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http://hdl.handle.net/11375/29702
Title: | Retrograde Transport of Radioiodinated Nerve Growth Factor via Tropomyosin Receptor Kinase A |
Authors: | Falah, Rojin |
Advisor: | Sadeghi, Saman Fahnestock, Margaret |
Department: | Chemical Biology |
Publication Date: | 2024 |
Abstract: | Clinical symptoms of Alzheimer’s disease (AD), like memory loss and cognitive decline, are rooted in neuronal loss and synaptic disruptions. Basal forebrain cholinergic neurons (BFCNs), crucial for learning and memory, deteriorate early in aging and AD. Cognitive decline in aging and AD correlates with reduced levels of tropomyosin receptor kinase A (TrkA), the nerve growth factor (NGF) receptor, in the cholinergic neurons, and impaired NGF axonal transport. Currently, TrkA levels can only be measured post-mortem. We proposed developing a TrkA-positron emission tomography (PET) tracer for in vivo measurement, potentially serving as an early diagnostic biomarker for aging and AD-associated cognitive decline. As part of our optimization efforts, we initially assessed the transport of iodinated NGF in mice. The primarily aim was to synthesize and characterize radioiodinated NGF and evaluate its uptake and retrograde transport from hippocampus to basal forebrain via BFCNs. The approach taken to track the radiotracer in the mouse brain involved the intracranial administration of 125I-NGF. Several optimization and preliminary steps were taken to establish a consistent method and coordinates to target the site of injection, hippocampal dentate gyrus (DG). NGF was radiolabeled with the isotope Iodine-125 (125I) using the direct labeling method with a radiochemical yield of 91.08 ± 1.20% and purified to a radiochemical purity of 99.17 ± 0.18%. Characterization methods such as iTLC and HPLC were employed to evaluate the purity of 125I-NGF. Additionally, stability tests were conducted to ensure the compound's stability before further use for in vitro and in vivo purposes. The specificity of 125I-NGF and its biological activity was validated by assaying in vitro receptor binding to TrkA-overexpressing PC12nnrB5 cells compared to TrkA-knockout PC12nnr cells. Stereotaxic intracranial administration of 1 µL (1.5-2 µCi) of 125I-NGF was conducted on young C57Bl/6 mice. After survival time of two and six hours post injection, mice were euthanized, and the brains were removed. Specific brain regions were sub-dissected including hippocampus, basal forebrain, cortex, midbrain, and cerebellum. Radioactivity of each brain region was measured by a gamma counter. Autoradiography was performed to analyze the localization of the radioligand in the brain. Consistency in delivering 125I-NGF into the hippocampus was confirmed through gamma measurements in mice euthanized prior to the injection. Comparison of the injected dose per gram of tissue (%ID/g) of basal forebrain from two-hour (mean: 3.190 ± 0.884) and six-hour (mean: 16.80 ± 2.683) intervals exhibited slight transportation to basal forebrain. Additionally, qualitative observation of autoradiography analysis revealed that the majority of the activity was found to remain in the site of injection in pre-euthanized mice and 2 hours post injection. Extended time points may be worth evaluating in the future to determine if they increase transport. The utilization of a healthy mouse model to demonstrate successful transport of 125I-NGF will facilitate future investigations into retrograde transport in aging and AD mouse models. These studies will allow for comparisons with healthy mice, shedding light on whether the absence of NGF transport, due to TrkA loss, contributes to memory loss and neurodegeneration in aging and neurodegenerative disorders. |
URI: | http://hdl.handle.net/11375/29702 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Falah_Rojin-2024April_MSc.pdf | 1.47 MB | Adobe PDF | View/Open |
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