Project

The Investigation of Specific Genetic Alleles and External Factors as a Determinant of Increased Neural Cell Cytotoxicity in Alzheimer 's Disease

The Investigation of Specific Genetic Alleles and External Factors as a Determinant of Increased Neural Cell Cytotoxicity in Alzheimer 's Disease VALA Sciences Cherie Handley Professional Master's Degree Program Cal State University San Marcos Objective Alzheimer's disease is a neurodegenerative disorder in the aged population in which memory is lost. It is a leading cause of dementia. Pathologically, the ailment is characterized by the aggregation of beta-amyloid plaques and tau neurofibrillary in the brain. There is no cure however researchers are investigating the cause of the disease, be it genetic or environmental, as a way to lead to a cure. Live cell imaging provides scientists with the ability to observe live cells via time-lapse photography. The objective of this project is to investigate the role of environmental compounds or genetics in the pathogenesis of the disease. The lab accomplishes this by developing a cell-based cytotoxicity assay using Kinetic Imaging Cytometers (KIC). These instruments are capable of imaging fluorescently labeled proteins and cell structures. Here we analyze cellular action potentials as well as calcium ion transients. Additionally, post-KIC we stain the cells using Immunocytochemistry (ICC) to observe cellular components of interest. The key to healthy neuronal cell function is to maintain cellular activities at metabolically non-disruptive levels. Our instrument provides a resource in studying the molecular and biochemical pathways for potential therapies. Methods We investigate the feasibility of developing our assay for use on various cell types as well as to observe the effects of potentially therapeutic or cytotoxic compounds. Upon completion of the feasibility studies our lab will optimize assay parameters, including controls. The lab sourced human induced pluripotent stem cells (hi-PSC) derived iGlutaneurons, excitatory neuronal cell lines. Additionally, we derived other cell types as needed. The cells are exposed to selected compounds at varying doses. We plan to assay isogenic cell lines to study differences among ApoE isotypes and their roles in neuropathology. VALA researchers measure cellular responses post-treatment via quantification of Calcium and Voltage fluorescent indicator signals followed by ICC. Results The mean peak area for Particulate Matter smaller than 2.5 micrometers, PM2.5, when exposed to ANOVA results, a p-value of 1.68E-03. Dunnet method indicates a statistically significant response at 100 ug/mL dose. Conclusions PM2.5 is significantly different among doses when neurons were exposed for 24 hours. The 24-hour exposure to PM2.5 results in increased Calcium signals in our neuronal cell line but eventually at the highest dose was below 0-dose control. There was no significant difference in action potentials. Treatments with ApoE3 and ApoE4 demonstrated a cytotoxic effect with increasing dose. Intracellular calcium eventually waned to below 0 treatment control at the highest dose. Analysis of the raw data is a work in progress. The threshold can vary between active and dead cells. We continue our on-going assay development.

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