A XAS Study of the Sulphur Environment Location in Human Neuromelanin and Synthetic Analogues P.R. Crippa, M. Eisner, S. Morante, F. Stellato, F. Vicentin, L. Zecca

Outline Parkinson’s Disease Neuromelanin X-Ray Absorption Spectroscopy Experiments Conclusions

Parkinson’s Disease Parkinsons’s Disease (PD): Progressive and fatal neurodegenerative disease Described in 1817 by James Parkinson Affects 1-2% of over 50 population <10% of PD is familial  majority of cases are sporadic 5 clearly defined genetic causes B. Thomas et al.(2007) Hum Mol Gen 16,R183.

Dopamine and acetylcholine are neurotrasmitters that control body movement Dopamine is produced in a small area in the base of the brain, called substantia nigra PD Pathogenesis In Parkinson’s disease NM pigmented neurons die synapse nerve terminal synaptic vesicle dopamine

Neuromelanin (NM): Dark pigment present in neurons of different brain areas Mixture of similar polymers which are made up of different structural units Accumulates with aging Contributes to the protection of neurons from oxidative processes Pigmented neurons are lost in Parkinson’s disease Relation between neuronal vulnerability and presence of NM still unclearNeuromelanin Neurons containing neuromelanin pigment

Polymeric compound composed by indolebenzothiazine groups XRD: multilayer (graphite-like) three dimensional structure with planar overlapped sheets consisting of cyclic molecules of indolebenzothiazine ring NM structure 15% Covalent bound peptide component 20% lipidic component Binds Fe and Zn Contains S NM L. Zecca et al.(2000) J Neurochem 74, 1758.

Sulphur Content Indolebenzothiazine groups contain S The peptidic part contains Cysteine (about 3% in weight) No Methionine detected Indolebenzothiazine S Cysteine

XAS Experiments X-ray Absorption Spectroscopy (XAS) study at the S K-edge Measurement of X-ray Absorption coefficient  (E) XAS features Selective for the absorber Local probe (~5 Å) No crystallization needed

Experimental Setup Incident energy selection X-ray mirror Synchrotron White beam X-ray source Monochromator Monochromatic beam Solid State Detector Ionization chambers Sample I F measurement I 0 and I measurement

EXAFS spectra are analyzed in terms of EXAFS region can be analyzed in the single scattering approximation: characteristic of atomic type indistinguishable for light atoms (N, O, C) introduce multiple scattering terms XAS spectrum. X-ray Absorption Near Edge Spectroscopy XANES region EXAFS region Extended X-ray Absorption Fine Structure  (E)  (k) E k XANES EXAFS

6 powder samples Human Neuromelanin (HNM) extracted from cerebellum 3 Synthetic Melanins prepared with different procedures 2 Model compounds (Cysteine and Trichochrome) Cerebellum Samples

Model Compounds Trichochrome S is present as heteroatom in aromatic rings Cysteine S is present in the amino acid side chain Cysteine Trichochrome

Synthetic Melanins Synthetic compounds similar to natural melanins Auto-oxidation Dopamine + Cysteine DAC Enzymatic Oxidation (With Tyrosinase) Dopamine + Cysteine DEC Dopa + Cysteine Pheomelanin Dopamine Dopa Cysteine Model of Synthetic Melanin

Spectra collected at the D04B bending magnet beam line of the Brazilian Synchrotron Light Laboratory Total Electron Yield (TEY) I 0 : incident current measured with a 0.75 μm carbon foil I: sample current collected with an electrometer   =I/I 0 Data Collection Synchrotron White beam Monochromator Monochromatic beam Carbon foil Electrometer Sample

Auger effect (Non-radiative de-excitation) -The photo-electron is emitted -The core hole is filled by an electron of an upper level -The energy is used up to eject an Auger electron -TEY: detection of all electrons emitted by the sample Total Electron Yield Fluorescence yield is low for low-Z elements (for S  F <0.1) X s, X A : emission probabilities of fluorescence photon and Auger electron

Results Model Compounds Cysteine – Trichochrome Significantly different spectral features Natural Melanin HNM different from both HNM & Model Compounds

Synthetic Melanins DAC DEC - Pheomelanin Similar spectral features Natural Melanin HNM Similar to Pheomelanin and DEC HNM & Synthetic Melanins

Difference Spectra D (E min > E 0 )D (E min < E 0 ) DAC-DEC DAC-Pheomelanin DEC-Pheomelanin HNM-DAC HNM-DEC HNM-Pheomelanin Qualitative findings are consistent with quantitative analysis of difference spectra

Data Analysis 1- Determination of edge energy E 0 2- Spectra shifted by E 0 3- Identification of two peaks (white line and first peak) in all spectra 4- P 1, P 2 : positions of the two peaks 5- A 1, A 2 : amplitudes of the two peaks E0E0 P1P1 P2P2

Data Analysis E 0 and P 1 are the same in all spectra P 2 is the same in all spectra but Cysteine SampleE0E0 P 1 =E 1 -E 0 P 2 =E 2 -E 0 Model Compounds Cysteine Trichochrome Synthetic Melanins DAC DEC Pheomelanin Natural Melanin HNM

CysteineTrichochromeDACDECPheoR HNM =64%36%0.8 HNM =28%72%0.4 HNM =0%100%0.9 HNM =35%65%1.6 HNM =10%90%2.2 HNM =61%39%0.9 HNM =25%75%8.2 Pheo =55%45%2.0 Fits of HNM are obtained minimizing Fit

—— HNM —— Fit HNM = 64% Cysteine + 36% Trichochrome

Pheomelanin = 55% Cysteine + 45% Trichochrome —— Pheomelanin —— Fit

Conclusions We have performed a structural study on natural Neuromelanin and Synthetic Analogues Identification of percentage of Trichochrome-like and a Cysteine-like components in Human Neuromelanin S structure is similar in Human Neuromelanin and Synthetic Melanins Pheomelanin and DEC