Matching Items (43)

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Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

Description

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis.

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  • 2017-03-27

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Layer specific and general requirements for ERK/MAPK signaling in the developing neocortex

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Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions

Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2[superscript +] neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability.

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  • 2016-02-05

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Hyperactive ERK/MAPK Regulates Cortical GABAergic Neuron Development

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Aberrant signaling through the canonical RAS/RAF/MEK/ERK (ERK/MAPK) pathway leads to the pathology of a group of neurodevelopmental disorders called RASopathies. RASopathies are caused by germline mutations in the ERK/MAPK pathway

Aberrant signaling through the canonical RAS/RAF/MEK/ERK (ERK/MAPK) pathway leads to the pathology of a group of neurodevelopmental disorders called RASopathies. RASopathies are caused by germline mutations in the ERK/MAPK pathway and have an incidence of approximately 1:2000 births. The majority of RASopathies stem from mutations that cause gain-of-function in the ERK/MAPK pathway. In this study, we have begun to unravel the roles that GABAergic interneurons play in the pathology of RASopathies. Our data demonstrate that gain-of-function ERK/MAPK signaling expressed in a GABAergic interneuron-specific fashion leads to forebrain hyperexcitability in mutant mice. Further, some GABAergic interneurons experience activated-caspase 3 mediated apoptosis in the embryonic subpallium, leading to a loss of PV-expressing interneurons in the somatosensory cortex. We found that pharmaceutical intervention during embryogenesis using a MEK1 inhibitor may be effective in preventing apoptosis of these neurons. Future work is still needed to understand the mechanism of the death of GABAergic interneurons and to further pursue therapeutic approaches. Taken together, this study suggests potential roles of cortical GABAergic interneurons in ERK/MAPK-linked pathologies and indicates possible approaches to provide therapy for these conditions.

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Date Created
  • 2019-05

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Early Life Stress: An Increased Risk of Schizophrenia through Activation of the Complement Component Pathway

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Schizophrenia is a debilitating psychiatric disorder with poorly understood genetic and environmental factors. An allelic variant of complement component 4 (C4), a protein first identified in innate immune response

Schizophrenia is a debilitating psychiatric disorder with poorly understood genetic and environmental factors. An allelic variant of complement component 4 (C4), a protein first identified in innate immune response is strongly associated with schizophrenia. In the brain, activity of C4 leads to dendritic pruning, a process that may be causal in disease progression. Environmental factors, such as early life exposure to significant stressors also associate with increased risk of schizophrenia in later life. My hypothesis is that these factors do not act independently, but rather in tandem to influence disease etiology.
This hypothesis is supported by previous studies demonstrating that stress-induced elevation of glucocorticoids increases the transcription of C4. I propose that activated glucocorticoid receptors directly increase C4 protein expression as a transcription factor activator. Additionally, I propose that activated glucocorticoid receptors inhibit the expression of the transcription factor nuclear factor-light-chain-enhancer of activated B cells (NF-κB), thereby leading to decreased expression of the C4 inhibitor CUB and Sushi multiple domains 1 (CSMD1).
Glucocorticoid receptors and C4 are richly expressed in the hippocampus, a region critical in memory consolidation, spatial, and declarative memory. I propose that stress-induced upregulation of C4 activity in the hippocampus promotes excessive synaptic pruning, contributing to specific deficits and hippocampal shrinkage seen in schizophrenia. Stress exposure during fetal development and adolescence likely acts through the proposed mechanisms to increase hippocampal C4 activity and subsequent schizophrenia risk. These mechanisms may reveal novel interactions between environmental and genetic risk factors in the etiology of schizophrenia through complement activation.

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  • 2017-05

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Effects of miR-495 in Cocaine Addiction: Viral Mediated Overexpression and Inhibition of miR-495 Affects Cocaine-Seeking Behavior

Description

MicroRNAs are small, non-coding transcripts that control gene expression by preventing mRNA from translating into proteins. They have been implicated to play a role in many drug addictions. We previously

MicroRNAs are small, non-coding transcripts that control gene expression by preventing mRNA from translating into proteins. They have been implicated to play a role in many drug addictions. We previously found that miR-495 targets several addiction-related genes (ARGs) and is highly expressed in the nucleus accumbens (NAc). We also found miR-495 is downregulated in the NAc following acute cocaine administration, and cocaine motivation measured by breakpoint on a progressive ratio schedule of cocaine reinforcement is decreased when miR-495 is overexpressed. In this study, we manipulated the endogenous levels of miR-495 by using a viral vector. Using an animal model, rats were first trained for self-administration on a fixed ratio (FR) schedule of reinforcement. After they were infused with a lentivirus to overexpress (LV-miR-495) or decrease (LV-Sponge) miR-495, in the NAc shell. The rats were then tested for extinction and reinstatement of cocaine-seeking behavior, which are measures of motivation for cocaine. We measured the relative levels of miR-495 in the NAc shell using qRT-PCR. Our results show that overexpression of miR-495 decreased cocaine-seeking behavior during extinction and cocaine reinstatement, as we hypothesized. Surprisingly, miR-495 LV-sponge also decreased cocaine-seeking behavior in extinction, not as we hypothesized. However, we found that LV-Sponge failed to significantly decrease levels of miR-495 as intended. In conclusion, understanding why LV-Sponge decreased, rather than increased, miR-495 will need further study, however, the results with LV-miR-495 extend previous findings that miR-495 plays a vital role in the molecular mechanism that influences motivation to seek cocaine.

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Date Created
  • 2017-05

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Effects of environmental enrichment on cocaine-seeking behavior in female rats and RNA expression in the Nucleus Accumbens

Description

Substance abuse disorder is a debilitating condition characterized by recurring drug-seeking behaviors and high rates of relapse. In male rats, this tendency to engage in drug-seeking behavior can be inhibited

Substance abuse disorder is a debilitating condition characterized by recurring drug-seeking behaviors and high rates of relapse. In male rats, this tendency to engage in drug-seeking behavior can be inhibited by environmental enrichment (EE) during abstinence. We have shown previously that cocaine-seeking behavior is associated with an increase in addiction-related genes such as Arc and CamkIIa and a decrease in the microRNA miR-495. We have also shown that miR-495 inhibits expression of Arc and CamkIIa post-transcriptionally. Therefore, we hypothesize that reduced cocaine-seeking behavior in EE female rats is associated with a downregulation of these addiction-related genes as well as an upregulation of miR-495 in the NAc shell. Based on previous studies that highlight differences between male and female motivation for cocaine, we also hypothesize that EE will not affect female motivation for cocaine as robustly as males. After acquiring cocaine through self-administration, females were assigned to either an enriched environment (EE) condition or an isolated condition, where they remained during abstinence. They were then given a one-hour cue-reactivity test, during which cocaine-seeking behavior differed significantly between the EE and isolated groups. We also found that the addiction-related genes Arc and CamkIIa were downregulated in the NAc core of EE females. Future research is needed to examine the role of miR-495 in these changes in behavior and gene expression. Overall, the results suggest that EE is protective against relapse to cocaine-seeking in females and may normalize the dysregulation of genes by cocaine.

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  • 2017-05

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Defining the effects of ERK/MAPK hyperactivation on the development of GABAergic

Description

Abstract: The RAS/RAF/MEK/ERK (RAS signaling cascade) pathway is a highly conserved biochemical signaling cascade that exists in every mammalian cell. The pathway is highly versatile in functionality due to hundreds

Abstract: The RAS/RAF/MEK/ERK (RAS signaling cascade) pathway is a highly conserved biochemical signaling cascade that exists in every mammalian cell. The pathway is highly versatile in functionality due to hundreds of substrates that regulate metabolism, apoptosis, and proliferation in both adult and developing tissues. The RAS signaling cascade has been examined in the context of cancers since mutations can lead to the disruption of the cell cycle and unregulated cellular proliferation. In addition, germline mutations in the pathway have been shown to cause a group of syndromes known as RASopathies. RASopathies are marked by facial defects, seizures, developmental delays, and cognitive dysfunction often due to enhanced activation of the RAS signaling cascade. Although there are noted factors that play roles in neurological disease, such as a hyperactivated RAS signaling cascade, the pathogenesis of neurological defects is not fully understood. The Newbern lab uses conditional mutagenesis to examine how hyperactivating the RAS/MAPK pathway affects GABAergic neurons in a cortical microcircuit, especially during development. Inhibitory neurons are implicated in seizures and epilepsy is common in RASopathies, thus GABAergic neurons are of particular interest (Rauen, 2013). Gain-of-function ERK was not found to significantly alter global locomotion or anxiety-like behaviors. Interestingly, the mutant mice exhibited freezing behavior in the first twenty-two seconds of the open field assay that appeared to be consistent with absence seizures. Direct EEG recordings confirmed spontaneous seizure activity and mutants had a reduced seizure threshold. We hypothesized that these deficits were due to altered GABAergic neuron number. Indeed, mutant mice exhibited a 30% reduction in total cortical GABAergic neuron number. This effect appeared to be cell subtype specific, where neurons expressing somatostatin (SST) existed in similar numbers among controls and mutants but a significant decrease in the number of those expressing parvalbumin (PV) was observed. I hypothesized that a recently identified GABAergic neuron expressing vasoactive intestinal polypeptide (VIP) would also be affected in such a manner that fewer VIP neurons exist in the mutants than the wildtype. Subsequent histological studies in these mice found there to be no significant difference in VIP populations. Selective affects seem to only have an effect on the development of PV neurons in the cortex. Further studies are underway to define the mechanism responsible for aberrant GABAergic neuron development.

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  • 2016-05

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Matrin 3 and Protein Localization in ALS

Description

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the deterioration of motor neurons. ALS affects about 1 in 20,000 people and leads to death within 2 to

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the deterioration of motor neurons. ALS affects about 1 in 20,000 people and leads to death within 2 to 5 years after diagnosis. There is currently no cure for ALS, but there are many genes known to be associated with ALS, such as SOD 1 and C9orf72. Recently, mutations in Matrin 3 were linked to ALS. While 15 mutations in Matrin 3 have been discovered, this study focuses on the four initial mutations, which are the Ser85Cys, Phe115Cys, Pro154Ser, and Thr622Ala mutations. This study attempts to understand the mechanism of how these mutations lead to ALS. The first aim focuses on the role of Matrin mutations in the mislocalization of TDP-43 from the nucleus to the cytoplasm, a pathological hallmark of ALS. We hypothesized expression of mutant Matrin 3 would lead to TDP-43 mislocalization, however the data did not support that hypothesis. The second aim of this study focuses on the mislocalization of TRanscription EXport (TREX) complex proteins within the nucleus. TREX proteins were studied based off of previous experiments suggesting that proteins within this complex bind to Matrin 3. The results showed differences in co-localization between each of these proteins and wild-type and mutant Matrin 3, confirming our earlier results. These findings can help increase our understanding of the mechanism of ALS while also setting the framework for future studies.

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  • 2016-12

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Dysregulated ERK/MAPK Signaling in RASopathy Animal Model Systems Leads to a Decrease in mTOR Expression and Activation of Translational Machinery

Description

The RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such,

The RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such, it plays an important role in development, specifically in development of the nervous system. Activation of ERK is indispensable for the differentiation of Embryonic Stem Cells (ESC) into neuronal precursors (Li z et al, 2006). ERK signaling has also shown to mediate Schwann cell myelination of the peripheral nervous system (PNS) as well as oligodendrocyte proliferation (Newbern et al, 2011). The class of developmental disorders that result in the dysregulation of RAS signaling are known as RASopathies. The molecular and cell-specific consequences of these various pathway mutations remain to be elucidated. While there is evidence for altered DNA transcription in RASopathies, there is little work examining the effects of the RASopathy-linked mutations on protein translation and post-translational modifications in vivo. RASopathies have phenotypic and molecular similarities to other disorders such as Fragile X Syndrome (FXS) and Tuberous Sclerosis (TSC) that show evidence of aberrant protein synthesis and affect related pathways. There are also well-defined downstream RAS pathway elements involved in translation. Additionally, aberrant corticospinal axon outgrowth has been observed in disease models of RASopathies (Xing et al, 2016). For these reasons, this present study examines a subset of proteins involved in translation and translational regulation in the context of RASopathy disease states. Results indicate that in both of the tested RASopathy model systems, there is altered mTOR expression. Additionally the loss of function model showed a decrease in rps6 activation. This data supports a role for the selective dysregulation of translational control elements in RASopathy models. This data also indicates that the primary candidate mechanism for control of altered translation in these modes is through the altered expression of mTOR.

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Date Created
  • 2017-05

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Assessing the Role of the Transcription Factor EGR3 in Activity-Induced DNA Damage Response

Description

Immediate early genes (IEGs) are rapidly activated in response to an environmental stimulus, and most code for transcription factors that mediate processes of synaptic plasticity, learning, and memory. EGR3, an

Immediate early genes (IEGs) are rapidly activated in response to an environmental stimulus, and most code for transcription factors that mediate processes of synaptic plasticity, learning, and memory. EGR3, an immediate early gene transcription factor, is a mediator of biological processes that are disrupted in patients with schizophrenia (SCZ). A microarray experiment conducted by our lab revealed that Egr3 also regulates genes involved in DNA damage response. A recent study revealed that physiological neuronal activity results in the formation of DNA double-stranded breaks (DSBs) in the promoters of IEGs. Additionally, they showed that these DSBs are essential for inducing the expression of IEGs, and failure to repair these DSBs results in the persistent expression of IEGs. We hypothesize that Egr3 plays a role in repairing activity- induced DNA DSBs, and mice lacking Egr3 should have an abnormal accumulation of these DSBs. Before proceeding with that experiment, we conducted a preliminary investigation to determine if electroconvulsive stimulation (ECS) is a reliable method of inducing activity- dependent DNA damage, and to measure this DNA damage in three subregions of the hippocampus: CA1, CA3, and dentate gyrus (DG). We asked the question, are levels of DNA DSBs different between these hippocampal subregions in animals at baseline and following electroconvulsive stimulation (ECS)? To answer this question, we quantified γ-H2AX, a biomarker of DNA DSBs, in the hippocampal subregions of wildtype mice. Due to technical errors and small sample size, we were unable to substantiate our preliminary findings. Despite these shortcomings, our experimental design can be modified in future studies that investigate the role of Egr3 in activity-induced DNA damage repair.

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Date Created
  • 2020-05