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
• r 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+

100 200 300 400 500 600 5 10 15 20

RFU [Dopamine] mM

Interaction between NM and Dopamine Under White Light

NM + Dopamine NM NM + Dopamine (Overnight)

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)

100 200 300 400 500 600 700 800 900 5 10 15 20

RFU [MPTP] mM

Effect of Altering [MPTP] on Interaction between MPTP and NM Under UV Light

NM + MPTP MPTP NM

100 200 300 400 500 600 700 0.1 0.2 0.3 0.4 0.5 0.6

RFU [NM]

Effect of Altering [NM] on Interaction between MPTP and NM Under White Light

NM + MPTP MPTP NM

Nicotine

• Correlation between smokers and PD

(Nefzger, 1968)

• Neuroprotective effects (Singh, 2010)

(By Harbin)

1000 2000 3000 4000 5000 6000 0.2 0.4 0.6 0.8 1 1.2

RFU [Nic] mM

Interaction between Nicotine and NM Under White Light

Nicotine Nic + NM NM

20 40 60 80 100 120 140 160 180 200

1 2 3 4

RFU

[MPTP] mM

Competitive Binding of Nicotine and MPTP to NM Under UV Light

NM + Nic + MPTP MPTP NM + MPTP

500 1000 1500 2000 2500 3000 0.1 0.2 0.3 0.4 0.5 0.6 0.7

RFU [Nicotine] mM

Competitive Binding of Nicotine and MPTP with Neuromelanin Under White Light

NM + MPTP + Nicotine NM + MPTP NM + Nicotine

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 Caffeine

(Singh, 2010)

• Shown to have neuroprotective properties
• Altering [Nicotine]
• Further characterize interaction between

dopamine and neuromelanin

References

1. Bush WD, G. J. (2006). “The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface”. PNAS, 14785‐14789. 2. By BruceBlaus. When using this image in external sources it can be cited as:Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal

• f

Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. ‐ Own work, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=27924394 3. By Harbin ‐ Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=5714122 4. By Harbin ‐ Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=1629854 5. By NIH ‐ http://www.drugabuse.gov/pubs/teaching/largegifs/slide‐2.gif, Public Domain, https://commons.wikimedia.org/w/index.php?curid=37682508 6. D’Amato, Robert, Guillermo Alexander, Robert Schwartzman, et al. (1987). “Neuromelanin: A role in MPTP‐induced neurotoxicity”. Life Sciences 40 (8): 705‐712. 7. D’Amato, ZP Lipman, and SH Snyder. (1986). “Selectivity of the parkinsonian neurotoxin MPTP: toxic metabolite MPP+ binds to neuromelanin”. Science 231(4741): 987‐989. 8. Di Monte, D.A and S.A Jewell. (2‐14). “MPTP Neurotoxicity”. Encyclopedia of Neurological Sciences 2: 131‐134. 9. Dzierzega‐Lecznar A, K. S. (2004). “GC/MS Analysis of Thermally Degraded Neuromelanin from the Human Substantia Nigra”. J Am Soc Mass Spectrom, 920‐926. 10. Liu Y, S. J. (2003). “Isolation and Biophysical Studies of Natural Eumelanins: Applications of Imaging Technologies and Ultrafast Spectroscopy”. Pigment Cell Res, 606‐618. 11. McGeer PL, McGeer EG. (2008). “Glial reactions in Parkinson’s disease”. Mov Disord 23, 474‐483. 12. Nefzger MD, Quadfasel FA, and Karl VC. (1968). “A retrospective study of smoking in Parkinson’s disease”. American Journal of Epidemiology 88: 149‐158. 13. Singh, Kavita, Seema Singh, Naveen Kumar Singhal, et al. (2010). “Nicotine and caffeine‐mediated changes in gene expression patterns of MPTP‐lesioned mouse striatum: Implications in neuroprotection mechanism”. Chemico‐Biological Interactions 185 (2): 81‐93. 14. Thermo Scientific. (2013). “NanoDrop 3300 Fluorospectrometer”. Thermo Fisher Scientific Inc. Web. 15. W, C. (2004). “The Chemical Structure of Melanin”. Pigment Cell Res, 422‐424. 16. Wakamatsu K, F. K. (2003). “The structure of neuromelanin as studied by chemical degradative methods”. J Neurochem, 1015‐ 1023. 17. Zecca L, T. D. (2001). “Substantia nigra neuromelanin: structure, synthesis, and molecular behavior”. Mol Pathol, 414‐418. 18. Zecca, L. et al. (2002). “Interaction of Human Substantia Nigra Neuromelanin with Lipids and Peptides”. Journal of Neurochemistry 74 (4): 1758‐1765. 19. Zecca L, Z. F. (2006). “A proposed dual role of neuromelanin in the pathogenesis of Parkinson’s disease”. NEUROLOGY, S8‐S11.