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Intracellular signalling during murine oocyte growthHurtubise, Patricia. January 2000 (has links)
No description available.
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The unfolded protein response couples neuronal identity to circuit formation in the developing mouse olfactory systemShayya, Hani January 2023 (has links)
Complex genetic mechanisms both endow developing neuronal subtypes with distinct molecular identities and translate those identities into the signatures of cell surface axon guidance molecules that direct neural circuit assembly. The final steps of this process, where axon guidance molecules instruct circuit outcomes, are well-understood. However, the upstream identity molecules that define guidance molecule signatures, and the molecular mechanisms by which cell type identity is transformed into these signatures, remain enigmatic.
The murine olfactory system contains nearly 1,5000 olfactory sensory neuron (OSN) subtypes which are intermixed in the olfactory epithelium (OE). Each OSN subtype expresses a unique olfactory receptor (OR) protein which both tunes its response properties to odorants in the environment and acts as an identity molecule that ensures all axons of a given OSN type converge to a single set of target glomeruli in the olfactory bulb (OB). Using a combination of bioinformatic and mouse genetic approaches, we have discovered an unanticipated role for endoplasmic reticulum stress (ER stress) and the unfolded protein response (UPR) in the translation of OR identity to OSN axon guidance molecule expression and glomerular targeting.
We find that slight differences in OR amino acid sequences lead to differential activation of the ER stress sensor PERK in different OSN subtypes. Graded patterns of the UPR are then interpreted through a master regulator transcription factor, Ddit3, which controls a set of stress-responsive axon guidance molecules that orchestrate the process of glomerular segregation in the OB. Our results define a novel paradigm for axon guidance in which graded activation of a canonical stress response pathway is leveraged towards the conversion of discrete neuronal identities into discrete circuit formation outcomes. These findings may be widely relevant for the formation of neural circuits across a variety of systems.
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Transcriptomic and Functional Analysis of Neuronal Activity and DiseaseKrizay, Daniel Kyle January 2022 (has links)
Advances in sequencing technologies have sparked the discovery of new genetic etiologies for neurological and neurodevelopmental disorders. As new disease-causing mutations are unveiled, questions into the specific mechanisms of pathogenicity and potential therapeutic approaches arise. To address these questions, in vivo and in vitro models have been generated and analyzed; but how best to utilize these models, and how well they recapitulate the human brain, is still not fully understood. Within the work discussed in this thesis, we address this problem through the transcriptomic and functional interrogation of these models in the context of neurodevelopment and disease.
In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing to examine the longitudinal transcriptomic profiles of neuronal network establishment and maturation in ex vivo mouse cortex- and hippocampus-derived cultures. Our data highlights unique developmental transcriptomic profiles for individual genes, disease gene subclasses, and biological processes, and discusses cell population-specific divergent transcriptomic profiles between genes associated with neurological diseases, focusing on epilepsy and autism spectrum disorder. We also compared the data from our ex vivo system to transcriptomic data collected from in vivo neonatal and adult mouse brains and human cortical organoids, highlighting the importance of the generation and consideration of system-specific transcriptomic datasets when looking into a gene, disease, or biological process of interest, and serves as a vital resource for researchers.
In Chapter 3, we propose a high-throughput drug discovery paradigm utilizing the application of transcriptome reversal for neurodevelopmental disorder-associated genes that affect the transcriptome. This approach describes the idea that if gene dysregulation is causal for the pathogenicity of a disease, then correcting the transcriptional signature should have a therapeutic effect. We demonstrated that small-molecule induced gene expression changes vary between both cell lines and neural cell populations, and highlight both the importance of selecting the appropriate model of disease and creating cell population-specific signatures for compounds and disease.
In Chapter 4, we focus on the utilization of multi-electrode arrays for the electrophysiological characterization of primary cortical networks derived from mouse models of epileptic encephalopathy. This technique allows for the analysis of numerous neuronal and network synchronization metrics for spontaneous longitudinal activity and responses to external stimuli in the form of electrical stimulation and compound addition. In particular, mouse models with mutations in the genes Grin2a, Gnb1, and Scn1a were analyzed. We discovered significant hyperexcitability, bursting, and synchrony phenotypes, and discuss how acute and chronic compound addition can be used to interrogate biological pathways and reverse disease activity signatures.
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Epitranscriptomic Alterations in Alzheimer’s Disease: The Role of MicroRNA Methylation in the Regulation of Tau ProteostasisKim, Yoon Anna January 2021 (has links)
The imbalance in the levels of certain microRNAs (miRNAs) in Alzheimer’s disease (AD) brains promotes alterations in tau proteostasis and neurodegeneration. However, potential mechanisms governing how specific miRNAs are dysregulated in AD brains are still under investigation. Epitranscriptomics is a mode of post-transcriptional regulation that can control brain functions during development and adulthood. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases that has the ability to modify mammalian non-coding RNAs. Importantly, autosomal-recessive loss of function mutations in NSun2 have been associated with neurological abnormalities in humans. Here, we report that dysregulation of NSun2 can induce alterations in tau phosphorylation by modulating the levels of miR-125b, a main player in tau pathology. We were able to provide supporting evidence by utilizing several model systems such as Drosophila, human induced pluripotent stem cell (iPSC) derived neurons, rat primary neuronal cultures and mice. Our Western blot analysis not only shows that NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex, but also NSun2 protein expression levels are downregulated in post-mortem brain tissues from AD patients. Remarkably, we also found decreased NSun2 protein levels in AD mice and human cellular models.
To prove these observed alterations were unique to AD, we further evaluated brain tissues from other tauopathies. Strikingly, NSun2 protein levels were similar between tauopathy cases and controls indicating that dysregulation of NSun2 might be unique to AD cases. Further, we investigated the pathological role of NSun2 by utilizing a well-established Drosophila melanogaster model of tau-induced toxicity. We found that a reduction of NSun2 protein levels exacerbated tau toxicity while overexpression of NSun2 partially abrogated toxicity proving bidirectionality. We used a lentiviral system to knock down NSun2 expression in iPSC derived neuronal cultures. Western blot analysis and immunofluorescence staining showed a significant change in tau phosphorylation levels. To investigate what could be triggering observed alterations in NSun2 levels, we performed experiments in rat primary hippocampal neurons. We found that the treatment with oligomeric amyloid-beta A caused a decrease in NSun2 protein levels and at the same time, increased tau phosphorylation levels in primary hippocampal neurons. Lastly, we performed RNA immunoprecipitation coupled with qPCR and histological analysis using NSun2 conditional knockout (KO) mice and observed that NSun2 deficiency promoted aberrant levels of m6A methylated miR-125b and tau hyperphosphorylation. Altogether, our study demonstrates that neuronal NSun2 deficiency in AD promotes neurodegeneration by altering tau phosphorylation and tau toxicity through an epitranscriptomic regulatory mechanism and highlights a potential novel therapeutic target.
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Therapeutic strategies targeting FUS toxicity in amyotrophic lateral sclerosis: from a novel mouse model of disease to a first-in-human studyKorobeynikov, Vlad January 2021 (has links)
Fused in sarcoma (FUS) is an RNA binding protein involved in DNA repair and RNA metabolism, including mRNA transcription, splicing, transport and translation. FUS is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which pathogenic mutations in FUS cause neurodegeneration in ALS-FUS, we generated a series of FUS knock-in mouse lines that express the equivalent of the ALS-associated mutant proteins FUSP525L and FUSΔEX14 at physiological levels from the FUS locus. We demonstrate that heterozygous mutant FUS mice show progressive, age-dependent loss of vulnerable subpopulations of spinal motor neurons. While ALS-associated mutations in FUS lead to partial loss of function, we provide genetic evidence that the motor neuron phenotype observed is a consequence of a dose-dependent gain of function, associated with the insolubility of FUS and related RNA binding proteins (RBPs).
Furthermore, we show that motor neuron degeneration is driven by cell autonomous mechanisms, associated with mutant FUS-independent inflammatory changes. In this faithful mouse model of ALS-FUS, we demonstrate that an antisense oligonucleotide (ASO) targeting the FUS transcript (ION363) results in the efficient silencing of both wild type and mutant FUS alleles, and that postnatal reduction of FUS protein levels in the brain and spinal cord delays disease onset in this mouse model of ALS-FUS. In a first-in-human trial of ION363, we demonstrate that repeated, intrathecal injections of this candidate therapeutic in an ALS patient with a FUSP525L mutation leads to the efficient silencing of both wild type and mutant FUS in the central nervous system, and a reduction in the burden of FUS aggregates that are a pathological hallmark of ALS-FUS. In mouse genetic and human clinical studies, we provide evidence in support of a therapeutic strategy by which silencing of the FUS gene may be used to prevent or delay disease onset in pre-symptomatic carriers of pathogenic FUS mutations, or to slow disease progression in symptomatic ALS- and FTD-FUS patients. In addition, we use this newly generated model to investigate the role of potential modifiers of FUS toxicity, including hnRNP U and UPF1, and study the role of chronic neuroinflammation in the disease progression that could lead to the development of novel therapeutics to provide immediate clinical benefit to patients with ALS-FUS.
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Genetic Correlation between Alcohol Preference and Motor Impulsivity with Genetically Selected High-Alcohol and Low-Alcohol Preferring Lines of MiceNovotney, Devon Michael 20 September 2012 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol related problems and abuse continue to be serious problems in the U.S. today affecting nearly 17.6 million Americans. Understanding of the specific genes and related behaviors associated with alcohol use may provide substantial preventative measures for those who are at an increased risk. Genetically selected lines such as the high-alcohol preferring (HAP) and low-alcohol preferring (LAP) mice have been created to examine which endophenotypes co-segregate with alcohol preference. One behavioral trait that has been commonly associated with alcohol related problems is impulsivity. Impulsivity is the inability to withhold a response (motor impulsivity) or to act without forethought (cognitive impulsivity). The latter comprises much of the research and literature today using delay discounting models to tease out differences in subject’s wiliness to discount larger reinforcers for smaller immediate reinforcers. This study utilized relatively two newer paradigms associated with motor impulsivity in attempt to test differences in response disinhibition between two independent replicate HAP and LAP lines. It is hypothesized that the genes responsible for alcohol preference would be genetically correlated with motor impulsivity as HAP mice would display a greater degree of response disinhibition.
Two independent replicates consisting of 48 mice (24 HAP II and 24 LAP II, representing the 37th generation; 24 HAP III and 24 LAP III, representing the 13th generation) were tested in two separate identical experiments. Each experiment was comprised of three phases. Phase I utilized a fixed interval (FI) 120s procedure for 30 days. After the 30 days of FI exposure mice were immediately moved to phase II for 10 days which implored a differential reinforcement of low rate procedure (DRL) at a time interval of 20s. Phase III used the same procedures as Phase II except the DRL was increased to 32s.
As hypothesized, there was a moderate genetic correlation between alcohol preference and impulsivity as the HAP II mice displayed greater response disinhibition throughout all three phases compared to the LAP II mice. No differences were observed amongst the replicate III mice in any of the three phases.
The findings from this study provide additional support that a genetic correlation between alcohol preference and impulsivity exists as seen in the delay discounting literature. Though this was observed in only one of the two replicates, interpretations must be taken at caution as the replicate III mice are still in the early stages of selection. It is possible at this stage in the selection process that increases in alcohol over successive generations are associated with selecting for taste until a threshold is met where selection shifts to pharmacologic drinking relevance. Until later generations of replicate III mice are studied where pharmacologic drinking occurs, conclusions from this study provide a moderate genetic correlation between alcohol preference and impulsivity.
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The impact of exogenous TGFβ1 on male reproductive function.McGrath, Leanne Jane January 2008 (has links)
The TGFβ family of cytokines are potent signalling molecules that regulate tissue development, inflammation and immunity. Previous studies in mice with a null mutation in the Tgfb1 gene (TGFβ1-/- mice) implicate a key role for TGFβ1 in male reproductive function. These mice show profound infertility due to an inability to copulate successfully, associated with reduced testosterone and sperm production. The focus of this project was to 1) further characterize mechanisms underpinning reproductive deficiency in male TGFβ1-/- mice, 2) identify a reliable physiological marker of TGFβ1 availability in vivo, and 3) to determine whether exogenous TGFβ1 administration influences TGFβ1 availability and restores fertility. To investigate the causes of unsuccessful copulation by TGFβ1-/- mice, penis morphometry was examined. Penile organ structure, as assessed by scanning electron microscopy, was comparable between genotypes however a superfluous epidermal covering that impeded penile spine protrusion was evident in TGFβ1-/- mice. The epidermal covering was not due to increased epithelial cell proliferation, as measured by Brdu labelling and immunohistology. Behavioural observations of erectile activity showed that TGFβ1-/- mice achieved spontaneous erections albeit at reduced frequency compared to TGFβ1+/+ mice. The efficacy of exogenous TGFβ1 replacement was evaluated by first identifying measures of in vivo TGFβ1 availability and/or function and selecting an effective route of administration. Serum TGFβ1 and testosterone levels were reliable discriminators of TGFβ1 genotype. Gene expression and phagocytic function of peritoneal macrophages revealed no differences between genotypes. Exogenous sources of TGFβ1 for replacement studies included colostrum, naturally occurring in breast milk and recombinant human latent TGFβ1 (rhLTGFβ1). Colostrum did not increase circulating levels and rhTGFβ1 injection caused only transient elevation of serum levels. Thus mini-osmotic pumps were used to deliver a constant supply of cytokine to TGFβ1-/- mice. The fertility status of TGFβ1-/- mice receiving exogenous TGFβ1 was investigated. Reproductive behaviour in response to normal receptive female mice was assessed twice during treatment, on day 7 and day 14. Blood, liver and reproductive tissues were collected at sacrifice. Circulating TGFβ1 was increased in TGFβ1 treated TGFβ1-/- mice above TGFβ1-/- control levels, although this did not affect circulating testosterone. Erectile activity and sperm production were unchanged. Videotaping behaviour with estrous females revealed that the TGFβ1+/+ mice successfully mounted and intromitted, unlike the TGFβ1-/- controls. The TGFβ1-/- mice receiving exogenous TGFβ1 displayed moderately enhanced mounting and intromission behaviour although this remained less frequent than in the TGFβ1+/+ controls. Ejaculation behaviour was not observed in any TGFβ1-/- mice regardless of TGFβ1 replacement, compared to TGFβ1+/+ controls where >90% mice displayed ejaculated. Modest improvement in the copulation activity of the TGFβ1-/- mice receiving exogenous TGFβ1 suggests that systemic TGFβ1 availability can influence reproductive performance in male TGFβ1-/- mice. However since fertility was not restored, locally produced TGFβ1 in the reproductive tract and/or hypothalamic pituitary axis are also implicated in regulating fertility. These findings advance our knowledge of the role of the TGFβ1 cytokine in male reproductive physiology and may have relevance for devising new treatments for infertility and erectile dysfunction in men. / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2008
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Adolescent and Adult Two-Bottle Choice Ethanol Drinking and Adult Impulsivity in Genetically Selected High-Alcohol Preferring MiceO'Tousa, David Scott 20 September 2012 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Abuse of alcohol during adolescence continues to be a problem, and it has been shown that earlier onset of drinking predicts increased alcohol abuse problems later in life. High levels of impulsivity have been demonstrated to be characteristic of alcoholics, and impulsivity has also been shown to predict later alcohol use in teenage subjects, showing that impulsivity may be an inherent underlying biological process that precedes the development of alcohol use disorders. These experiments examined adolescent drinking in a high-drinking, relatively impulsive mouse population, and assessed its effects on adult drinking and adult impulsivity.
Experiment 1: Selectively bred High-Alcohol Preferring (HAP II) mice, which are shown to be highly impulsive, were given either alcohol (free choice access) or water only for two weeks during middle adolescence or adulthood. All mice were given free choice access to alcohol following 30 days without access, in adulthood. Experiment 2: Adolescent HAP II mice drank alcohol and water, or water alone, for two weeks, and were then trained to perform a delay discounting task as adults to measure impulsivity. In each experiment, effects of volitional ethanol consumption on later behavior were assessed. We expected adolescent alcohol exposure to increase subsequent drinking and impulsivity.
Adolescent mice consumed significant quantities of ethanol, reaching average blood ethanol concentrations (BECs) of 142 mg/dl in Experiment 1 and 108 mg/dl in Experiment 2. Adult mice reached average BECs of 154 mg/dl in Experiment 2. Mice pre-exposed to alcohol in either adolescence or adulthood showed a transient increase in ethanol consumption, but we observed no differences in impulsivity in adult mice as a function of whether mice drank alcohol during adolescence.
These findings indicate that HAP II mice drink intoxicating levels of alcohol during both adolescence and adulthood, and that this volitional intake has long-term effects on subsequent drinking behavior. Nonetheless, this profound exposure to alcohol during adolescence does not increase impulsivity in adulthood, indicating that long-term changes in drinking are mediated by mechanisms other than impulsivity. Importantly, this research demonstrates that the HAP II mouse is a good candidate for a model of heavy adolescent alcohol consumption.
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