Browsing by Subject "Mice, Transgenic"
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Item Analysis of Circadian Rhythm Using a Novel SCN-Specific Cre Transgenic Mouse Line(2010-07-12) Chang, Alexander S.; Yanagisawa, MasashiThe neurons that make up the suprachiasmatic nucleus (SCN) temporally organize behavior into circadian cycles of activity and rest. When dissociated, these neurons individually oscillate with various period, phase, and amplitude. These conflicting results can be reconciled if inter-neuronal networking in the SCN is required for a consolidated behavioral circadian rhythm. To test this hypothesis, a novel SCN-specific Cre transgenic mouse line, named NMS-Cre, was developed by inserting a bicistronic Cre expression cassette at the 3â-untranslated region of the Neuromedin S (NMS) gene. By crossing NMS-Cre line to a lox-STOP-lox diphtheria toxin receptor line, behavioral circadian rhythm was disrupted upon intraperitoneal injection of diphtheria toxin. A histological examination showed that diphtheria toxin injection eliminated ~85% of NMS-Cre containing neurons at the SCN. Next, I generated NMS-Cre mediated Bmal1 conditional knockout animals to study behavioral rhythm output when most of the SCN neurons are without a molecular oscillator. The NMS-Cre(+);Bmal1flox/flox animals have essentially normal circadian rhythm of locomotor activity. Then, I generated NMS-Cre mediated Vesicular GABA Transporter (VGAT) conditional knockout animals because GABA has long been suspected to play a role in behavioral circadian rhythm. The NMS-Cre;VGATflox/flox animals performed normally in behavioral circadian rhythm parameter such as free-running period, robustness, and phase response curve. These in vivo data demonstrated a model that intra-SCN neuronal network is required for behavioral circadian rhythm, and can be a conduit that mediates molecular clock outputs from a small number of SCN neurons. Despite the fact that virtually all SCN neurons are GABAergic, GABA is an unlikely transmitter for this intra-SCN networking. Finally, NMS knockout animals have a well-consolidated behavioral circadian rhythm. However, when subjected to photic phase advancement, NMS knockout animals shifted their activity onset time quicker than wild type control animals. In situ hybridization results ruled out that an altered response to light stimuli or dampened molecular clock oscillation in the SCN as the cause for the rapid phase shift. NMS knockout animals switching from constant illumination to constant dark environment are unable to return to the typical less than twenty-four hour free-running period. Therefore, NMS is involved in a circadian pacemaker function.Item Constitutive Overexpression of Acyloxyacyl Hydrolase in Mus Musculus(2009-01-14) Ojogun, Noredia I.; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is a highly conserved host lipase that selectively removes the secondary acyl chains from lipid A, the bioactive center of Gram negative bacterial lipopolysaccharide (LPS). Deacylated LPS has a marked reduction in bioactivity and antagonizes the LPS signaling pathway. Thus, AOAH deacylation of LPS may represent a mechanism by which animals control responses to Gram-negative bacteria. Prior to the experiments described in this study, mice deficient in AOAH were found to be susceptible to the long-term effects of LPS. Aoah-/- mice developed long-lasting hepatomegaly, exaggerated antibody responses, and prolonged immunosuppression in response to small doses of LPS. In the studies described here, AOAH was overexpressed in mice by using CD68 promoter sequences which have been shown by others to drive transgene expression in macrophages. CD68p-AOAH transgenic mice had constitutive overexpression of AOAH in macrophages, dendritic cells and tissues rich in these cells (liver, spleen and lung). They also secreted the enzyme into blood and deacylated LPS at a faster rate both in vitro and in vivo. Importantly, constitutive overexpression of AOAH did not interfere with the initial pro-inflammatory responses to LPS, in keeping with prior observations that AOAH-mediated inactivation of LPS occurs over several hours and does not moderate acute reactions to LPS in vivo. The protective role of constitutive AOAH overexpression was determined by two test systems. First, after an intraperitoneal dose of LPS, CD68p-AOAH transgenic mice returned to their pre-challenge weights more rapidly than did the wildtype mice. Secondly, CD68p-AOAH transgenic mice were less susceptible to LPS and Gram-negative bacteria induced hepatosplenomegaly. These results suggest that overexpression of AOAH in macrophages could accelerate recovery from Gram-negative bacterial infections in animals, including humans.Item Neural Stem Cells in Brain Tumor Development(2009-09-04) Llaguno, Sheila R. Alcantara; Parada, Luis F.Malignant astrocytomas are highly invasive and incurable brain tumors. Mouse models that genetically resemble the human disease are valuable tools in understanding the pathogenesis of these malignancies. We previously reported mouse models based on conditional inactivation of the human astrocytoma-relevant tumor suppressors Nf1, p53 and Pten. Through somatic loss of heterozygosity, these mice develop varying grades of astrocytic malignancy with 100% penetrance. Studies on our tumor suppressor mouse models indicated a central role for neural stem cells and stem cell-like cancer cells in malignant astrocytoma formation. Using stereotactic viral cre-mediated approach, we demonstrate that targeting of tumor suppressor inactivating mutations in the subventricular zone (SVZ) where neural stem and progenitor cells reside is both necessary and sufficient to induce astrocytoma formation. We also show evidence of spontaneous differentiation and infiltration of these cancer-initiating cells in situ during tumor development. These studies have so far shown that neural stem cells or its progeny can give rise to astrocytomas. Neural stem cells, which have unlimited self-renewal potential, produce transit amplifying cells, or progenitor cells, which undergo limited mitoses before differentiating into more mature cell types. By genetically targeting transit amplifying cells using the Ascl1-creERT2 transgenic mouse, we show that tumor suppressor inactivation in the progenitor compartment alone induces malignant astrocytoma formation. Defects in proliferation, differentiation, and migration are likewise found several months prior to advanced disease. This establishes both neural stem and progenitor cells as cells of origin of malignant astrocytomas in our tumor suppressor mouse models. In another study, we isolated and characterized a population of stem cell-like cancer cells from murine astrocytomas that are enriched for tumor cells compared to primary tumor tissue, exhibit aberrant stem cell properties, and are tumorigenic in vivo. We demonstrate resistance to a known chemotherapeutic agent and the migratory capacity of these cells. We also investigated the mechanisms involved in astrocytoma progression and maintenance by gene expression analysis. Genomic profiling of tumor-derived neurosphere-forming cells from conditional astrocytoma mouse models show prominent dysregulation of genes involved in neurodevelopmental processes and transcriptional regulation, particularly the hox transcription factors, in high-grade astrocytomas. Taken together, we have demonstrated that neural stem and progenitor cells are the origins of malignant astrocytoma in tumor suppressor mouse models. We have established a system by which molecular mechanisms of tumor development can be further investigated and performed genomic profiling of tumor-derived neurosphere-forming cells, suggesting a possible role for homeobox transcription factors in malignant astrocytoma formation. These mouse models thus represent powerful tools in understanding various aspects of cancer development that otherwise cannot be explored in humans. Further studies will provide a better understanding of the biology of these tumors and will hopefully pave the way for more effective therapeutic approaches for these devastating diseases.Item The Role of TNF Signaling in Regulating Beta-Amyloid Burden in the 3xTgAD Mouse Model of Alzheimer's Disease(2008-05-13) McAlpine, Fiona E.; Tansey, MalĂș G.Microglial activation and overproduction of inflammatory mediators in the CNS have been implicated in Alzheimer's Disease (AD), but the precise nature of the key molecular mediators of neurotoxicity that directly contribute to neurodegeneration or loss of specific neuronal populations is less clear. The pro-inflammatory cytokine Tumor Necrosis Factor (TNF) has been implicated in AD by its elevated presence in serum and post-mortem brains of patients with AD. To test the hypothesis that TNF-dependent neuroinflammation and neurotoxicity contributes to the increased microglial burden and exacerbated pathology observed in the hippocampus and cortex of 3xTgAD mice (transgenic mouse expressing human familial AD mutations in APP, PS1, and a familial frontotemporal dementia mutation in tau) after chronic systemic LPS exposure, we inhibited TNF signaling with novel engineered dominant negative TNF inhibitors (DN-TNF) selective for soluble TNF (solTNF) or lentiviral-derived DN-TNF to achieve long-term inhibition of TNF activity and halt or delay the early stages of amyloid-associated neuropathology. In vitro, cells infected with lenti-pro-DN-TNF-IRES-GFP produce sufficient levels of DN-TNF protein to block nuclear translocation of p65 in response to stimulation by TNF. Infection with lenti-DN-TNF also blocks solTNF-induced activation of primary microglia. Results from in vivo studies indicate that short-term pharmacological inhibition as well as long-term lentiviral-driven inhibition of soluble TNF signaling decreases the accumulation of intraneuronal full length APP in hippocampus and cortex induced by chronic systemic inflammation. To our knowledge, this is the first study that selectively inhibits soluble TNF signaling in an acute manner using a pharmacologic agent, thereby directly linking endogenous TNF activity in vivo to accumulation of APP in a model of Alzheimer's disease. Targeted inhibition of soluble TNF in the central nervous system may represent a new therapeutic approach to slow the appearance of amyloid pathology, cognitive deficits, and possibly the progressive loss of neurons in AD.