Neurodegenerative diseases have been declared the ‘coming epidemic of the 21st century’ and remain largely underfunded research outside of philanthropic organizations. However, the challenge of therapeutic development is more fundamental stemming from the lack of human physiologically-relevant research models. A prime example of this can be found in Neuromelanin (NM), the dark pigment which comprises the substantia nigra pars compacta (SNpc). Found predominantly in primates, NM binds neurotoxic compounds and transition metals. Extraction of NM from the cortex is labor intensive, protein contaminated and often provides low yields. On the other hand, synthetic melanins possess limited metal binding characteristic of true biological melanin. Researchers have even ventured so far as to extract NM from humans for use in animal cell culture models.
Using our existing 3D brain model we incorporated a NM analogue to dopaminergic cultures to add physiological relevance. Here we describe extraction, characterization and incorporation of sepia melanin as a functional replacement for NM in our 3D cultures. It was found that this NM analogue chelated transition metals unlike synthetic melanin. The iron-precipitate (Fe-NM) exhibited tailorable properties of particle diameter and ellipticity with ultrasonic dissociation. Furthermore, the product produced peroxides (H2O2) via Fenton chemistry (n = 24, p < 0.001, Peroxide Assay) and depleted antioxidants and nutrients from cell culture (n = 8, p, 0.05, GSSH Assay).
The 3D model was made ‘human relevant’ by making use of Lund’s Human Mesencephalon (LUHMES) dopaminergic (DA) cells. We investigated the effects of this extracted NM and its precipitate on electrical activity by Local Field Potential (LFP) measurements and correlated the results against metabolic assays. We concluded that NM reliably reduces the activity of these neurons roughly two fold (n = 22, p < 0.05, individual trials). Additionally, free-radicals generated by the precipitate depleted cell nutrients/antioxidants and increased the carbonylated proteins (~8 Fold Increase, p < 0.05) suggesting oxidative damage to the proteome.
NM plays a fundamental role in disease progression. Theory regarding means by which NM may expedite neurodegeneration /dysregualtion within the human cortex is elaborated on including: (1) neurotransmitter oxidation (2) Neuromodulation (3) metallostasis. Future directions describing further experiments on neuro-inflammation, materials advancements, methods standardization and bioreactor integration are provided.
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|Advisor:||Kaplan, David L.|
|Commitee:||Greenblatt, David J., Koppes, Abigail N., Nieland, Thomas J., Omenetto, Fiorenzo G., Pothos, Emmanuel N.|
|School:||Sackler School of Graduate Biomedical Sciences (Tufts University)|
|Department:||Pharmacology & Experimental Therapeutics|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 80/07(E), Dissertation Abstracts International|
|Subjects:||Neurosciences, Pharmacology, Biomedical engineering|
|Keywords:||3D tissue models, Chelation, Dopaminergic neurons, Local field potential, Neuromelanin, Reactive oxygen species|
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