Competitive Binding of Parkinsonianinducing Neurotoxins to - PowerPoint PPT Presentation

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 (By NIH)

Neuromelanin (NM) • Dopaminergic neurons pigmented with neuromelanin • Hypothesized to be responsible for aiding in the controlled release of dopamine (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) • Fe 3+

Interaction between NM and Dopamine Under White Light 600 NM + Dopamine 500 NM NM + Dopamine 400 (Overnight) RFU 300 200 100 0 0 5 10 15 20 [Dopamine] mM

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) (By Harbin)

Effect of Altering [MPTP] on Interaction between MPTP and NM Under UV Light 900 800 NM + MPTP MPTP 700 NM 600 500 RFU 400 300 200 100 0 0 5 10 15 20 [MPTP] mM

Effect of Altering [NM] on Interaction between MPTP and NM Under White Light 700 600 500 400 RFU NM + MPTP 300 MPTP NM 200 100 0 0 0.1 0.2 0.3 0.4 0.5 0.6 [NM]

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

Interaction between Nicotine and NM Under White Light 6000 Nicotine Nic + NM 5000 NM 4000 RFU 3000 2000 1000 0 0 0.2 0.4 0.6 0.8 1 1.2 [Nic] mM

Competitive Binding of Nicotine and MPTP to NM Under UV Light 200 180 NM + Nic + MPTP 160 140 MPTP 120 RFU 100 NM + MPTP 80 60 40 20 0 0 1 2 3 4 [MPTP] mM

Competitive Binding of Nicotine and MPTP with Neuromelanin Under White Light 3000 2500 2000 RFU 1500 NM + MPTP + Nicotine NM + MPTP 1000 NM + Nicotine 500 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 [Nicotine] mM

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

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 C affeine (Singh, 2010) • Shown to have neuroprotective properties • Altering [Nicotine] • Further characterize interaction between dopamine and neuromelanin

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