Aubrey Hernandez Dr. Robert Haining Spring 2016 Competitive Binding of Parkinsonian-inducing Neurotoxins to Neuromelanin Aubrey Hernandez Dr. Robert Haining Spring 2016

Parkinson’s Disease (PD) 7-10 million worldwide living with PD (Parkinson’s Disease Foundation, 2016) 1 million Americans (PD Foundation, 2016) 60,000 diagnosed each year (PD Foundation, 2016) Age: 50+ (PD Foundation, 2016) Symptoms: Tremors, bradykinesia, akinesia, cognition, emotions, postural instability (NIH, 2016)

Parkinson’s Disease (PD) Death of dopaminergic neurons in substantia nigra Dopamine can no longer reach the striatum Causes problems with controlled movements More specific on biology of PD (By NIH)

Neuromelanin (NM) Dopaminergic neurons pigmented with neuromelanin Hypothesized to be responsible for aiding in the controlled release of dopamine How does NM tie in (By BruceBlaus)

Research Questions Do neurotoxins interact directly with neuromelanin? Can the interaction between neuromelanin and neurotoxins be disrupted by nicotine? Is neuromelanin directly involved in the controlled release of dopamine?

Methodology NanoDrop 3300 (Thermo Scientific, 2013) Measures relative fluorescence under either white, UV or blue light RFU = Relative Fluorescence Unit Source of Neuromelanin Synthesized in the lab following protocol from Zecca’s Neuromelanin Interaction with Lipids and Peptides (Zecca, 2002) Fe3+

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Drug-induced Parkinson’s disease Converted to MPP+ Dopaminergic uptake system/neuromelanin (D’Amato, 1986) Nicotine (By Harbin)

Effect of Altering [NM] on Interaction between MPTP and NM Working on ways to quantitate it right now by the amount of iron it binds Effect of Altering [NM] on Interaction between MPTP and NM

Nicotine Correlation between smokers and PD (Nefzger, 1968) Neuroprotective effects (Singh, 2010) (By Harbin)

Conclusion Presence of NM causes MPTP’s RFU to disappear Indicates interaction Presence of nicotine causes NM RFU to increase dramatically

Conclusion Addition of nicotine to MPTP+NM solution causes a release of MPTP back into solution Shows nicotine can disrupt the interaction between MPTP and NM Addition of MPTP to nicotine+NM solution doesn’t cause a change in RFU Shows MPTP cannot disrupt the interaction between nicotine and NM

Further Experimentation MPP+ rather than MPTP 6-hydroxy dopamine (6-OHDA) Chloroquine (D’Amato, 1987) and Caffeine (Singh, 2010) Shown to have neuroprotective properties Altering [Nicotine] Further characterize interaction between dopamine and neuromelanin

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