Biological applications of boronate optical probes, neurotoxins and antioxidants in neuronal cell culture
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Abstract
The neurochemistry of hydrogen peroxide (H2 O2) in living systems has received increased attention owing to its contributions as a marker for oxidative stress and damage events associated with neurodegeneration. H2 O2 is a major reactive oxygen species (ROS) in living organisms, yet its production, accumulation and trafficking within neuronal cells is poorly understood. Using the H2 O2 selective optical probe Peroxyfluor-1 (PF1), we determined its ability to operate within neuronal cell lines (RN46A-B14 and SKNSH) and primary cortical neurons.
In collaboration with Professor Bryan K Yamamoto at the BU Medical Campus, we used PF1 to study the role of H2 O2 in serotonin (5-HT) neurons in the presence of the well-known neurotoxin and drug of abuse, 3,4methylenedioxymethamphetamine (MDMA). We found that MDMA creates the pro-oxidant environment to non-enzymatically hydroxylate L-tyrosine in the 5-HT cell line RN46A-B14. Dopamine (DA) is formed by endogenous amino acid decarboxylase (AADC) and is further metabolized by monoamine oxidase B (MAO-B) to produce H2O2 as a by-product.
Next, we investigated oxidative stress in a dopaminergic cell line (SKNSH). We found significant increases in H2 O2 formation upon treatment with the current drug therapy for Parkinson's disease (PD), L-3,4 dihydroxyphenylalanine (L-dopa). L-dopa results in the AADC conversion of L-dopa to DA in the brain. A replenishment of DA in the surviving neurons temporarily alleviates symptoms of the disease; however, the therapy is not curative, and dopaminergic cells continue to die in PD patients receiving L-dopa. The proposed mechanism for L-dopa induced cell death is by autoxidation into ROS and toxic quinones. We found that resveratrol and flavonoid antioxidants reduced L-dopa-induced H2O2 formation and cytotoxicity.
We also synthesized boronate polyphenols that resemble the antioxidant scaffolds with which we had great success as neuroprotectants. We envisioned that placing a boronate ester functionality on the antioxidant backbone would increase solubility, cell permeability and decrease H2O2 -induced cell death in SKNSH cells. Of the boronate coumarins, stilbenes and flavones we synthesized, we found that 7-boronatecoumarin and to a lesser extent (E)-3,5-dihydroxy, 4'-boronatestilbene were water soluble, cell permeable and preserved SKNSH cell viability in the presence of H2O2.
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Thesis (Ph.D.)--Boston University
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PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.