sodium/potassium‐transporting ATPase subunit alpha‐3 (uniprotkb:Q6PIC6), Sodium/potassium‐transporting ATPase subunit alpha‐1 (uniprotkb:Q8VDN2), Potassium‐transporting ATPase alpha chain 1 (uniprotkb:Q64436), Synaptophysin (uniprotkb:Q62277), Syntaxin‐1A (uniprotkb:O35526) and Dynamin‐1 (uniprotkb:P39053) by anti bait co‐immunoprecipitation (MI:0006)

Defective neuromuscular synaptogenesis in mice expressing constitutively active ErbB2 in skeletal muscle fibers

We overexpressed a constitutively active form of the neuregulin receptor ErbB2 (CAErbB2) in skeletal muscle fibers in vivo and in vitro by tetracycline-inducible expression. Surprisingly, CAErbB2 expression during embryonic development was lethal and impaired synaptogenesis yielding a phenotype with loss of synaptic contacts, extensive axonal sprouting, and diffuse distribution of acetylcholine receptor (AChR) transcripts, reminiscent of agrin-deficient mice. CAErbB2 expression in cultured myotubes inhibited the formation and maintenance of agrin-induced AChR clusters, suggesting a muscle- and not a nerve-origin for the defect in CAErbB2-expressing mice. Levels of tyrosine phosphorylated MuSK, the signaling component of the agrin receptor, were similar, while tyrosine phosphorylation of AChRbeta subunits was dramatically reduced in CAErbB2-expressing embryos relative to controls. Thus, a gain-of-function manipulation of ErbB2 signaling pathways renders an agrin-deficient-like phenotype that uncouples MuSK and AChR tyrosine phosphorylation.

Localization of PPAR isotypes in the adult mouse and human brain.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain.

Evidence for Muscle-Dependent Neuromuscular Synaptic Site Determination in Mammals

Recent evidence challenges the prevalent view that neural factors induce the formation of a de novo postsynaptic apparatus during development of the vertebrate neuromuscular junction. The latest experiments suggest an alternative model in which the muscle fiber induces a nascent postsynaptic apparatus and sets the location of the future synapse. On axonal contact, these sites, laid out in a prepattern in the central area of developing muscle fibers, mature into synapses by the combined action of neural factors such as agrin and ACh. We sought to test in mammals these two models of neuromuscular synaptogenesis. Previously, we showed that continuous prenatal muscle expression of constitutively active ErbB2 (CAErbB2) led to synaptic loss, exuberant axonal sprouting, and lethality at birth. Here, we transiently induced CAErbB2 during midgestation and examined synapse restoration after inducer withdrawal. Centrally enriched ACh receptor (AChR) transcription and clustering were abolished after transient CAErbB2 induction. After inducer withdrawal, synapses were restored but were distributed widely over the entire diaphragm muscle. Under the nerve-dependent model, this distribution is explained by the wide pattern of axonal sprouting triggered by CAErbB2. Yet, in the absence of the nerve, introduced in our animals by mating to Hb9+/− mice, a very similar, wide distribution of aneural AChR clusters resulted. Thus, transient expression of CAErbB2 in skeletal muscles leads to reprogramming of the endogenous muscle AChR prepattern. This, and not the nerve, seems primarily responsible for the widely distributed pattern of synapses in our experimental animals.

Inhibition of IKKβ reduces ethanol consumption in C57BL/6J mice

Abstract Proinflammatory pathways in neuronal and non-neuronal cells are implicated in the acute and chronic effects of alcohol exposure in animal models and humans. The nuclear factor-κB (NF-κB) family of DNA transcription factors plays important roles in inflammatory diseases. The kinase IKKβ mediates the phosphorylation and subsequent proteasomal degradation of cytosolic protein inhibitors of NF-κB, leading to activation of NF-κB. The role of IKKβ as a potential regulator of excessive alcohol drinking had not previously been investigated. Based on previous findings that the overactivation of innate immune/inflammatory signaling promotes ethanol consumption, we hypothesized that inhibiting IKKβ would limit/decrease drinking by preventing the activation of NF-κB. We studied the systemic effects of two pharmacological inhibitors of IKKβ, TPCA-1 and sulfasalazine, on ethanol intake using continuous- and limited-access, two-bottle choice drinking tests in C57BL/6J mice. In both tests, TPCA-1 and sulfasalazine reduced ethanol intake and preference without changing total fluid intake or sweet taste preference. A virus expressing Cre recombinase was injected into the nucleus accumbens and central amygdala to selectively knock down IKKβ in mice genetically engineered with a conditional Ikkb deletion (IkkbF/F). Although IKKβ was inhibited to some extent in astrocytes and microglia, neurons were a primary cellular target. Deletion of IKKβ in either brain region reduced ethanol intake and preference in the continuous access two-bottle choice test without altering the preference for sucrose. Pharmacological and genetic inhibition of IKKβ decreased voluntary ethanol consumption, providing initial support for IKKβ as a potential therapeutic target for alcohol abuse.

Schwann cell-derived neuregulin-2α can function as a cell-attached activator of muscle acetylcholine receptor expression

Here we show that neuregulin‐2 (Nrg‐2) α‐ and β‐isoforms can activate acetylcholine receptor (AChR) transcription as surface‐attached ligands. More importantly, we demonstrate that Schwann cells that express Nrg‐2α on their cell surface, the same Nrg‐2 isoform expressed by terminal Schwann cells at the neuromuscular junction, can induce AChR expression if brought into cell‐to‐cell contact with myotubes specifically expressing ErbB4. These Schwann cells, the D6P2T cell line, induce AChR expression apparently as well as 293T cells transfected with Nrg‐2β, the isoform with the highest AChR‐inducing activity when presented in a soluble form. These results provide a potential role for the previously reported, paradoxical perisynaptic accumulation of Nrg‐2α, the isoform with the least AChR‐inducing activity when presented in a soluble form. They also raise the possibility that Schwann cell‐derived Nrg‐2 could activate ErbB receptors on the synaptic sarcolemma and that this could account, at least in part, for the Nrg‐mediated regulation of AChR expression.

Stimulation of acetylcholine receptor transcription by neuregulin-2 requires an N-box response element and is regulated by alternative splicing.

The neuregulin (Nrg) family of growth/differentiation factors is encoded by at least four genes in the mammalian genome: nrg-1, nrg-2, nrg-3 and nrg-4. Nrg-1 and Nrg-2 share the highest homology within the family, and the primary RNA transcripts from their encoding genes are subjected to extensive alternative splicing. Although little is known about the biological function of Nrg-2-4, their structural similarity with Nrg-1 suggests that they could account for some of the activities presently attributed to Nrg-1. Thus, at the neuromuscular junction Nrg-1 has been a favored candidate for the signal that activates selective acetylcholine receptor (AChR) transcription in synaptic myonuclei. However, we have recently shown that like Nrg-1, Nrg-2 can also activate AChR transcription in cultured myotubes and accumulates at the synaptic site. Synapse-specific and Nrg-1-induced AChR transcription require an enhancer sequence, the N-box, which is also mutated in some patients with congenital myasthenia gravis. Here, we show that Nrg-2-induced AChR transcription requires an N-box motif and is regulated by alternative splicing. We also show that unique Nrg-2 isoforms are differentially distributed between spinal cord and skeletal muscle, the tissues that harbor the cellular components of the neuromuscular synapse.