Development and testing of Carbon Quantum Dot architectures to arrest neurotoxicant-insult- related outcomes - Exposure to pesticides, fungicides and herbicides is linked to neuronal injury, neuronal loss and the onset and progress of neurodegeneration. The parent R16 grant was based on preliminary data revealing that organo-acid- derived Carbon Quantum Dots (CQDs) resolve pesticide-associated neuronal dyshomeostasis. These data underscored the interference by CQDs in the soluble-to-toxic transformation of amyloidogenic proteins and their ability to scavenge free radicals and mitigate reactive oxygen species stress, an outcome of their sp2- hybridization-rich architectures. Since then, we have synthesized and characterized a number of other CQDs derived from blood-brain barrier penetrating organic acids and metabolites (caffeic acid, quinic acid, chlorogenic acid, and tyrosine) and established their ability to intervene experimentally, in vitro and in vivo in the trajectory to pesticide-driven neurodegeneration. Nevertheless, we observe CQD-specific differences in i) their ability to prevent fibril formation, ii) cytotoxicity iii) intracellular free radical scavenging ability iv) ability to rescue dopaminergic neurons from paraquat-associated ablation and v) restituting pesticide-exposure-associated loss of locomotion in nematodes. We hypothesize that a knowledge of the potential mechanism(s) by which CQDs intervene against xenobiotic insult-associated neurotoxicity in vitro and in vivo will facilitate their development, screening and transitioning to vertebrate models of pesticide-insult associated neurodegeneration. To bridge the mechanistic knowledge-gap we propose to image the intrinsically fluorescent CQDs in vitro and in vivo to determine their bioavailability, subcellular distribution and localization within SHSY-5Y cells and in the C. elegans nervous system. In combination with GFP-tagged α-synuclein expressing cells and transgenic worms (that express neurons and amyloid-forming proteins tagged with different colors), we will observe and identify where CQDs localize and their mechanism of action when the cell (and worm) is exposed to xenobiotic-insult. Outputs that will be quantitatively measured include (prevention of) amyloid-protein aggregation, reduction in the levels of free radicals and prevention of neuronal ablation. Furthermore, the use of confocal microscopy will allow us to examine pesticide-associated neurotoxicity, including neuronal development, neuronal absence or shrinkage, absence of cell bodies, and reduction in the fluorescence intensity and the effect of CQDs to overcome this toxicity. In conclusion, by examining and characterizing the nanoscale behavior of cell lines, neurons and molecules within live tissue in the presence of CQDs, we will reduce barriers in our understanding of the mechanistic role of CQDs in rescuing cell lines and organisms (C. elegans) from pesticide-associated neuronal dyshomeostasis. The data from this proposal will pave the way for their testing in vertebrate models of environmental toxicant-associated neurodegenerative outcomes. The proposed studies are relevant to public health given the incidence of Parkinson’s and Alzheimer’s diseases associated with pesticide exposure and are NIH mission-specific.
