Functional Effects of a Neuromelanin Analog on Dopaminergic Neurons in 3D Cell Culture: Biomaterials for Modeling Neurodegenerative Disease
Collins, William.
2019
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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 ... read morecomprises 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.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Pharmacology & Experimental Therapeutics.
Advisor: David Kaplan.
Committee: Emmanuel Pothos, David Greenblatt, Fiorenzo Omenetto, Thomas Nieland, and Abigail Koppes.
Keywords: Biomedical engineering, Neurosciences, and Pharmacology.read less - ID:
- ms35tn35c
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