Browsing by Subject "Gene expression profiling"

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    Mechanisms of specificity in neuronal activity-regulated gene transcription.
    (2012) Lyons, Michelle Renée
    In the nervous system, activity-regulated gene transcription is one of the fundamental processes responsible for orchestrating proper brain development–a process that in humans takes over 20 years. Moreover, activity-dependent regulation of gene expression continues to be important for normal brain function throughout life; for example, some forms of synaptic plasticity important for learning and memory are known to rely on alterations in gene transcription elicited by sensory input. In the last two decades, increasingly comprehensive studies have described complex patterns of gene transcription induced and/or repressed following different kinds of stimuli that act in concert to effect changes in neuronal and synaptic physiology. A key theme to emerge from these studies is that of specificity, meaning that different kinds of stimuli up- and down regulate distinct sets of genes. The importance of such signaling specificity in synapse-to-nucleus communication becomes readily apparent in studies examining the physiological effects of the loss of one or more forms of transcriptional specificity – often, such genetic manipulations result in aberrant synapse formation, neuronal cell death, and/or cognitive impairment in mutant mice. The two primary loci at which mechanisms of signaling specificity typically act are 1) at the synapse – in the form of calcium channel number, localization, and subunit composition – and 2) in the nucleus – in the form of transcription factor expression, localization, and post-translational modification. My dissertation research has focused on the mechanisms of specificity that govern the activity-regulated transcription of the gene encoding Brain-derived Neurotrophic Factor(Bdnf). BDNF is a secreted protein that has numerous important functions in nervous system development and plasticity, including neuronal survival, neurite outgrowth, synapse formation, and long-term potentiation. Due to Bdnf’s complex transcriptional regulation by various forms of neural stimuli, it is well positioned to function as a transducer through which altered neural activity states can lead to changes in neuronal physiology and synaptic function. In this dissertation, I show that different families of transcription factors, and even different isoforms or splice variants within a single family, can specifically regulate Bdnf transcription in an age- and stimulus-dependent manner. Additionally, I characterize another mechanism of synapse-to-nucleus signaling specificity that is dependent upon NMDA-type glutamate receptor subunit composition, and provide evidence that the effect this signaling pathway has on gene transcription is important for normal GABAergic synapse formation. Taken together, my dissertation research sheds light on several novel signaling mechanisms that could lend specificity to the activity-dependent transcription of Bdnf exon IV. My data indicate that distinct neuronal stimuli can differentially regulate the Calcium-Response Element CaRE1 within Bdnf promoter IV through activation of two distinct transcription factors: Calcium-Response Factor (CaRF) and Myocyte Enhancer Factor 2 (MEF2). Furthermore, individual members of the MEF2 family of transcription factors differentially regulate the expression of Bdnf, and different MEF2C splice variants are unequally responsive to L-type voltage-gated calcium channel activation. Additionally, I show here for the first time that the NMDA-type glutamate receptor subunit NR3A (also known as GluN3A) is capable of exerting an effect on NMDA receptor-dependent Bdnf exon IV transcription, and that changes in the expression levels of NR3A may function to regulate the threshold for activation of synaptic plasticity-inducing transcriptional programs during brain development. Finally, I provide evidence that the transcription factor CaRF might function in the regulation of homeostatic programs of gene transcription in an age- and stimulus-specific manner. Together, these data describe multiple novel mechanisms of specificity in neuronal activity-regulated gene transcription, some of which function at the synapse, others of which function in the nucleus.
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    The Role of Tumor Necrosis Factor-Stimulated Gene 6 Protein (TSG-6) in Osteoarthritis
    (2017) Chou, Chingheng

    In our studies, we developed a novel method for extracting high quality nucleic acid from joint tissue that relies on finely grinding the desired regions of the knee tibial plateau under liquid nitrogen. By controlling the depth of drill penetration we could reliably separate and isolate articular cartilage and site-matched subchondral bone with high fidelity. We also set up a model system based on specific regions of interest within a joint corresponding to a gradient of disease severities; this system can be used to represent early (relative intact), intermediate (mild to moderate damaged) and late (severe damaged) disease within the knee joints. We performed microarray profiling to identify disease relevant genes and pathways associated with knee OA progression in human articular cartilage. Tumor Necrosis Factor-Stimulated Gene 6 (TSG-6) was identified to be significantly associated with OA progression and validated in both medial and lateral compartment dominant OA samples.

    Next, we explored mechanisms underlying the association of TSG-6 with OA progression. We characterized the effects of TSG-6, in the presence and absence of inter-alpha-inhibitor (IαI), on anti-plasmin activity, evaluated for the in vivo presence of heavy chains (HCs) in cartilage matrix (evidence of TSG-6 activity), evaluated the gene expression of IαI components and TSG-6 in cartilage, and explored effects of TSG-6 on matrix assembly in vitro.

    TSG-6 synergized with IαI to inhibit plasmin activity by 39.3%; the presence of HA (full length or fragments) reduced the inhibitory effect. TSG-6 activity was highly correlated (R = 0.6392, P = 0.0006) with TSG-6 protein concentrations in synovial fluid (SF) from knee OA joints. TSG-6 protein and RNA were highly expressed in damaged cartilage from knee tibial and meniscal cartilage and chondrocytes treated with cytokines. The components of the IαI complex, ITIH1, ITIH2, and AMBP genes were either not expressed or expressed at a low level in intact or damaged cartilage of OA joints and chondrocytes. Rate limiting amounts of IαI were demonstrated by spiking-in exogenous IαI into cartilage extracts. This suggested that the source of IαI for TSG-6 mediated HC transfer onto damaged cartilage is likely the synovial fluid and not the cartilage itself. Interestingly, SF TSG-6 activity was significantly positively associated with the inflammatory reactants TIMP-1, A2M, VEGF, VCAM-1, ICAM, MMP-3, TNFR2, cytokines IL-6, IL-8, IL-18, quantity of activated macrophages in synovium and soluble macrophage-associated marker CD14 and number of low molecular weight (pro-inflammatory) hyaluronan (HA) molecules. Moreover, TSG-6 impaired HA-aggrecan assembly, but TSG-6 mediated HA-HC formation reduced this negative effect.

    IαI enhances the anti-protease activity of TSG6, preserves matrix assembly capabilities by enabling TSG6 transfer of HC to HA and mitigates inhibition of matrix assembly by TSG-6. During OA progression, inflammatory mediators increase production of TSG-6, but IαI originating outside cartilage acting in a rate-limiting manner and may only moderately interact with TSG-6 released from damaged cartilage. Along with previous work, these findings suggest that the net beneficial effect of TSG-6 is therefore dependent upon the availability of IαI in knee OA.