Down syndrome results from the trisomy of human chromosome 21 and is the most prevalent genetic cause of intellectual disability. Down syndrome results in various developmental changes that can be observed on a cellular level, including a change in the expression of certain proteins. Because receptor for activated C kinase 1 (RACK1) and amyloid precursor protein (APP) have both been implicated in the pathogenesis of Down syndrome and play a role in cell adhesion and motility, we hypothesized that their expression may vary in Down syndrome. Fibroblasts were obtained from a human diagnosed by karyotype for Down syndrome and a healthy human subject that was age, sex and race matched to the individual with Down syndrome. RACK1 and APP expression in these fibroblasts were labeled using quantitative immunocytochemistry. Preliminary results demonstrate that both RACK1 and APP are overexpressed in Down syndrome fibroblasts. Thus, it is likely that the overexpression of APP and RACK1 in Down syndrome may lead to inappropriate cell adhesion and motility, and contribute to the pathogenesis of this disorder. In future experiments, we will examine how RACK1 mRNA localization may be dysregulated in Down syndrome. In addition, the expression of APP and RACK1 in Down syndrome neurons, instead of fibroblasts, will be examined to further understand how these proteins contribute to the formation of brain connectivity. These experiments will further advance the understanding of Down syndrome, its connection to Alzheimer's disease, and aid in the development of treatments for these disorders.
RACK1 regulates axon outgrowth and point contact formation through local translation in developing neurons04/05/2018
During development, neurons must extend processes and make connections with their appropriate targets. This process is dependent on multiple molecular and cellular mechanisms, and if disrupted, neurodevelopmental disorders can result. We previously demonstrated that Receptor for activated C kinase (RACK1), a ribosomal scaffolding protein, regulates the adhesion and motility of developing neurons through its regulation of point contacts, adhesion points located in the tips of pathfinding axons. Furthemore, RACK1 regulates the local translation of β-actin mRNA, which is necessary for appropriate axon guidance. However, RACK1 has multiple signaling and ribosomal functions, and how the ribosomal binding function of RACK1 contributes to neural development is unknown, Thus, we specifically investigated the ribosomal binding function of RACK1 in point contact formation, axonal outgrowth, and local translation. We overexpressed RACK1-WT, RACK1-DE (a mutant form of RACK1 that cannot bind ribosomes) or a control construct in embryonic mouse cortical neurons. Immunocytochemistry was performed, followed by quantification of point contact formation, axon outgrowth, and local translation of β-actin. Overexpression of RACK1-DE inhibited BDNF-induced point contact formation, and also led to a significant decrease in axonal outgrowth. We are currently examining whether �-actin protein levels decrease following overexpression of RACK1-DE; this is expected because RACK1 mediates the local translation of �-actin. Together, these experiments show that local translation mediated by RACK1 regulates adhesion and axon outgrowth in the developing nervous system. We previously identified aberrant expression of RACK1 in Down syndrome, and thus these results have implications for the pathogenesis of this neurodevelopmental disorder.
Human Down syndrome fibroblasts exhibit changes in cell motility due to increased adhesion
Paige Cassidy1, Shelby Kelemen1, Sami Bailey B.S.1,Taylor Bumbledare1, Leah Kershner B.S.1, Kristy Welshhans Ph.D1,2
1Department of Biological Sciences, 2School of Biomedical Sciences
Kent State University, Kent, Ohio
Down syndrome is a common developmental disorder which results from the triplication of human chromosome 21. Intellectual disability is ubiquitous in Down syndrome, but our understanding of the cellular mechanisms underlying this phenotype are limited. Focal adhesions link the extracellular matrix to the intracellular cytoskeleton and regulate cellular motility. Focal adhesions are composed of and regulated by multiple proteins, including paxillin and RACK1. Age, sex and race matched human fibroblasts from an individual with Down syndrome and a healthy individual were plated onto coverslips, allowed to grow to 70% confluency, and then fixed. Immunocytochemistry was performed to stain for paxillin or RACK1, and the fluorescence intensity was quantified using FIJI software. We found that expression of paxillin and RACK1 is increased in human Down syndrome fibroblasts, as compared to control fibroblasts. This data suggests that there is increased adhesiveness in Down syndrome cells, which likely contributes to the cellular abnormalities that are characteristic of this disorder. We are currently investigating how this increase in focal adhesions lead to changes in cellular motility in Down syndrome. These results have implications for not only fibroblasts, but also neurons, because many of the same mechanisms underlie motility of these diverse cell types. Thus, one of the mechanisms contributing to the intellectual disability phenotype of Down syndrome may be changes in adhesion during neural development, which leads to inappropriate neuron migration and axon guidance. These experiments will increase our understanding of Down syndrome and help inform the development of treatments for this disorder.