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Bryan Soth Abstracts for Research Symposium - RACK1 Rescue Project
Down syndrome occurs due to triplication of human chromosome 21, and results in many phenotypic defects including slow wound healing, intellectual disability and reduced muscle tone. Here we focus on the role of adhesions in this disorder, which are involved in tissue development, cell migration, and memory formation. Previous work from our lab demonstrates that fibroblasts from individuals with Down syndrome have increased focal adhesions, which are multi-protein complexes that link the extracellular matrix to the intracellular cytoskeleton. We also found that the area, perimeter and motility of Down syndrome fibroblasts is altered. Taken together, these data suggest that increased adhesion may lead to reduced cellular motility in Down syndrome. Here we show that RACK1, a scaffolding protein and member of the focal adhesion complex, is overexpressed in Down syndrome fibroblasts. We also demonstrate that RACK1 can be knocked down with shRNA in Down syndrome fibroblasts to levels comparable to apparently healthy control cells. We are currently investigating whether knocking down RACK1 to control values is able to rescue the phenotypic defects present in Down syndrome fibroblasts, such as cell size, cell shape, and motility. Furthermore, substrate type is known to have a profound effect on protein expression and differentiation, and we have preliminary data that the substrate affects the expression level of RACK1 differentially in Down syndrome fibroblasts. Examining the role of RACK1 in Down syndrome is relevant for understanding the phenotypic defects of individuals with this disorder and may also provide insight into therapeutic and clinical treatments. Identification of Opioid Receptors in Mixed Cortical Cultures
Introduction: The six layers of the rat cerebral cortex consists of around twenty-one million cells which can be sub-divided into distinct neuronal and glial populations (e.g. GABAnergic, Glutaminergic, Astrocytic). These neuronal populations are differentially targeted by both licit and illicit drugs to alter cellular states, excitability, and/or behavioral outcome. Opioids alter cortical excitability through inhibition of GABAnergic inhibitory interneurons leading to disinhibition of glutaminergic neurons and downstream excitation of dopaminergic neurons. Resulting in altered reward evaluation and opioid induce analgesia. Objective: To use Immunocytochemistry - Immunofluorescence to identify the key cellular targets of opioids in the cortex. Methods: Cortical neurons were isolated from (P0) rat pups, cultured on glass coverslips and allowed to form mature synapses over 12 days. On days 13-20 cultures were Immuno-labeled with neuronal (Anti-NeuN), Astrocytic (Anti-GFAP), GABAnergic (Anti-GAD67) and Mu1 Opioid receptor (Anti-OPRM1) conjugated antibodies and imaged at 405nm, 488nm, 550nm, and 633nm respectively on an inverted confocal microscope. Results: Opioid receptors were found to be co-localized with GAD67 and NeuN labeling with little to no detectable expression on astrocytes or other neuronal subpopulations. Conclusion: These data support previous evidence that opioids selectively act on specific subpopulations of cortical neurons that suppress and limit cortical excitability.- Image
Methods to Create a Multi Modal Imaging Probe
Magnetic resonance imaging (MRI) scans use a combination of radio waves and magnetic fields to create an image of tissues within the body. While MRI is proven effective it only has limited use as to which tissue structures can be accurately resolved. Using contrast agents in MRI, it is possible to gain enhanced detail in acquired images to help resolve disease symptoms, tissue activity, and improve the signal of tissues during a scan. Contrast agents are used in patients with Multiple Sclerosis to detect lesions in brain tissue. Two issues with current contrast agents is their lack of specificity and toxicity at relatively low doses. Recently, a new a gadolinium-based nanoparticle (GdNP) has been developed. GdNP is approximately ten times stronger than typical contrast agents allowing it to provide the same contrast enhancement with ten percent of the typically required concentration. Targeting agents further decrease the number of molecules needed for desired image enhancement by binding to specific areas of the tissues being studied. This research is based around developing and evaluating nanoparticle-targeting agents as contrast enhancing probes with tissue specificity. The albumin binding protein, Evans blue (EB), have been attached to contrast enhancing nanoparticles. EB targets serum albumin (SA) in the blood vessels when injected intraperitoneally. We present in vivo MRI and microscopic data detailing development and usage of this new probe designed to enhance signal from the neurovasculature of a mouse brain. The Effects of Human Chorionic Gonadotropin on the Onset and Severity of Kiantic-acid Induced Seizure Activity
Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein involved in reproductive function known to bind the luteinizing hormone receptor (LHR). LHR activation has been shown to inhibit function at high doses. Importantly, in the APP/PS1 Alzheimer’s disease mouse model, known to have increased susceptibility to seizures, we have observed that mortality due to seizures is significantly reduced in animals treated with intracerebral (ICV) hCG. Therefore, to more directly address if hCG has anti-convulsant effects we used a model of epilepsy (kiantic acid administration) to induce convulsions in the presence and absence of chronic ICV hCG treatment in WT and APP/PS1 mice. The seizure induction and severity were scored using a seizure scale and neuronal changes/damage was determined by Tunnel staining. Our preliminary data in APP/PS1 suggests hCG may reduce susceptibility and severity of seizures in this model of epilepsy.