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

OutlineOutline

• Parkinson’s Disease

• Neuromelanin

• X-Ray Absorption Spectroscopy Experiments

• Conclusions

Parkinson’s DiseaseParkinson’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 unclear

NeuromelaninNeuromelanin

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

NMNM

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

SS

Cysteine

XAS ExperimentsXAS 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

IF measurement

I0 and I measurement

μ(E)d0eII(E) :LawBeer -Lambert

EXAFS spectra are analyzed in terms of

k

kkk

0

0

2

0Em2k

EXAFS region can be analyzed in the single scattering approximation:

)k(kR2sinee),k(AkR

NS)k( ii

)k(R2

i

k2i2

i

i20

i2i

2

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

TrichochromeS is present as heteroatom in aromatic rings

CysteineS 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

DopamineDopa

Cysteine

Model of Synthetic Melanin

•Spectra collected at the D04B

bending magnet beam line of the

Brazilian Synchrotron Light Laboratory

•Total Electron Yield (TEY)

I0: incident current measured

with a 0.75 μm carbon foil

I: sample current collected

with an electrometer

=I/I0

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)

Xs, XA: emission probabilities of fluorescence photon and Auger electron

AF

FF XX

Xη

Results

0

-4 0 4 8

CysteineTrichochromeHNM

E-E0

(eV)

Model Compounds

Cysteine – Trichochrome

Significantly different spectral features

Natural Melanin

HNM

different from both

HNM & Model Compounds

0

-4 0 4 8

DACDECPheomelaninHNM

E-E0 (eV)

Synthetic Melanins

DAC

DEC - Pheomelanin

Similar spectral features

Natural Melanin

HNM

Similar to Pheomelanin and DEC

HNM & Synthetic Melanins

Difference Spectra

D (Emin> E0) D (Emin< E0)

DAC-DEC 0.10 0.17

DAC-Pheomelanin 0.09 0.17

DEC-Pheomelanin 0.03 0.10

HNM-DAC 0.08 0.15

HNM-DEC 0.04 0.10

HNM-Pheomelanin 0.02 0.06

MAX

min

E

E

mn dE(E)μ(E)μD

Qualitative findings are consistent with

quantitative analysis of difference spectra

0

-5 0 5 10

DAC-DECDAC-PheomelaninDEC-PheomelaninHNM-DACHNM-DECHNM-Pheomelanin

E (eV)

-5 0 5

CysteineTrichochromeHNMDACDECPheomelanin

E-E0(eV)

Data Analysis

1- Determination of edge energy E0

2- Spectra shifted by E0

3- Identification of two peaks (white line and first peak) in all spectra

4- P1, P2: positions of the two peaks

5- A1, A2: amplitudes of the two peaks

E0

P1

P2

Data Analysis

E0 and P1 are the same in all spectra

P2 is the same in all spectra but Cysteine

Sample E0 P1=E1-E0 P2=E2-E0

Model Compounds

Cysteine 2470.5 1.0 6.0

Trichochrome 2470.5 1.5 8.5

Synthetic Melanins

DAC 2470.5 1.5 9.0

DEC 2471.0 1.0 8.0

Pheomelanin 2471.5 1.0 8.0

Natural Melanin

HNM 2471.0 1.0 8.0

Cysteine Trichochrome DAC DEC Pheo RHNM = 64% 36% 0.8HNM = 28% 72% 0.4HNM = 0% 100% 0.9HNM = 35% 65% 1.6HNM = 10% 90% 2.2HNM = 61% 39% 0.9HNM = 25% 75% 8.2Pheo = 55% 45% 2.0

(E)p)μ(1(E)pμp)(E,μ where

(E))(σ

(E)μp)(E,μ

2N

1R(p)

EXPm

EXPl

TH

E2EXP

2EXPTH

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